@article{azcarate-peril_roach_marsh_chey_sandborn_ritter_savaiano_klaenhammer_2021, title={A double-blind, 377-subject randomized study identifies Ruminococcus, Coprococcus, Christensenella, and Collinsella as long-term potential key players in the modulation of the gut microbiome of lactose intolerant individuals by galacto-oligosaccharides}, volume={13}, ISSN={["1949-0984"]}, DOI={10.1080/19490976.2021.1957536}, abstractNote={Background. Our recent publication (Chey et al., Nutrients 2020) showed that a 30-day administration of pure galacto-oligosaccharides (GOS) significantly reduced symptoms and altered the fecal microbiome in patients with lactose intolerance (LI). Results. In this addendum, we performed an in-depth analysis of the fecal microbiome of the 377 LI patients randomized to one of two GOS doses (Low, 10-15 grams/day or High, 15-20 grams/day), or placebo in a multi-center, double-blinded, placebo-controlled trial. Sequencing of 16S rRNA amplicons was done on GOS or placebo groups at weeks zero (baseline), four (end of treatment), nine, 16 and 22. Taxa impacted by treatment and subsequent dairy consumption included lactose-fermenting species of Bifidobacterium, Lactobacillus, Lactococcus, and Streptococcus. Increased secondary fermentation microorganisms included Coprococcus and Ruminococcus species, Blautia producta, and Methanobrevibacterium. Finally, tertiary fermenters that use acetate to generate butyrate were also increased, including Faecalibacterium prausnitzii, Roseburia faecis, and C. eutactus. Conclusions. Results confirmed and expanded data on GOS microbiome modulation in LI individuals. Microbiome analysis at 16 and 22 weeks after treatment further suggested relatively long-term benefits when individuals continued consumption of dairy products.}, number={1}, journal={GUT MICROBES}, author={Azcarate-Peril, M. A. and Roach, J. and Marsh, A. and Chey, William D. and Sandborn, William J. and Ritter, Andrew J. and Savaiano, Dennis A. and Klaenhammer, T. R.}, year={2021}, month={Jan} }
 @article{goh_barrangou_klaenhammer_2021, title={In Vivo Transcriptome of Lactobacillus acidophilus and Colonization Impact on Murine Host Intestinal Gene Expression}, volume={12}, ISSN={["2150-7511"]}, url={https://doi.org/10.1128/mBio.03399-20}, DOI={10.1128/mBio.03399-20}, abstractNote={To date, our basis for comprehending the probiotic mechanisms of Lactobacillus acidophilus , one of the most widely consumed probiotic microbes, was largely limited to in vitro functional genomic studies. Using a germfree murine colonization model, in vivo -based transcriptional studies provided the first view of how L. acidophilus survives in the mammalian gut environment, including gene expression patterns linked to survival, efficient nutrient acquisition, stress adaptation, and host interactions.}, number={1}, journal={MBIO}, publisher={American Society for Microbiology}, author={Goh, Yong Jun and Barrangou, Rodolphe and Klaenhammer, Todd R.}, editor={Huffnagle, Gary B.Editor}, year={2021} }
 @article{klaenhammer_2019, title={Get Cultured: Eat Bacteria}, volume={10}, ISSN={["1941-1413"]}, DOI={10.1146/annurev-food-032818-121826}, abstractNote={The Klaenhammer group at North Carolina State University pioneered genomic applications in food microbiology and beneficial lactic acid bacteria used as starter cultures and probiotics. Dr. Todd Klaenhammer was honored to be the first food scientist elected to the National Academy of Sciences (2001). The program was recognized with the highest research awards presented by the American Dairy Science Association (Borden Award 1996), the Institute of Food Technologists (Nicholas Appert Medal, 2007), and the International Dairy Federation (Eli Metchnikoff Award in Biotechnology, 2010) as well as with the Outstanding Achievement Award from the University of Minnesota (2001) and the Oliver Max Gardner Award (2009) for outstanding research across the 16-campus University of North Carolina system. Dr. Klaenhammer is a fellow of the American Association for the Advancement of Science, the American Dairy Science Association, and the Institute of Food Technology. Over his career, six of his PhD graduate students were awarded the annual Kenneth Keller award for the outstanding PhD dissertation that year in the College of Agriculture and Life Sciences. He championed the use of basic microbiology and genomic approaches to set a platform for translational applications of beneficial microbes in foods and their use in food preservation and probiotics and as oral delivery vehicles for vaccines and biotherapeutics. Dr. Klaenhammer was also a founding and co-chief editor of the Annual Review of Food Science and Technology.}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 10}, author={Klaenhammer, Todd Robert}, year={2019}, pages={1–20} }
 @article{stout_sanozky-dawes_goh_crawley_klaenhammer_barrangou_2018, title={Deletion-based escape of CRISPR-Cas9 targeting in Lactobacillus gasseri}, volume={164}, ISSN={["1465-2080"]}, DOI={10.1099/mic.0.000689}, abstractNote={Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52–70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1–15 spacers, and systematically included the leader-proximal targeting spacer. This study shows that deletions of spacers within CRISPR arrays constitute a key escape mechanism to evade CRISPR targeting, while preserving the functionality of the CRISPR-Cas system. This mechanism enables cells to maintain an active immune system, but allows the uptake of potentially beneficial plasmids. Our study revealed the co-occurrence of other genomic mutations associated with various phenotypes, showing how this selection process uncovers population diversification.}, number={9}, journal={MICROBIOLOGY-SGM}, publisher={Microbiology Society}, author={Stout, Emily A. and Sanozky-Dawes, Rosemary and Goh, Yong Jun and Crawley, Alexandra B. and Klaenhammer, Todd R. and Barrangou, Rodolphe}, year={2018}, month={Sep}, pages={1098–1111} }
 @article{bumgardner_zhang_lavoy_andre_frank_kajikawa_klaenhammer_dean_2018, title={Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform}, volume={13}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0196950}, abstractNote={Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1β in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.}, number={5}, journal={PLOS ONE}, author={Bumgardner, Sara A. and Zhang, Lin and LaVoy, Alora S. and Andre, Barbara and Frank, Chad B. and Kajikawa, Akinobu and Klaenhammer, Todd R. and Dean, Gregg A.}, year={2018}, month={May} }
 @article{daughtry_johanningsmeier_sanozky-dawes_klaenhammer_barrangou_2018, title={Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber}, volume={280}, ISSN={["1879-3460"]}, DOI={10.1016/j.ijfoodmicro.2018.04.044}, abstractNote={Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm–11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials.}, journal={INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY}, publisher={Elsevier BV}, author={Daughtry, Katheryne V and Johanningsmeier, Suzanne D. and Sanozky-Dawes, Rosemary and Klaenhammer, Todd R. and Barrangou, Rodolphe}, year={2018}, month={Sep}, pages={46–56} }
 @article{moller_goh_rasmussen_cypryk_celebioglu_klaenhammer_svensson_abou hachem_2017, title={An Extracellular Cell-Attached Pullulanase Confers Branched alpha-Glucan Utilization in Human Gut Lactobacillus acidophilus}, volume={83}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00402-17}, abstractNote={ABSTRACT Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 ( La Pul13_14), and its role in branched α-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by La Pul13_14 and is abolished in a mutant strain lacking a functional La Pul13_14 gene. Hydrolysis kinetics of recombinant La Pul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest K m reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by β-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut. IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.}, number={12}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Moller, Marie S. and Goh, Yong Jun and Rasmussen, Kasper Bowig and Cypryk, Wojciech and Celebioglu, Hasan Ufuk and Klaenhammer, Todd R. and Svensson, Birte and Abou Hachem, Maher}, year={2017}, month={Jun} }
 @article{stout_klaenhammer_barrangou_2017, title={CRISPR-Cas Technologies and Applications in Food Bacteria}, volume={8}, ISSN={["1941-1421"]}, DOI={10.1146/annurev-food-072816-024723}, abstractNote={Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.}, number={1}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 8}, publisher={Annual Reviews}, author={Stout, Emily and Klaenhammer, Todd and Barrangou, Rodolphe}, year={2017}, pages={413–437} }
 @article{selle_goh_johnson_sarah_andersen_barrangou_klaenhammer_2017, title={Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus}, volume={8}, DOI={10.3389/fmicb.2017.00553}, abstractNote={Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and Manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated Manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of LTA removal on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.}, journal={Frontiers in Microbiology}, publisher={Frontiers Media SA}, author={Selle, Kurt and Goh, Yong J. and Johnson, Brant R. and Sarah, O’Flaherty and Andersen, Joakim M. and Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2017}, month={Apr} }
 @article{theilmann_goh_nielsen_klaenhammer_barrangou_abou hachem_2017, title={Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals}, volume={8}, ISSN={["2150-7511"]}, url={https://doi.org/10.1128/mBio.01421-17}, DOI={10.1128/mbio.01421-17}, abstractNote={Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host.IMPORTANCE Thousands of therapeutically active plant-derived compounds are widely present in berries, fruits, nuts, and beverages like tea and wine. The bioactivity and bioavailability of these compounds, which are typically glycosylated, are altered by microbial bioconversions in the human gut. Remarkably, little is known about the bioconversion of PGs by the gut microbial community, despite the significance of this metabolic facet to human health. Our work provides the first molecular insights into the metabolic routes of diet relevant and therapeutically active PGs by Lactobacillus acidophilus and related human gut lactobacilli. This taxonomic group is adept at metabolizing the glucoside moieties of select PG and externalizes their aglycones. The study highlights an important role of lactobacilli in the bioconversion of dietary PG and presents a framework from which to derive molecular insights into their metabolism by members of the human gut microbiota.}, number={6}, journal={MBIO}, publisher={American Society for Microbiology}, author={Theilmann, Mia C. and Goh, Yong Jun and Nielsen, Kristian Fog and Klaenhammer, Todd R. and Barrangou, Rodolphe and Abou Hachem, Maher}, editor={Martens, Eric and McFall-Ngai, Margaret J.Editors}, year={2017} }
 @article{johnson_o'flaherty_goh_carroll_barrangou_klaenhammer_2017, title={The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM}, volume={8}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2017.01185}, abstractNote={Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (∆prtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ∆prtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ∆prtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ∆prtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms’ immunomodulatory properties and interactions with some intestinal epithelial cell components.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Johnson, Brant R. and O'Flaherty, Sarah and Goh, Yong Jun and Carroll, Ian and Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2017}, month={Jun} }
 @article{johnson_klaenhammer_2016, title={AcmB Is an S-Layer-Associated beta-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM}, volume={82}, ISSN={["1098-5336"]}, DOI={10.1128/aem.02025-16}, abstractNote={ABSTRACT Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β- N -acetylglucosaminidase encoded by the gene lba0176 ( acmB ), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus . Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the Δ acmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the Δ acmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro . These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.}, number={18}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Johnson, Brant R. and Klaenhammer, Todd R.}, year={2016}, month={Sep}, pages={5687–5697} }
 @article{johnson_hymes_sanozky-dawes_henriksen_barrangou_klaenhammer_2016, title={Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli}, volume={82}, ISSN={["1098-5336"]}, url={https://doi.org/10.1128/AEM.01968-15}, DOI={10.1128/aem.01968-15}, abstractNote={ABSTRACT The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus , L. helveticus , L. crispatus , L. amylovorus , L. gallinarum , L. delbrueckii subsp. bulgaricus , L. gasseri , and L. johnsonii . While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus , which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.}, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Johnson, Brant R. and Hymes, Jeffrey and Sanozky-Dawes, Rosemary and Henriksen, Emily DeCrescenzo and Barrangou, Rodolphe and Klaenhammer, Todd R.}, editor={Nojiri, H.Editor}, year={2016}, month={Jan}, pages={134–145} }
 @article{list_mashayekhi_bertini_nevers_2016, title={Development of a Transportation System Simulation Manual Framework From Theory to Practice}, ISSN={["2169-4052"]}, DOI={10.3141/2561-06}, abstractNote={After several decades of advances, simulation has become an important tool in the modeling of transportation systems and is widely applied in practice. Guides have been created by organizations in several countries, and dozens of papers have been published in scientific journals on the theory and application of transport simulation; these works are aimed at guiding practitioners in the use of simulation tools. However, transport simulation still lacks a unified and comprehensive guide for use in practice. The lack of such a document leads to conflicts between modelers, agencies, and decision makers and allows inappropriate use of the models. The outcome is often inaccurate results, inefficient use of resources, and conflict. This paper reviews and analyzes the existing transportation simulation guides. It identifies gaps and limitations and proposes an outline for a comprehensive simulation manual that is based on stakeholder input. Review of the existing guidance documents reveals that almost all these documents focus on traffic operations, and they provide either broad guidelines for building simulation models or advice on using a specific software product. Other issues, particularly those related to topics such as safety assessments, environmental impacts, public transportation, pedestrians, bicycles, simulation algorithms, agent-based simulation, and multimodal simulation, are addressed in only a cursory fashion. This paper proposes a possible structure for a transportation system simulation manual that would cover the limitations and gaps in the existing literature. The proposed document would consist of five volumes: concepts, model building, verification and validation, results analysis, and case studies and supplementary materials.}, number={2561}, journal={TRANSPORTATION RESEARCH RECORD}, author={List, George and Mashayekhi, Mehdi and Bertini, Robert L. and Nevers, Brandon}, year={2016}, pages={45–52} }
 @article{celebioglu_ejby_majumder_kobler_goh_thorsen_schmidt_o'flaherty_abou hachem_lahtinen_et al._2016, title={Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic}, volume={16}, ISSN={["1615-9861"]}, DOI={10.1002/pmic.201500212}, abstractNote={Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.}, number={9}, journal={PROTEOMICS}, author={Celebioglu, Hasan Ufuk and Ejby, Morten and Majumder, Avishek and Kobler, Carsten and Goh, Yong Jun and Thorsen, Kristian and Schmidt, Bjarne and O'Flaherty, Sarah and Abou Hachem, Maher and Lahtinen, Sampo J. and et al.}, year={2016}, month={May}, pages={1361–1375} }
 @article{hymes_johnson_barrangou_klaenhammer_2016, title={Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM}, volume={82}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00024-16}, abstractNote={ABSTRACT Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB -deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro . Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group.}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Hymes, Jeffrey P. and Johnson, Brant R. and Barrangou, Rodolphe and Klaenhammer, Todd R.}, editor={Dudley, E. G.Editor}, year={2016}, month={May}, pages={2676–2685} }
 @article{douglas_azcarate-peril_klaenhammer_2016, title={Genomic evolution of lactic acid bacteria: From single gene function to the pan-genome}, DOI={10.1002/9781118868386.ch3}, journal={Biotechnology of Lactic Acid Bacteria: Novel Applications, 2nd Edition}, author={Douglas, G. L. and Azcarate-Peril, M. A. and Klaenhammer, T. R.}, year={2016}, pages={32–54} }
 @article{durmaz_hu_aroian_klaenhammer_2016, title={Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic}, volume={82}, ISSN={["1098-5336"]}, DOI={10.1128/aem.02365-15}, abstractNote={ABSTRACT The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis , showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.}, number={4}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Durmaz, Evelyn and Hu, Yan and Aroian, Raffi V. and Klaenhammer, Todd R.}, year={2016}, month={Feb}, pages={1286–1294} }
 @article{o'flaherty_klaenhammer_2016, title={Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus}, volume={82}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01533-16}, abstractNote={ABSTRACT Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid-based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating the antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including Lactobacillus acidophilus , have shown potential for mucosal vaccine delivery, as L. acidophilus is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells, including dendritic cells. In this study, we successfully expressed C. botulinum and B. anthracis antigens in the probiotic L. acidophilus strain NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of L. acidophilus .}, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2016}, month={Oct}, pages={6091–6101} }
 @misc{hymes_klaenhammer_2016, title={Stuck in the Middle: Fibronectin-Binding Proteins in Gram-Positive Bacteria}, volume={7}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2016.01504}, abstractNote={Fibronectin is a multidomain glycoprotein found ubiquitously in human body fluids and extracellular matrices of a variety of cell types from all human tissues and organs, including intestinal epithelial cells. Fibronectin plays a major role in the regulation of cell migration, tissue repair, and cell adhesion. Importantly, fibronectin also serves as a common target for bacterial adhesins in the gastrointestinal tract. Fibronectin-binding proteins (FnBPs) have been identified and characterized in a wide variety of host-associated bacteria. Single bacterial species can contain multiple, diverse FnBPs. In pathogens, some FnBPs contribute to virulence via host cell attachment, invasion, and interference with signaling pathways. Although FnBPs in commensal and probiotic strains are not sufficient to confer virulence, they are essential for attachment to their ecological niches. Here we describe the interaction between human fibronectin and bacterial adhesins by highlighting the FnBPs of Gram-positive pathogens and commensals. We provide an overview of the occurrence and diversity of FnBPs with a focus on the model pathogenic organisms in which FnBPs are most characterized. Continued investigation of FnBPs is needed to fully understand their divergence and specificity in both pathogens and commensals.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Hymes, Jeffrey P. and Klaenhammer, Todd R.}, year={2016}, month={Sep} }
 @article{selle_klaenhammer_barrangou_2015, title={CRISPR-based screening of genomic island excision events in bacteria}, volume={112}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1508525112}, abstractNote={Significance The development of Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated genes (CAS)–based technology for targeted genome editing has revolutionized molecular biology approaches, but significant and outstanding gaps exist for applications in bacteria, the native hosts of these adaptive immune systems. This study shows that CRISPR-Cas systems can be directed to target and delete genomic islands that are flanked by insertion-sequence elements and devoid of essential genes. Naturally occurring minor subpopulations harboring deletions in genomic islands were identified and readily isolated using CRISPR-Cas screening. Promising applications of this approach can define minimal bacterial genomes, determine essential genes, and characterize genetically heterogeneous bacterial populations.}, number={26}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Selle, Kurt and Klaenhammer, Todd R. and Barrangou, Rodolphe}, year={2015}, month={Jun}, pages={8076–8081} }
 @article{sun_harris_mccann_guo_argimon_zhang_yang_jeffery_cooney_kagawa_et al._2015, title={Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera}, volume={6}, ISSN={["2041-1723"]}, DOI={10.1038/ncomms9322}, abstractNote={Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.}, journal={NATURE COMMUNICATIONS}, publisher={Springer Nature}, author={Sun, Zhihong and Harris, Hugh M. B. and McCann, Angela and Guo, Chenyi and Argimon, Silvia and Zhang, Wenyi and Yang, Xianwei and Jeffery, Ian B. and Cooney, Jakki C. and Kagawa, Todd F. and et al.}, year={2015}, month={Sep} }
 @article{goh_klaenhammer_2015, title={Genetic Mechanisms of Prebiotic Oligosaccharide Metabolism in Probiotic Microbes}, volume={6}, ISSN={["1941-1421"]}, DOI={10.1146/annurev-food-022814-015706}, abstractNote={Recent insights into the relationship between the human gut and its resident microbiota have revolutionized our appreciation of this symbiosis and its impact on health and disease development. Accumulating evidence on probiotic and prebiotic interventions has demonstrated promising effects on promoting gastrointestinal health by modulating the microbiota toward the enrichment of beneficial microorganisms. However, the precise mechanisms of how prebiotic nondigestible oligosaccharides are metabolized by these beneficial microbes in vivo remain largely unknown. Genome sequencing of probiotic lactobacilli and bifidobacteria has revealed versatile carbohydrate metabolic gene repertoires dedicated to the catabolism of various oligosaccharides. In this review, we highlight recent findings on the genetic mechanisms involved in the utilization of prebiotic fructooligosaccharides, β-galactooligosaccharides, human milk oligosaccharides, and other prebiotic candidates by these probiotic microbes.}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 6}, author={Goh, Yong Jun and Klaenhammer, Todd R.}, year={2015}, pages={137–156} }
 @article{kajikawa_zhang_lavoy_bumgardner_klaenhammer_dean_2015, title={Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0141713}, abstractNote={Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.}, number={10}, journal={PLOS ONE}, author={Kajikawa, Akinobu and Zhang, Lin and LaVoy, Alora and Bumgardner, Sara and Klaenhammer, Todd R. and Dean, Gregg A.}, year={2015}, month={Oct} }
 @article{sanozky-dawes_selle_o'flaherty_klaenhammer_barrangou_2015, title={Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri}, volume={161}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.000129}, abstractNote={Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5′-NTAA-3′ flanking the 3′ end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.}, journal={MICROBIOLOGY-SGM}, author={Sanozky-Dawes, Rosemary and Selle, Kurt and O'Flaherty, Sarah and Klaenhammer, Todd and Barrangou, Rodolphe}, year={2015}, month={Sep}, pages={1752–1761} }
 @article{lightfoot_selle_yang_goh_sahay_zadeh_owen_colliou_li_johannssen_et al._2015, title={SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis}, volume={34}, ISSN={["1460-2075"]}, DOI={10.15252/embj.201490296}, abstractNote={Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.}, number={7}, journal={EMBO JOURNAL}, author={Lightfoot, Yaima L. and Selle, Kurt and Yang, Tao and Goh, Yong Jun and Sahay, Bikash and Zadeh, Mojgan and Owen, Jennifer L. and Colliou, Natacha and Li, Eric and Johannssen, Timo and et al.}, year={2015}, month={Apr}, pages={881–895} }
 @article{call_goh_selle_klaenhammer_o'flaherty_2015, title={Sortase-deficient lactobacilli: effect on immunomodulation and gut retention}, volume={161}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.000007}, abstractNote={Surface proteins of probiotic microbes, including Lactobacillus acidophilus and Lactobacillus gasseri, are believed to promote retention in the gut and mediate host–bacterial communications. Sortase, an enzyme that covalently couples a subset of extracellular proteins containing an LPXTG motif to the cell surface, is of particular interest in characterizing bacterial adherence and communication with the mucosal immune system. A sortase gene, srtA, was identified in L. acidophilus NCFM (LBA1244) and L. gasseri ATCC 33323 (LGAS_0825). Additionally, eight and six intact sortase-dependent proteins were predicted in L. acidophilus and L. gasseri, respectively. Due to the role of sortase in coupling these proteins to the cell wall, ΔsrtA deletion mutants of L. acidophilus and L. gasseri were created using the upp-based counterselective gene replacement system. Inactivation of sortase did not cause significant alteration in growth or survival in simulated gastrointestinal juices. Meanwhile, both ΔsrtA mutants showed decreased adhesion to porcine mucin in vitro. Murine dendritic cells exposed to the ΔsrtA mutant of L. acidophilus or L. gasseri induced lower levels of pro-inflammatory cytokines TNF-α and IL-12, respectively, compared with the parent strains. In vivo co-colonization of the L. acidophilus ΔsrtA mutant and its parent strain in germ-free 129S6/SvEv mice resulted in a significant one-log reduction of the ΔsrtA mutant population. Additionally, a similar reduction of the ΔsrtA mutant was observed in the caecum. This study shows for the first time that sortase-dependent proteins contribute to gut retention of probiotic microbes in the gastrointestinal tract.}, journal={MICROBIOLOGY-SGM}, author={Call, Emma K. and Goh, Yong Jun and Selle, Kurt and Klaenhammer, Todd R. and O'Flaherty, Sarah}, year={2015}, month={Feb}, pages={311–321} }
 @article{sanders_klaenhammer_ouwehand_pot_johansen_heimbach_marco_tennila_ross_franz_et al._2014, title={Effects of genetic, processing, or product formulation changes on efficacy and safety of probiotics}, volume={1309}, ISSN={["0077-8923"]}, DOI={10.1111/nyas.12363}, abstractNote={Commercial probiotic strains for food or supplement use can be altered in different ways for a variety of purposes. Production conditions for the strain or final product may be changed to address probiotic yield, functionality, or stability. Final food products may be modified to improve flavor and other sensory properties, provide new product formats, or respond to market opportunities. Such changes can alter the expression of physiological traits owing to the live nature of probiotics. In addition, genetic approaches may be used to improve strain attributes. This review explores whether genetic or phenotypic changes, by accident or design, might affect the efficacy or safety of commercial probiotics. We highlight key issues important to determining the need to re-confirm efficacy or safety after strain improvement, process optimization, or product formulation changes. Research pinpointing the mechanisms of action for probiotic function and the development of assays to measure them are greatly needed to better understand if such changes have a substantive impact on probiotic efficacy.}, journal={ANNALS REPORTS}, author={Sanders, Mary Ellen and Klaenhammer, Todd R. and Ouwehand, Arthur C. and Pot, Bruno and Johansen, Eric and Heimbach, James T. and Marco, Maria L. and Tennila, Julia and Ross, R. Paul and Franz, Charles and et al.}, year={2014}, pages={1–18} }
 @misc{johnson_klaenhammer_2014, title={Impact of genomics on the field of probiotic research: historical perspectives to modern paradigms}, volume={106}, ISSN={["1572-9699"]}, DOI={10.1007/s10482-014-0171-y}, abstractNote={For thousands of years, humans have safely consumed microorganisms through fermented foods. Many of these bacteria are considered probiotics, which act through diverse mechanisms to confer a health benefit to the host. However, it was not until the availability of whole-genome sequencing and the era of genomics that mechanisms of probiotic efficacy could be discovered. In this review, we explore the history of the probiotic concept and the current standard of integrated genomic techniques to discern the complex, beneficial relationships between probiotic microbes and their hosts.}, number={1}, journal={ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY}, author={Johnson, Brant R. and Klaenhammer, Todd R.}, year={2014}, month={Jul}, pages={141–156} }
 @misc{goh_klaenhammer_2014, title={Insights into glycogen metabolism in Lactobacillus acidophilus: impact on carbohydrate metabolism, stress tolerance and gut retention}, volume={13}, ISSN={["1475-2859"]}, DOI={10.1186/s12934-014-0094-3}, abstractNote={In prokaryotic species equipped with glycogen metabolism machinery, the co-regulation of glycogen biosynthesis and degradation has been associated with the synthesis of energy storage compounds and various crucial physiological functions, including global cellular processes such as carbon and nitrogen metabolism, energy sensing and production, stress response and cell-cell communication. In addition, the glycogen metabolic pathway was proposed to serve as a carbon capacitor that regulates downstream carbon fluxes, and in some microorganisms the ability to synthesize intracellular glycogen has been implicated in host persistence. Among lactobacilli, complete glycogen metabolic pathway genes are present only in select species predominantly associated with mammalian hosts or natural environments. This observation highlights the potential involvement of glycogen biosynthesis in probiotic activities and persistence of intestinal lactobacilli in the human gastrointestinal tract. In this review, we summarize recent findings on (i) the presence and potential ecological distribution of glycogen metabolic pathways among lactobacilli, (ii) influence of carbon substrates and growth phases on glycogen metabolic gene expression and glycogen accumulation in L. acidophilus, and (iii) the involvement of glycogen metabolism on growth, sugar utilization and bile tolerance. Our present in vivo studies established the significance of glycogen biosynthesis on the competitive retention of L. acidophilus in the mouse intestinal tract, demonstrating for the first time that the ability to synthesize intracellular glycogen contributes to gut fitness and retention among probiotic microorganisms.}, journal={MICROBIAL CELL FACTORIES}, author={Goh, Yong Jun and Klaenhammer, Todd R.}, year={2014}, month={Nov} }
 @article{barrangou_klaenhammer_2014, title={Microbiology bacteria get vaccinated}, volume={513}, number={7517}, journal={Nature}, author={Barrangou, R. and Klaenhammer, T. R.}, year={2014}, pages={175–176} }
 @article{moller_goh_viborg_andersen_klaenhammer_svensson_abou hachem_2014, title={Recent insight in alpha-glucan metabolism in probiotic bacteria}, volume={69}, ISSN={["1336-9563"]}, DOI={10.2478/s11756-014-0367-7}, abstractNote={α-Glucans from bacterial exo-polysaccharides or diet, e.g., resistant starch, legumes and honey are abundant in the human gut and fermentation of resistant fractions of these α-glucans by probiotic lactobacilli and bifidobacteria impacts human health positively. The ability to degrade polymeric α-glucans is confined to few strains encoding extracellular amylolytic activities of glycoside hydrolase (GH) family 13. Debranching pullulanases of the subfamily GH13_14 are the most common extracellular GH13 enzymes in lactobacilli, whereas corresponding enzymes are mainly α-amylases and amylopullulanases in bifidobacteria. Extracellular GH13 enzymes from both genera are frequently modular and possess starch binding domains, which are important for efficient catalysis and possibly to mediate attachment of cells to starch granules. α-1,6-Linked glucans, e.g., isomalto-oligosaccharides are potential prebiotics. The enzymes targeting these glucans are the most abundant intracellular GHs in bifidobacteria and lactobacilli. A phosphoenolpyruvate-dependent phosphotransferase system and a GH4 phospho-α-glucosidase are likely involved in metabolism of isomaltose and isomaltulose in probiotic lactobacilli based on transcriptional analysis. This specificity within GH4 is unique for lactobacilli, whereas canonical GH13 31 α-1,6-glucosidases active on longer α-1,6-gluco-oligosaccharides are ubiquitous in bifidobacteria and lactobacilli. Malto-oligosaccharide utilization operons encode more complex, diverse, and less biochemically understood activities in bifidobacteria compared to lactobacilli, where important members have been recently described at the molecular level. This review presents some aspects of α-glucan metabolism in probiotic bacteria and highlights vague issues that merit experimental effort, especially oligosaccharide uptake and the functionally unassigned enzymes, featuring in this important facet of glycan turnover by members of the gut microbiota.}, number={6}, journal={BIOLOGIA}, author={Moller, Marie S. and Goh, Yong Jun and Viborg, Alexander H. and Andersen, Joakim M. and Klaenhammer, Todd R. and Svensson, Birte and Abou Hachem, Maher}, year={2014}, month={Jun}, pages={713–721} }
 @article{baugher_durmaz_klaenhammer_2014, title={Spontaneously Induced Prophages in Lactobacillus gasseri Contribute to Horizontal Gene Transfer}, volume={80}, ISSN={["1098-5336"]}, DOI={10.1128/aem.04092-13}, abstractNote={Lactobacillus gasseri is an endogenous species of the human gastrointestinal tract and vagina. With recent advances in microbial taxonomy, phylogenetics, and genomics, L. gasseri is recognized as an important commensal and is increasingly being used in probiotic formulations. L. gasseri strain ADH is lysogenic and harbors two inducible prophages. In this study, prophage adh was found to spontaneously induce in broth cultures to populations of ∼ 10(7) PFU/ml by stationary phase. The adh prophage-cured ADH derivative NCK102 was found to harbor a new, second inducible phage, vB_Lga_jlb1 (jlb1). Phage jlb1 was sequenced and found to be highly similar to the closely related phage LgaI, which resides as two tandem prophages in the neotype strain L. gasseri ATCC 33323. The common occurrence of multiple prophages in L. gasseri genomes, their propensity for spontaneous induction, and the high degree of homology among phages within multiple species of Lactobacillus suggest that temperate bacteriophages likely contribute to horizontal gene transfer (HGT) in commensal lactobacilli. In this study, the host ranges of phages adh and jlb1 were determined against 16 L. gasseri strains. The transduction range and the rate of spontaneous transduction were investigated in coculture experiments to ascertain the degree to which prophages can promote HGT among a variety of commensal and probiotic lactobacilli. Both adh and jlb1 particles were confirmed to mediate plasmid transfer. As many as ∼10(3) spontaneous transductants/ml were obtained. HGT by transducing phages of commensal lactobacilli may have a significant impact on the evolution of bacteria within the human microbiota.}, number={11}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Baugher, J. L. and Durmaz, E. and Klaenhammer, T. R.}, year={2014}, month={Jun}, pages={3508–3517} }
 @article{goh_klaenhammer_2013, title={A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon}, volume={89}, ISSN={["1365-2958"]}, DOI={10.1111/mmi.12338}, abstractNote={Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.}, number={6}, journal={MOLECULAR MICROBIOLOGY}, author={Goh, Yong Jun and Klaenhammer, Todd R.}, year={2013}, month={Sep}, pages={1187–1200} }
 @misc{selle_klaenhammer_2013, title={Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health}, volume={37}, ISSN={["1574-6976"]}, DOI={10.1111/1574-6976.12021}, abstractNote={Certain lactic acid bacteria (LAB) have the capacity to occupy mucosal niches of humans, including the oral cavity, gastrointestinal tract, and vagina. Among commensal, LAB are species of the acidophilus complex, which have proven to be a substantial reservoir for microorganisms with probiotic attributes. Specifically, Lactobacillus gasseri is an autochthonous microorganism which has been evaluated for probiotic activity based on the availability of genome sequence and species-specific adaptation to the human mucosa. Niche-related characteristics of L. gasseri contributing to indigenous colonization include tolerance of low pH environments, resistance to bile salts, and adhesion to the host epithelium. In humans, L. gasseri elicits various health benefits through its antimicrobial activity, bacteriocin production, and immunomodulation of the innate and adaptive systems. The genomic and empirical evidence supporting use of L. gasseri in probiotic applications is substantiated by clinical trial data displaying maintenance of vaginal homeostasis, mitigation of Helicobacter pylori infection, and amelioration of diarrhea.}, number={6}, journal={FEMS MICROBIOLOGY REVIEWS}, author={Selle, Kurt and Klaenhammer, Todd R.}, year={2013}, month={Nov}, pages={915–935} }
 @article{johnson_selle_o'flaherty_goh_klaenhammer_2013, title={Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM}, volume={159}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.070755-0}, abstractNote={Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.}, journal={MICROBIOLOGY-SGM}, author={Johnson, Brant and Selle, Kurt and O'Flaherty, Sarah and Goh, Yong Jun and Klaenhammer, Todd}, year={2013}, month={Nov}, pages={2269–2282} }
 @article{savaiano_ritter_klaenhammer_james_longcore_chandler_walker_foyt_2013, title={Improving lactose digestion and symptoms of lactose intolerance with a novel galacto-oligosaccharide (RP-G28): a randomized, double-blind clinical trial}, volume={12}, ISSN={["1475-2891"]}, DOI={10.1186/1475-2891-12-160}, abstractNote={Lactose intolerance (LI) is a common medical problem with limited treatment options. The primary symptoms are abdominal pain, diarrhea, bloating, flatulence, and cramping. Limiting dairy foods to reduce symptoms contributes to low calcium intake and the risk for chronic disease. Adaptation of the colon bacteria to effectively metabolize lactose is a novel and potentially useful approach to improve lactose digestion and tolerance. RP-G28 is novel galacto-oligosaccharide (GOS) being investigated to improve lactose digestion and the symptoms of lactose intolerance in affected patients. A randomized, double-blind, parallel group, placebo-controlled study was conducted at 2 sites in the United States. RP-G28 or placebo was administered to 85 patients with LI for 35 days. Post-treatment, subjects reintroduced dairy into their daily diets and were followed for 30 additional days to evaluate lactose digestion as measured by hydrogen production and symptom improvements via a patient-reported symptom assessment instrument. Lactose digestion and symptoms of LI trended toward improvement on RP-G28 at the end of treatment and 30 days post-treatment. A reduction in abdominal pain was also demonstrated in the study results. Fifty percent of RP-G28 subjects with abdominal pain at baseline reported no abdominal pain at the end of treatment and 30 days post treatment (p = 0.0190). RP-G28 subjects were also six times more likely to claim lactose tolerance post-treatment once dairy foods had been re-introduced into their diets (p = 0.0389). Efficacy trends and favorable safety/tolerability findings suggest that RP-G28 appears to be a potentially useful approach for improving lactose digestion and LI symptoms. The concurrent reduction in abdominal pain and improved overall tolerance could be a meaningful benefit to lactose intolerant individuals. ClinicalTrials.gov NCT01113619 .}, journal={NUTRITION JOURNAL}, author={Savaiano, Dennis A. and Ritter, Andrew J. and Klaenhammer, Todd R. and James, Gareth M. and Longcore, Amy T. and Chandler, Justin R. and Walker, W. Allan and Foyt, Howard L.}, year={2013}, month={Dec} }
 @article{petschow_dore_hibberd_dinan_reid_blaser_cani_degnan_foster_gibson_et al._2013, title={Probiotics, prebiotics, and the host microbiome: the science of translation}, volume={1306}, DOI={10.1111/nyas.12303}, abstractNote={Recent advances in our understanding of the community structure and function of the human microbiome have implications for the potential role of probiotics and prebiotics in promoting human health. A group of experts recently met to review the latest advances in microbiota/microbiome research and discuss the implications for development of probiotics and prebiotics, primarily as they relate to effects mediated via the intestine. The goals of the meeting were to share recent advances in research on the microbiota, microbiome, probiotics, and prebiotics, and to discuss these findings in the contexts of regulatory barriers, evolving healthcare environments, and potential effects on a variety of health topics, including the development of obesity and diabetes; the long-term consequences of exposure to antibiotics early in life to the gastrointestinal (GI) microbiota; lactose intolerance; and the relationship between the GI microbiota and the central nervous system, with implications for depression, cognition, satiety, and mental health for people living in developed and developing countries. This report provides an overview of these discussions.}, journal={ANNALS REPORTS}, author={Petschow, Bryon and Dore, Joel and Hibberd, Patricia and Dinan, Timothy and Reid, Gregor and Blaser, Martin and Cani, Patrice D. and Degnan, Fred H. and Foster, Jane and Gibson, Glenn and et al.}, year={2013}, pages={1–17} }
 @misc{abou hachem_andersen_barrangou_moller_fredslund_majumder_ejby_lahtinen_jacobsen_lo leggio_et al._2013, title={Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria}, volume={31}, ISSN={["1029-2446"]}, DOI={10.3109/10242422.2013.828048}, abstractNote={In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota.In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics.Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.}, number={4}, journal={BIOCATALYSIS AND BIOTRANSFORMATION}, publisher={Informa UK Limited}, author={Abou Hachem, Maher and Andersen, Joakim M. and Barrangou, Rodolphe and Moller, Marie S. and Fredslund, Folmer and Majumder, Avishek and Ejby, Morten and Lahtinen, Sampo J. and Jacobsen, Susanne and Lo Leggio, Leila and et al.}, year={2013}, month={Aug}, pages={226–235} }
 @misc{call_klaenhammer_2013, title={Relevance and application of sortase and sortase-dependent proteins in lactic acid bacteria}, volume={4}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2013.00073}, abstractNote={Lactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria found in a vast array of environments including dairy products and the human gastrointestinal tract (GIT). In both niches, surface proteins play a crucial role in mediating interactions with the surrounding environment. The sortase enzyme is responsible for covalently coupling a subset of sortase-dependent proteins (SDPs) to the cell wall of Gram-positive organisms through recognition of a conserved C-terminal LPXTG motif. Genomic sequencing of LAB and annotation has allowed for the identification of sortase and SDPs. Historically, sortase and SDPs were predominately investigated for their role in mediating pathogenesis. Identification of these proteins in LAB has shed light on their important roles in mediating nutrient acquisition through proteinase P as well as positive probiotic attributes including adhesion, mucus barrier function, and immune signaling. Furthermore, sortase expression signals in LAB have been exploited as a means to develop oral vaccines targeted to the GIT. In this review, we examine the collection of studies which evaluate sortase and SDPs in select species of dairy-associated and health promoting LAB.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Call, Emma K. and Klaenhammer, Todd R.}, year={2013}, month={Apr} }
 @article{andersen_barrangou_abou hachem_lahtinen_goh_svensson_klaenhammer_2013, title={Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04}, volume={14}, ISSN={["1471-2164"]}, DOI={10.1186/1471-2164-14-312}, abstractNote={Abstract Background Probiotic bifidobacteria in combination with prebiotic carbohydrates have documented positive effects on human health regarding gastrointestinal disorders and improved immunity, however the selective routes of uptake remain unknown for most candidate prebiotics. The differential transcriptomes of Bifidobacterium animalis subsp. lactis Bl-04, induced by 11 potential prebiotic oligosaccharides were analyzed to identify the genetic loci involved in the uptake and catabolism of α- and β-linked hexoses, and β-xylosides. Results The overall transcriptome was modulated dependent on the type of glycoside (galactosides, glucosides or xylosides) utilized. Carbohydrate transporters of the major facilitator superfamily (induced by gentiobiose and β-galacto-oligosaccharides (GOS)) and ATP-binding cassette (ABC) transporters (upregulated by cellobiose, GOS, isomaltose, maltotriose, melibiose, panose, raffinose, stachyose, xylobiose and β-xylo-oligosaccharides) were differentially upregulated, together with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77. Sequence analysis of the identified solute-binding proteins that determine the specificity of ABC transporters revealed similarities in the breadth and selectivity of prebiotic utilization by bifidobacteria. Conclusion This study identified the differential gene expression for utilization of potential prebiotics highlighting the extensive capabilities of Bifidobacterium lactis Bl-04 to utilize oligosaccharides. Results provide insights into the ability of this probiotic microbe to utilize indigestible carbohydrates in the human gastrointestinal tract.}, number={1}, journal={BMC GENOMICS}, publisher={Springer Nature}, author={Andersen, Joakim M. and Barrangou, Rodolphe and Abou Hachem, Maher and Lahtinen, Sampo J. and Goh, Yong Jun and Svensson, Birte and Klaenhammer, Todd R.}, year={2013}, month={May} }
 @article{khazaie_zadeh_khan_bere_gounari_dennis_blatner_owen_klaenhammer_mohamadzadeh_2012, title={Abating colon cancer polyposis by Lactobacillus acidophilus deficient in lipoteichoic acid}, volume={109}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1207230109}, abstractNote={An imbalance of commensal bacteria and their gene products underlies mucosal and, in particular, gastrointestinal inflammation and a predisposition to cancer. Lactobacillus species have received considerable attention as examples of beneficial microbiota. We have reported previously that deletion of the phosphoglycerol transferase gene that is responsible for lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus (NCK2025) rendered this bacterium able to significantly protect mice against induced colitis when delivered orally. Here we report that oral treatment with LTA-deficient NCK2025 normalizes innate and adaptive pathogenic immune responses and causes regression of established colonic polyps. This study reveals the proinflammatory role of LTA and the ability of LTA-deficient L. acidophilus to regulate inflammation and protect against colonic polyposis in a unique mouse model.}, number={26}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Khazaie, Khashayarsha and Zadeh, Mojgan and Khan, Mohammad W. and Bere, Praveen and Gounari, Fotini and Dennis, Kirsten and Blatner, Nichole R. and Owen, Jennifer L. and Klaenhammer, Todd R. and Mohamadzadeh, Mansour}, year={2012}, month={Jun}, pages={10462–10467} }
 @article{carroll_ringel-kulka_siddle_klaenhammer_ringel_2012, title={Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage}, volume={7}, DOI={10.1371/journal.pone.0046953}, abstractNote={The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.}, number={10}, journal={PLoS One}, author={Carroll, I. M. and Ringel-Kulka, T. and Siddle, J. P. and Klaenhammer, T. R. and Ringel, Y.}, year={2012} }
 @article{kajikawa_zhang_long_nordone_stoeker_lavoy_bumgardner_klaenhammer_dean_2012, title={Construction and Immunological Evaluation of Dual Cell Surface Display of HIV-1 Gag and Salmonella enterica Serovar Typhimurium FliC in Lactobacillus acidophilus for Vaccine Delivery}, volume={19}, ISSN={["1556-6811"]}, DOI={10.1128/cvi.00049-12}, abstractNote={ABSTRACT Oral vaccines that elicit a mucosal immune response may be effective against human immunodeficiency virus type 1 (HIV-1) because its transmission occurs mainly at the mucosa. The aim of this study was to construct recombinant Lactobacillus for oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinant Lactobacillus acidophilus strain expressing the HIV-1 Gag on the bacterial cell surface was established by fusion with the signal peptide and anchor motif of a mucus binding protein (Mub) from L. acidophilus with or without coexpression of Salmonella enterica serovar Typhimurium flagellin (FliC) fused to a different Mub signal peptide and anchor. Using HEK293 cells engineered to express Toll-like receptor 5 (TLR5), the biological activity of FliC on the bacterial cell surfaces was determined. The surface-exposed flagellin retained its TLR5-stimulating activity, suggesting that the recombinant strain with Gag and FliC dual display might provide a different immunopotency than the strain expressing only Gag. The immunological properties of the recombinant strains were assessed by coculture with human myeloid dendritic cells (DCs). The heterologous antigens on the cell surface affected maturation and cytokine responses of DCs. Acquired immune responses were also investigated by intragastric immunization of mice. The enzyme-linked immunosorbent spot assay showed induction of gamma interferon-producing cells at local mucosa after immunization of mice with the Gag-producing strain. Meanwhile, the immunization with L. acidophilus displaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the Gag-displaying L. acidophilus elicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.}, number={9}, journal={CLINICAL AND VACCINE IMMUNOLOGY}, author={Kajikawa, Akinobu and Zhang, Lin and Long, Julie and Nordone, Shila and Stoeker, Laura and LaVoy, Alora and Bumgardner, Sara and Klaenhammer, Todd and Dean, Gregg}, year={2012}, month={Sep}, pages={1374–1381} }
 @article{o'flaherty_klaenhammer_2012, title={Influence of Exposure Time on Gene Expression by Human Intestinal Epithelial Cells Exposed to Lactobacillus acidophilus}, volume={78}, ISSN={["0099-2240"]}, DOI={10.1128/aem.00504-12}, abstractNote={ABSTRACT Analysis of global temporal gene expression by human intestinal cells when exposed to Lactobacillus acidophilus revealed induction of immune-related pathways and NF-κB target genes after a 1-h exposure, compared to a 4- or 8-h exposure. Additionally, an L. acidophilus derivative expressing covalently bound flagellin resulted in increased induction of il8 , cxc1 , and cxcl2 compared to the parent L. acidophilus .}, number={14}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2012}, month={Jul}, pages={5028–5032} }
 @article{abou hachem_fredslund_andersen_larsen_majumder_ejby_van zanten_lahtinen_barrangou_klaenhammer_et al._2012, title={Raffinose family oligosaccharide utilisation by probiotic bacteria: insight into substrate recognition, molecular architecture and diversity of GH36 alpha-galactosidases}, volume={30}, ISSN={["1024-2422"]}, DOI={10.3109/10242422.2012.674717}, abstractNote={The organisation of genes conferring utilisation of raffinose family oligosaccharides (RFOs) has been analysed in several probiotic bacteria from the Bifidobacterium and Lactobacillus genera. Glycoside hydrolase family 36 (GH36) α-galatosidase encoding genes occur together with sugar transport systems of the glycoside–pentoside–hexuronide cation symporter family (GPH), sugar phosphotransferase systems (PTSs) or ATP-binding cassette systems (ABCs) highlighting the diversity of RFO uptake. The GH36 genes are often clustered together with sucrose hydrolases or phosphorylases ensuring the degradation of RFO to monosaccharides. Differential proteomics and transcriptomics data from our laboratories implicated ABC transporters in the uptake of RFO in both Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Interestingly, only one of three GH36 encoding genes in B. animalis subsp. lactis Bl-04 was upregulated upon growth on RFO, suggesting that the other two gene products may have different specificities. The structure of the GH36 homotetrameric α-galactosidase from L. acidophilus NCFM (LaMel36A) was determined in complex with galactose bound in the active site to 1.58 Å. Differences in the N- and C-terminal domains of the LaMel36A monomer distinguished it from the monomeric TmGalA from Thermotoga maritima providing a structural rationale for the observed difference in oligomeric states of the two enzymes. Tetramerisation of LaMel36A creates a narrow and deep active site pocket between three monomers, which explains the preference of tetrameric GH36 enzymes for RFO and their lack of activity on polymeric galacto(gluco)mannan. Finally, GH36 was divided into four subgroups based on active site motifs, which illuminates functional and structural diversity in the family and aids further annotation of emerging sequences.}, number={3}, journal={BIOCATALYSIS AND BIOTRANSFORMATION}, author={Abou Hachem, Maher and Fredslund, F. and Andersen, J. M. and Larsen, R. Jonsgaard and Majumder, A. and Ejby, M. and Van Zanten, G. and Lahtinen, S. J. and Barrangou, R. and Klaenhammer, T. and et al.}, year={2012}, month={May}, pages={316–325} }
 @article{klaenhammer_kleerebezem_kopp_rescigno_2012, title={The impact of probiotics and prebiotics on the immune system}, volume={12}, ISSN={["1474-1741"]}, DOI={10.1038/nri3312}, abstractNote={Here,Nature Reviews Immunologyasks four experts to share their thoughts on the ability of probiotics and prebiotics to modulate the immune system and influence disease. Can these products have a therapeutic application for inflammatory diseases? Probiotics and prebiotics are increasingly being added to foodstuffs with claims of health benefits. Probiotics are live microorganisms that are thought to have beneficial effects on the host, whereas prebiotics are ingredients that stimulate the growth and/or function of beneficial intestinal microorganisms. But can these products directly modulate immune function and influence inflammatory diseases? Here, Nature Reviews Immunology asks four experts to discuss these issues and provide their thoughts on the future application of probiotics as a disease therapy.}, number={10}, journal={NATURE REVIEWS IMMUNOLOGY}, author={Klaenhammer, Todd R. and Kleerebezem, Michiel and Kopp, Matthias Volkmar and Rescigno, Maria}, year={2012}, month={Oct}, pages={728–734} }
 @article{andersen_barrangou_abou hachem_lahtinen_goh_svensson_klaenhammer_2012, title={Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM}, volume={7}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0044409}, abstractNote={The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.}, number={9}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Andersen, Joakim Mark and Barrangou, Rodolphe and Abou Hachem, Maher and Lahtinen, Sampo J. and Goh, Yong-Jun and Svensson, Birte and Klaenhammer, Todd R.}, editor={Gibas, CynthiaEditor}, year={2012}, month={Sep} }
 @article{gordon_klaenhammer_2011, title={A rendezvous with our microbes}, volume={108}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1101958108}, abstractNote={On November 2–3, 2009 an international group of scientists representing multiple disciplines gathered to consider the current state of our understanding of the symbiotic and beneficial relationships between microbes and humans and to define the challenges, gaps in knowledge, and opportunities that this exciting field of study now offers.

A number of adjectives come to mind when describing the subject of microbes and health, ranging from ancient and historic , to integrative and interdisciplinary , to timely and pressing . Coexistence and coevolution with microbes has been a theme of life on Earth for all metazoans past and present. Historically, the discovery that microbes are an integral part of us was made as soon as Antonie van Leeuwenhoek peered through his microscope and examined dental plaque sampled from himself and others (without institutional review board approval!). His sense of awe and his early appreciation of the diversity our microbial partners were evident in the words he chose for a letter written to the Royal Society of London in September 1683:

> “Though my teeth are kept usually very clean, nevertheless, when I view them in a magnifying glass, I find growing between them a little white matter as thick as wetted flower: in this substance though I could not perceive any motion, I judged there might probably be living creatures. I therefore took some of this flower and mixed it either with pure rain water wherein were no animals, or else with some of my spittle (having no air bubbles to cause a motion in it) and then to my great surprise perceived that the aforesaid matter maintained very many small living animals, which moved themselves very extravagantly. ….The spittle of an old man that had lived soberly, had no animals in it; but the substance upon and between his teeth … 

[↵][1]1To whom correspondence should be addressed. E-mail: jgordon{at}wustl.edu.

 [1]: #xref-corresp-1-1}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Gordon, Jeffrey I. and Klaenhammer, Todd R.}, year={2011}, month={Mar}, pages={4513–4515} }
 @article{stoeker_nordone_gunderson_zhang_kajikawa_lavoy_miller_klaenhammer_dean_2011, title={Assessment of Lactobacillus gasseri as a Candidate Oral Vaccine Vector}, volume={18}, ISSN={["1556-6811"]}, DOI={10.1128/cvi.05277-11}, abstractNote={Lactobacillus species are commensal bacteria that have long been recognized as probiotic microbes and are generally regarded as safe (GRAS) for human consumption. We have investigated the use of L. gasseri as a vaccine vector for oral immunization against mucosal pathogens. Recent research has shown that the immune response to different lactobacilli can vary widely depending on the species or subspecies of Lactobacillus being studied. While some lactobacilli seem to induce oral tolerance, others induce an adaptive immune response. This study characterized the systemic and mucosal immune response to wild-type and genetically modified L. gasseri. L. gasseri primarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile of L. gasseri and the immunogenicity of the vector, a plasmid containing fliC, the gene encoding bacterial flagellin, was introduced which resulted in the strong activation of TLR5. The treatment of human myeloid dendritic cells with recombinant lactobacilli expressing flagellin triggered phenotypic maturation and the release of proinflammatory cytokines. In contrast, bacterial treatment also resulted in a statistically significant increase in IL-10 production. In vivo studies established that treatment with L. gasseri led to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modified L. gasseri led to a significant decrease in the percentage of FoxP3(+) colonic lymphocytes. Taken together, these data clarify the interaction of L. gasseri with the host immune system and support further investigation of the in vivo immunogenicity of L. gasseri expressing both flagellin and candidate vaccine antigens.}, number={11}, journal={CLINICAL AND VACCINE IMMUNOLOGY}, author={Stoeker, Laura and Nordone, Shila and Gunderson, Sara and Zhang, Lin and Kajikawa, Akinobu and LaVoy, Alora and Miller, Michael and Klaenhammer, Todd R. and Dean, Gregg A.}, year={2011}, month={Nov}, pages={1834–1844} }
 @article{douglas_klaenhammer_2011, title={Directed Chromosomal Integration and Expression of the Reporter Gene gusA3 in Lactobacillus acidophilus NCFM}, volume={77}, ISSN={["0099-2240"]}, DOI={10.1128/aem.06028-11}, abstractNote={ABSTRACT Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β- d -glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3 -negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.}, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Douglas, Grace L. and Klaenhammer, Todd R.}, year={2011}, month={Oct}, pages={7365–7371} }
 @article{kajikawa_nordone_zhang_stoeker_lavoy_klaenhammer_dean_2011, title={Dissimilar Properties of Two Recombinant Lactobacillus acidophilus Strains Displaying Salmonella FliC with Different Anchoring Motifs}, volume={77}, ISSN={["0099-2240"]}, DOI={10.1128/aem.05153-11}, abstractNote={ABSTRACT Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro . The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.}, number={18}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Kajikawa, Akinobu and Nordone, Shila K. and Zhang, Lin and Stoeker, Laura L. and LaVoy, Alora S. and Klaenhammer, Todd R. and Dean, Gregg A.}, year={2011}, month={Sep}, pages={6587–6596} }
 @article{altermann_klaenhammer_2011, title={Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis}, volume={6}, ISSN={["1555-8932"]}, DOI={10.1007/s12263-010-0191-9}, abstractNote={Lactic acid bacteria (LAB) have been used in fermentation processes for centuries. More recent applications including the use of LAB as probiotics have significantly increased industrial interest. Here we present a comparative genomic analysis of four completely sequenced Lactobacillus strains, isolated from the human gastrointestinal tract, versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Lactobacillus acidophilus NCFM, Lactobacillus johnsonii NCC533, Lactobacillus gasseri ATCC33323, and Lactobacillus plantarum WCFS1are all considered probiotic and widely used in industrial applications. Using Differential Blast Analysis (DBA), each genome was compared to the respective remaining three other Lactobacillus and 25 other LAB genomes. DBA highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Initial comparative analyses highlighted a significant number of genes involved in cell adhesion, stress responses, DNA repair and modification, and metabolic capabilities. Furthermore, the range of the recently identified potential autonomous units (PAUs) was broadened significantly, indicating the possibility of distinct families within this genetic element. Based on in silico results obtained for the model organism L. acidophilus NCFM, DBA proved to be a valuable tool to identify new key genetic regions for functional genomics and also suggested re-classification of previously annotated genes.}, number={3}, journal={GENES AND NUTRITION}, author={Altermann, Eric and Klaenhammer, Todd R.}, year={2011}, month={Aug}, pages={319–340} }
 @misc{baugher_klaenhammer_2011, title={Invited review: Application of omics tools to understanding probiotic functionality}, volume={94}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2011-4384}, abstractNote={The human gut microbiota comprises autochthonous species that colonize and reside at high levels permanently and allochthonous species that originate from another source and are transient residents of the human gut. The interactions between bacteria and the human host can be classified as a continuum from symbiosis and commensalism (mutualism) to pathogenesis. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Recent advances in omics tools and sequencing techniques have furthered our understanding of probiotic functionality and the specific interactions between probiotics and their human hosts. Although it is known that not all probiotics use the same mechanisms to confer benefits on hosts, some specific mechanisms of action have been revealed through omic investigations. These include competitive exclusion, bacteriocin-mediated protection against intestinal pathogens, intimate interactions with mucin and the intestinal epithelium, and modulation of the immune system. The ability to examine fully sequenced and annotated genomes has greatly accelerated the application of genetic approaches to elucidate many important functional roles of probiotic microbes.}, number={10}, journal={JOURNAL OF DAIRY SCIENCE}, author={Baugher, J. L. and Klaenhammer, T. R.}, year={2011}, month={Oct}, pages={4753–4765} }
 @misc{guarner_sanders_gibson_klaenhammer_cabana_scott_reid_delzenne_fahey_hill_2011, title={Probiotic and prebiotic claims in Europe: seeking a clear roadmap}, volume={106}, ISSN={["1475-2662"]}, DOI={10.1017/s0007114511002248}, abstractNote={An abstract is not available for this content. As you have access to this content, full HTML content is provided on this page. A PDF of this content is also available in through the ‘Save PDF’ action button.}, number={11}, journal={BRITISH JOURNAL OF NUTRITION}, author={Guarner, Francisco and Sanders, Mary Ellen and Gibson, Glenn and Klaenhammer, Todd and Cabana, Michael and Scott, Karen and Reid, Gregor and Delzenne, Nathalie M. and Fahey, George C., Jr. and Hill, Colin}, year={2011}, month={Dec}, pages={1765–1767} }
 @article{mohamadzadeh_pfeiler_brown_zadeh_gramarossa_managlia_bere_sarraj_khan_pakanati_et al._2011, title={Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid}, volume={108}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1005066107}, abstractNote={Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile -induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4 + T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4 + CD45RB high T cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4 + FoxP3 + T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Mohamadzadeh, Mansour and Pfeiler, Erika A. and Brown, Jeffrey B. and Zadeh, Mojgan and Gramarossa, Matthew and Managlia, Elizabeth and Bere, Praveen and Sarraj, Bara and Khan, Mohammad W. and Pakanati, Krishna Chaitanya and et al.}, year={2011}, month={Mar}, pages={4623–4630} }
 @article{o'flaherty_klaenhammer_2011, title={The Impact of Omic Technologies on the Study of Food Microbes}, volume={2}, ISBN={["978-0-8243-4902-8"]}, ISSN={["1941-1421"]}, DOI={10.1146/annurev-food-030810-110338}, abstractNote={The advent of the molecular biology era in the 1950s and the subsequent emergence of new technologies positively impacted on all areas of biology. New discoveries in molecular biology and experimental tools were developed over the next 60 years that have revolutionized the study of food microbiology. Previously, food microbiology relied on classic microbiology techniques, which had remained relatively unchanged since the discoveries of Louis Pasteur in the 1800s. More recently, new advances resulting in “omic” technologies have exploded the areas of genomics, transcriptomics, and proteomics and revealed many fundamental processes driven by both pathogens and commensals. This review outlines advances in omic technologies and how these have impacted food microbiology through providing examples of recently published landmark work.}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 2}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2011}, pages={353–371} }
 @article{andersen_barrangou_abou hachem_lahtinen_goh_svensson_klaenhammer_2011, title={Transcriptional and functional analysis of galactooligosaccharide uptake by lacS in Lactobacillus acidophilus}, volume={108}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1114152108}, abstractNote={Probiotic microbes rely on their ability to survive in the gastrointestinal tract, adhere to mucosal surfaces, and metabolize available energy sources from dietary compounds, including prebiotics. Genome sequencing projects have proposed models for understanding prebiotic catabolism, but mechanisms remain to be elucidated for many prebiotic substrates. Although β-galactooligosaccharides (GOS) are documented prebiotic compounds, little is known about their utilization by lactobacilli. This study aimed to identify genetic loci in Lactobacillus acidophilus NCFM responsible for the transport and catabolism of GOS. Whole-genome oligonucleotide microarrays were used to survey the differential global transcriptome during logarithmic growth of L. acidophilus NCFM using GOS or glucose as a sole source of carbohydrate. Within the 16.6-kbp gal-lac gene cluster, lacS , a galactoside-pentose-hexuronide permease-encoding gene, was up-regulated 5.1-fold in the presence of GOS. In addition, two β-galactosidases, LacA and LacLM, and enzymes in the Leloir pathway were also encoded by genes within this locus and up-regulated by GOS stimulation. Generation of a lacS -deficient mutant enabled phenotypic confirmation of the functional LacS permease not only for the utilization of lactose and GOS but also lactitol, suggesting a prominent role of LacS in the metabolism of a broad range of prebiotic β-galactosides, known to selectively modulate the beneficial gut microbiota.}, number={43}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Andersen, Joakim M. and Barrangou, Rodolphe and Abou Hachem, Maher and Lahtinen, Sampo and Goh, Yong Jun and Svensson, Birte and Klaenhammer, Todd R.}, year={2011}, month={Oct}, pages={17785–17790} }
 @article{duong_miller_barrangou_azcarate-peril_klaenhammer_2010, title={Construction of vectors for inducible and constitutive gene expression in Lactobacillus}, volume={4}, DOI={10.1111/j.1751-7915.2010.00200.x}, abstractNote={Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.}, number={3}, journal={Microbial Biotechnology}, publisher={Wiley-Blackwell}, author={Duong, Tri and Miller, Michael J. and Barrangou, Rodolphe and Azcarate-Peril, M. Andrea and Klaenhammer, Todd R.}, year={2010}, month={Sep}, pages={357–367} }
 @article{goh_klaenhammer_2010, title={Functional Roles of Aggregation-Promoting-Like Factor in Stress Tolerance and Adherence of Lactobacillus acidophilus NCFM}, volume={76}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00030-10}, abstractNote={ABSTRACT Aggregation-promoting factors (Apf) are secreted proteins that have been associated with a diverse number of functional roles in lactobacilli, including self-aggregation, the bridging of conjugal pairs, coaggregation with other commensal or pathogenic bacteria, and maintenance of cell shape. In silico genome analysis of Lactobacillus acidophilus NCFM identified LBA0493 as a 696-bp apf gene that encodes a putative 21-kDa Apf protein. Transcriptional studies of NCFM during growth in milk showed apf to be one of the most highly upregulated genes in the genome. In the present study, reverse transcriptase-quantitative PCR (RT-QPCR) analysis revealed that the apf gene was highly induced during the stationary phase compared to that during the logarithmic phase. To investigate the functional role of Apf in NCFM, an Δ apf deletion mutant was constructed. The resulting Δ apf mutant, NCK2033, did not show a significant difference in cell morphology or growth compared to that of the NCFMΔ upp reference strain, NCK1909. The autoaggregation phenotype of NCK2033 in planktonic culture was unaffected. Additional phenotypic assays revealed that NCK2033 was more susceptible to treatments with oxgall bile and sodium dodecyl sulfate (SDS). Survival rates of NCK2033 decreased when stationary-phase cells were exposed to simulated small-intestinal and gastric juices. Furthermore, NCK2033 in the stationary phase showed a reduction of in vitro adherence to Caco-2 intestinal epithelial cells, mucin glycoproteins, and fibronectin. The data suggest that the Apf-like proteins may contribute to the survival of L. acidophilus during transit through the digestive tract and, potentially, participate in the interactions with the host intestinal mucosa.}, number={15}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Goh, Yong Jun and Klaenhammer, Todd R.}, year={2010}, month={Aug}, pages={5005–5012} }
 @article{sarah j. o'flaherty_klaenhammer_2010, title={Functional and phenotypic characterization of a protein from Lactobacillus acidophilus involved in cell morphology, stress tolerance and adherence to intestinal cells}, volume={156}, ISSN={["1465-2080"]}, DOI={10.1099/mic.0.043158-0}, abstractNote={Structural components of the cell surface have an impact on some of the beneficial attributes of probiotic bacteria. In silico analysis of the L. acidophilus NCFM genome sequence revealed the presence of a putative cell surface protein that was predicted to be a myosin cross-reactive antigen (MCRA). As MCRAs are conserved among many probiotic bacteria, we used the upp-based counterselective gene replacement system, designed recently for use in L. acidophilus, to determine the functional role of this gene (LBA649) in L. acidophilus NCFM. Phenotypic assays were undertaken with the parent strain (NCK1909) and deletion mutant (NCK2015) to assign a function for this gene. The growth of NCK2015 (ΔLBA649) was reduced in the presence of lactate, acetate, porcine bile and salt. Adhesion of NCK2015 to Caco-2 cells was substantially reduced for both stationary-phase (∼45 % reduction) and exponential-phase cells (∼50 % reduction). Analysis of NCK2015 by scanning electron microscopy revealed a longer cell morphology after growth in MRS broth compared to NCK1909. These results indicate a role for LBA649 in stress tolerance, cell wall division and adherence to Caco-2 cells.}, journal={MICROBIOLOGY-SGM}, author={Sarah J. O'Flaherty and Klaenhammer, Todd R.}, year={2010}, month={Nov}, pages={3360–3367} }
 @article{douglas_klaenhammer_2010, title={Genomic Evolution of Domesticated Microorganisms}, volume={1}, ISBN={["978-0-8243-4901-1"]}, ISSN={["1941-1413"]}, DOI={10.1146/annurev.food.102308.124134}, abstractNote={Strains of lactic acid bacteria, yeasts, and molds have been selected over thousands of years based on the unique sensory attributes they provide to food fermentations. Over the centuries they have evolved to their domesticated roles, leading to genome decay, loss of pathways, acquisition of genomic elements, and beneficial mutations that provide an advantage in their nutrient-rich food environments. This review highlights the evolutionary traits influenced by the domestication process as these microbes adapted to nutrient-rich foods developed by humans.}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 1}, author={Douglas, Grace L. and Klaenhammer, Todd R.}, year={2010}, pages={397–414} }
 @article{damelin_mavri-damelin_klaenhammer_tiemessen_2010, title={Plasmid Transduction Using Bacteriophage Phi adh for Expression of CC Chemokines by Lactobacillus gasseri ADH}, volume={76}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00139-10}, abstractNote={Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.}, number={12}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Damelin, Leonard H. and Mavri-Damelin, Demetra and Klaenhammer, Todd R. and Tiemessen, Caroline T.}, year={2010}, month={Jun}, pages={3878–3885} }
 @article{mohamadzadeh_durmaz_zadeh_pakanati_gramarossa_cohran_klaenhammer_2010, title={Targeted expression of anthrax protective antigen by Lactobacillus gasseri as an anthrax vaccine}, volume={5}, ISSN={["1746-0921"]}, DOI={10.2217/fmb.10.78}, abstractNote={Aim: Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice. Materials & methods: Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection. Results: Oral application of L. gasseri expressing the PA–DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expression vaccine strategy reduced the number of inoculations and length of the boosting period, leading to animal protection via efficacious bacterial adjuvanticity and safe oral delivery of this vaccine to mucosal immune cells, including dendritic cells. Conclusion: Lactobacillus-based delivery offers tremendous practical advantages. Recombinant antigens such as PA would not require chemical coupling agents, and the recombinant bacteria can be administered orally where upon both mucosal and systemic immune responses are elicited.}, number={8}, journal={FUTURE MICROBIOLOGY}, author={Mohamadzadeh, Mansour and Durmaz, Evelyn and Zadeh, Mojgan and Pakanati, Krishna Chaitanya and Gramarossa, Matthew and Cohran, Valeria and Klaenhammer, Todd R.}, year={2010}, month={Aug}, pages={1289–1296} }
 @article{o'flaherty_klaenhammer_2010, title={The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host}, volume={20}, ISSN={["1879-0143"]}, DOI={10.1016/j.idairyj.2009.11.011}, abstractNote={Recent research efforts have focused on understanding the interactions of probiotic bacteria with commensal gut bacteria and the human host as a means to determine mechanisms of probiotic functionality that contribute to their beneficial attributes. Our growing understanding of the intrinsic interactions between probiotic and commensal bacteria and between the milieu of bacteria and the host tissues of the gastrointestinal tract (GIT) has been facilitated by the use of ‘omic’ technologies. Surveys of bacterial inhabitants in the GIT using sequencing technologies have demonstrated the complexities of this human organ which varies between different populations and individuals, such as diet. In addition, transcriptomics have rapidly facilitated an insight into the complex communication between bacteria (commensal and probiotic) and the GIT. This review outlines the recent important advances in this exciting area of research, which has led to a greater understanding of the critical interface between gut microbiota, probiotic bacteria and the host.}, number={4}, journal={INTERNATIONAL DAIRY JOURNAL}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2010}, month={Apr}, pages={262–268} }
 @misc{barrangou_azcarate-peril_altermann_duong_klaenhammer_2009, title={Compositions comprising promoter sequences and methods of use}, volume={7,495,092}, number={2009 Feb. 24}, author={Barrangou, R. and Azcarate-Peril, A. and Altermann, E. and Duong, T. and Klaenhammer, T. R.}, year={2009} }
 @article{mohamadzadeh_duong_sandwick_hoover_klaenhammer_2009, title={Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge}, volume={106}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0900029106}, abstractNote={Efficient vaccines potentiate antibody avidity and increase T cell longevity, which confer protection against microbial lethal challenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via specific dendritic cell-targeting peptides to dendritic cells (DCs), which reside in the periphery and mucosal surfaces, thus directing and regulating acquired immunity. The efficiency of oral delivery of L. acidophilus expressing a PA-DCpep fusion was evaluated in mice challenged with lethal B. anthracis Sterne. Vaccination with L. acidophilus expressing PA-DCpep induced robust protective immunity against B. anthracis Sterne compared with mice vaccinated with L. acidophilus expressing PA-control peptide or an empty vector. Additionally, serum anti-PA titers, neutralizing PA antibodies, and the levels of IgA-expressing cells were all comparable with the historical recombinant PA plus aluminum hydroxide vaccine administered s.c. Collectively, development of this strategy for oral delivery of DC-targeted antigens provides a safe and protective vaccine via a bacterial adjuvant that may potentiate mucosal immune responses against deadly pathogens.}, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Mohamadzadeh, M. and Duong, T. and Sandwick, S. J. and Hoover, T. and Klaenhammer, T. R.}, year={2009}, month={Mar}, pages={4331–4336} }
 @article{goh_azcarate-peril_o'flaherty_durmaz_valence_jardin_lortal_klaenhammer_2009, title={Development and Application of a upp-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM}, volume={75}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.02502-08}, abstractNote={ABSTRACT In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp -encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the Δ upp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus . The resulting Δ slpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus .}, number={10}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Goh, Yong Jun and Azcarate-Peril, M. Andrea and O'Flaherty, Sarah and Durmaz, Evelyn and Valence, Florence and Jardin, Julien and Lortal, Sylvie and Klaenhammer, Todd R.}, year={2009}, month={May}, pages={3093–3105} }
 @misc{ventura_o'flaherty_claesson_turroni_klaenhammer_sinderen_paul w. o'toole_2009, title={Genome-scale analyses of health-promoting bacteria: probiogenomics}, volume={7}, ISSN={["1740-1534"]}, DOI={10.1038/nrmicro2047}, number={1}, journal={NATURE REVIEWS MICROBIOLOGY}, author={Ventura, Marco and O'Flaherty, Sarah and Claesson, Marcus J. and Turroni, Francesca and Klaenhammer, Todd R. and Sinderen, Douwe and Paul W. O'Toole}, year={2009}, month={Jan}, pages={61–U77} }
 @misc{goh_klaenhammer_2009, title={Genomic features of Lactobacillus species}, volume={14}, journal={Frontiers in Bioscience}, author={Goh, Y. J. and Klaenhammer, T. R.}, year={2009}, pages={1362–1386} }
 @misc{schroeter_klaenhammer_2009, title={Genomics of lactic acid bacteria}, volume={292}, ISSN={["1574-6968"]}, DOI={10.1111/j.1574-6968.2008.01442.x}, abstractNote={Lactic acid bacteria (LAB) are found to occupy a variety of ecological niches including fermented foods as well as mucosal surfaces of humans and other vertebrates. This review is based on the genomic content of LAB that is responsible for the functional and ecological diversity of these bacteria. These genomes reveal an ongoing process of reductive evolution as the LAB have specialized to different nutritionally rich environments. Species-to-species variation in the number of pseudogenes as well as genes directing nutrient uptake and metabolism reflects the adaptation of LAB to food matrices and the gastrointestinal tract. Although a general trend of genome reduction was observed, certain niche-specific genes appear to be recently acquired and appear on plasmids or adjacent to prophages. Recent work has improved our understanding of the genomic content responsible for various phenotypes that continue to be discovered, as well as those that have been exploited by man for thousands of years.}, number={1}, journal={FEMS MICROBIOLOGY LETTERS}, author={Schroeter, Joel and Klaenhammer, Todd}, year={2009}, month={Mar}, pages={1–6} }
 @article{gunderson_nordone_lavoy_zhang_klaenhammer_dean_2009, title={Immunogenicity of Lactobacillus gasseri-FliC as an oral mucosal vaccine adjuvant for HIV}, volume={6}, ISSN={["1742-4690"]}, DOI={10.1186/1742-4690-6-s3-p163}, abstractNote={Background Transmission of HIV-1 across mucosal surfaces is the most prevalent mode of viral infection. Therefore, a successful vaccine must induce broad anti-viral immunity at the mucosal surface. In the present study, we investigated the immunogenicity of a novel Lactobacillus gasseri mucosal vaccine vector for use in oral delivery of HIV antigens. L. gasseri was genetically engineered to express Salmonella spp. flagellin (L. gasseri-FliC) – the agonist for Toll-like receptor 5 (TLR5) and a potent activator of innate immune cells.}, journal={RETROVIROLOGY}, author={Gunderson, S. and Nordone, S. and LaVoy, A. and Zhang, L. and Klaenhammer, T. and Dean, G.}, year={2009} }
 @misc{klaenhammer_russell_altermann_buck_2009, title={Lactobacillus acidophillus nucleic acid sequences encoding cell surface protein homologues and uses therefore}, volume={7,538,209}, number={2009 May 26}, author={Klaenhammer, T. R. and Russell, W. M. and Altermann, E. and Buck, B. L.}, year={2009} }
 @misc{klaenhammer_altermann_azcarate-peril_mcauliffe_russell_2009, title={Lactobacillus acidophilus nucleic acid sequences encoding stress-related proteins and uses therefor}, volume={7,608,700}, number={1999 Oct. 27}, author={Klaenhammer, T. R. and Altermann, E. and Azcarate-Peril, M. A. and McAuliffe, O. and Russell, W. M.}, year={2009} }
 @misc{klaenhammer_russell_altermann_azcarate-peril_2009, title={Nucleic acid sequences encoding two-component sensing and regulatory proteins, antimicrobial proteins and uses therefor}, volume={7,550,576}, number={2009 Jun 23}, author={Klaenhammer, T. R. and Russell, W. M. and Altermann, E. and Azcarate-Peril, A.}, year={2009} }
 @article{engelbrektson_korzenik_pittler_sanders_klaenhammer_leyer_kitts_2009, title={Probiotics to minimize the disruption of faecal microbiota in healthy subjects undergoing antibiotic therapy}, volume={58}, ISSN={["1473-5644"]}, DOI={10.1099/jmm.0.47615-0}, abstractNote={A novel combination of culturing and DNA-based terminal restriction fragment length polymorphism (TRFLP) analysis was used to investigate the effect of probiotics on antibiotic-induced gut microbiota alterations to determine if a probiotic preparation containing bifidobacteria and lactobacilli, taken during and after antibiotic therapy, can minimize antibiotic disturbance of faecal microbiota. Healthy subjects administered amoxicillin/clavulanate were randomized and concomitantly received a placebo or probiotic mixture. The primary end point was similarity of faecal microbiota as determined by culturing and TRFLP from subjects taking probiotics compared to those taking a placebo measured by comparing data from baseline to post-treatment for each subject. TRFLP analysis revealed a high subject to subject variation in the baseline faecal microbiota. The most common antibiotic-induced disturbance was a relative increase in Clostridium , Eubacterium , Bacteroides and Enterobacteraceae . The mean similarity to the baseline increased over time in both treatment groups, although the probiotic group was less disturbed according to both TRFLP and culture data. The culture method revealed that post-antibiotic faecal microbiota in probiotic-consuming subjects were more similar to the baseline microbiota than the control group ( P =0.046). Changes in Enterobactereaceae ( P =0.006) and Bifidobacterium ( P =0.030) counts were significantly different between the groups. Analysis of TRFLP data reinforced the trend between groups but was not statistically significant ( P =0.066). This study indicates this mixture of probiotics promotes a more rapid return to pre-antibiotic baseline faecal bacterial microbiota.}, number={5}, journal={JOURNAL OF MEDICAL MICROBIOLOGY}, author={Engelbrektson, Anna and Korzenik, Joshua R. and Pittler, Arlyn and Sanders, Mary E. and Klaenhammer, Todd R. and Leyer, Gregory and Kitts, Christopher L.}, year={2009}, month={May}, pages={663–670} }
 @article{pfeiler_klaenhammer_2009, title={Role of Transporter Proteins in Bile Tolerance of Lactobacillus acidophilus}, volume={75}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00495-09}, abstractNote={ABSTRACT Lactobacillus acidophilus NCFM derivatives containing deletion mutations in the transporter genes LBA0552, LBA1429, LBA1446, and LBA1679 exhibited increased sensitivity to bile. These strains showed unique patterns of sensitivity to a variety of inhibitory compounds, as well as differential accumulations of ciprofloxacin and taurocholate.}, number={18}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Pfeiler, Erika A. and Klaenhammer, Todd R.}, year={2009}, month={Sep}, pages={6013–6016} }
 @article{buck_azcarate-peril_klaenhammer_2009, title={Role of autoinducer-2 on the adhesion ability of Lactobacillus acidophilus}, volume={107}, ISSN={["1365-2672"]}, DOI={10.1111/j.1365-2672.2009.04204.x}, abstractNote={Lactobacilli adhere to the intestinal epithelium and this intimate association likely promotes retention in the gastrointestinal tract and communication with the immune system. The aim of this study was to investigate whether or not the quorum-sensing signalling molecule, autoinducer (AI)-2, was produced by Lactobacillus acidophilus and affected adherence to intestinal epithelial cells.Microarray analysis of concentrated cells of L. acidophilus NCFM revealed several genes involved in a classic stress response and potentially adhesion. Putative genes linked to the synthesis of the interspecies signalling molecule, AI-2, were overexpressed. Examination of the NCFM genome revealed the complete pathway for AI-2 synthesis. AI-2 activity from NCFM was detected using the Vibrio harveyi BB170 assay system. Using site-specific integration, an isogenic mutation was created in luxS and the resulting mutant did not produce AI-2. In addition to some minor metabolic effects, the luxS mutation resulted in 58% decrease in adherence to Caco-2 cells.L. acidophilus NCFM encodes the genes for synthesis of the quorum-sensing signal, AI-2, and produces this molecule during planktonic growth.The ability to produce AI-2 affects the ability of L. acidophilus to attach to intestinal epithelial cells.}, number={1}, journal={JOURNAL OF APPLIED MICROBIOLOGY}, author={Buck, B. L. and Azcarate-Peril, M. A. and Klaenhammer, T. R.}, year={2009}, month={Jul}, pages={269–279} }
 @article{azcarate-peril_tallon_klaenhammer_2009, title={Temporal gene expression and probiotic attributes of Lactobacillus acidophilus during growth in milk}, volume={92}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2008-1457}, abstractNote={Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt and used in dietary supplements. In this study, a whole genome microarray was employed to monitor gene expression of L. acidophilus NCFM cells propagated in 11% skim milk during early, mid and late logarithmic phase, and stationary phase. Approximately 21% of 1,864 open reading frames were differentially expressed at least in one time point. Genes differentially expressed in skim milk included several members of the proteolytic enzyme system. Expression of prtP (proteinase precursor) and prtM (maturase) increased over time as well as several peptidases and transport systems. Expression of Opp1 (oligopeptide transport system 1) was highest at 4 h, whereas gene expression of Opp2 increased over time reaching its highest level at 12 h, suggesting that the 2 systems have different specificities. Expression of a 2-component regulatory system, previously shown to regulate acid tolerance and proteolytic activity, also increased during the early log and early stationary phases of growth. Expression of the genes involved in lactose utilization increased immediately (5 min) upon exposure to milk. The acidification activity, survival under storage conditions, and adhesion to mucin and Caco-2 tissue culture cells of selected mutants containing insertionally inactivated genes differentially expressed in the wild-type strain during growth in milk were examined for any potential links between probiotic properties and bacterial growth and survival in milk. Some of the most interesting genes found to be expressed in milk were correlated with signaling (autoinducer-2) and adherence to mucin and intestinal epithelial cells, in vitro.}, number={3}, journal={JOURNAL OF DAIRY SCIENCE}, author={Azcarate-Peril, M. A. and Tallon, R. and Klaenhammer, T. R.}, year={2009}, month={Mar}, pages={870–886} }
 @article{stoeker_nordone_lavoy_tallon_klaenhammer_dean_2009, title={Toll-like receptor activation profiles of wild-type, recombinant, and mutant Lactobacillus: implications for vaccine design}, volume={6}, ISSN={["1742-4690"]}, DOI={10.1186/1742-4690-6-s3-p133}, journal={RETROVIROLOGY}, author={Stoeker, L. and Nordone, S. and LaVoy, A. and Tallon, R. and Klaenhammer, T. and Dean, G.}, year={2009} }
 @article{azcarate-peril_altermann_goh_tallon_sanozky-dawes_pfeiler_o'flaherty_buck_dobson_duong_et al._2008, title={Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism}, volume={74}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00054-08}, abstractNote={ABSTRACT This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.}, number={15}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Azcarate-Peril, M. Andrea and Altermann, Eric and Goh, Yong Jun and Tallon, Richard and Sanozky-Dawes, Rosemary B. and Pfeiler, Erika A. and O'Flaherty, Sarah and Buck, B. Logan and Dobson, Alleson and Duong, Tri and et al.}, year={2008}, month={Aug}, pages={4610–4625} }
 @article{klaenhammer_altermann_pfeiler_buck_goh_o'flaherty_barrangou_duong_2008, title={Functional genomics of probiotic Lactobacilli}, volume={42}, ISSN={["0192-0790"]}, DOI={10.1097/MCG.0b013e31817da140}, abstractNote={Lactic acid bacteria (LAB) have been used in fermentation processes for millennia. Recent applications such as the use of living cultures as probiotics have significantly increased industrial interest. Related bacterial strains can differ significantly in their genotype and phenotype, and features from one bacterial strain or species cannot necessarily be applied to a related one. These strain or family-specific differences often represent unique and applicable traits. Since 2002, the complete genomes of 13 probiotic LABs have been published. The presentation will discuss these genomes and highlight probiotic traits that are predicted, or functionally linked to genetic content. We have conducted a comparative genomic analysis of 4 completely sequenced Lactobacillus strains versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Using Differential Blast Analysis, each genome is compared with 3 other Lactobacillus and 25 other LAB genomes. Differential Blast Analysis highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Lactobacillus-specific genes include mucus-binding proteins involved in cell-adhesion and several transport systems for carbohydrates and amino acids. Comparative genomic analysis has identified gene targets in Lactobacillus acidophilus for functional analysis, including adhesion to mucin and intestinal epithelial cells, acid tolerance, bile tolerance, and quorum sensing. Whole genome transcriptional profiling of L. acidophilus, and isogenic mutants thereof, has revealed the impact of varying conditions (pH, bile, carbohydrates) and food matrices on the expression of genes important to probiotic-linked mechanisms.}, number={8}, journal={JOURNAL OF CLINICAL GASTROENTEROLOGY}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Klaenhammer, Todd R. and Altermann, Eric and Pfeiler, Erika and Buck, Brock Logan and Goh, Yong-Jun and O'Flaherty, Sarah and Barrangou, Rodolphe and Duong, Tri}, year={2008}, month={Sep}, pages={S160–S162} }
 @article{durmaz_miller_azcarate-peril_toon_klaenhammer_2008, title={Genome sequence and characteristics of Lrm1, a prophage from industrial Lactobacillus rhamnosus strain M1}, volume={74}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00010-08}, abstractNote={ABSTRACT Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.}, number={15}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Durmaz, Evelyn and Miller, Michael J. and Azcarate-Peril, M. Andrea and Toon, Stephen P. and Klaenhammer, Todd R.}, year={2008}, month={Aug}, pages={4601–4609} }
 @misc{klaenhammer_altermann_barrangou_russell_duong_2008, title={Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor}, volume={7,459,289}, number={2008 Dec. 2}, author={Klaenhammer, T. R. and Altermann, E. and Barrangou, R. and Russell, W. M. and Duong, T.}, year={2008} }
 @misc{klaenhammer_altermann_buck_russell_2008, title={Lactobacillus acidophilus nucleic acid sequences encoding cell surface protein homologues and uses therefore}, volume={7,348,420}, number={2008 Mar. 25}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Altermann, E. and Buck, B. L. and Russell, W. M.}, year={2008} }
 @misc{klaenhammer_altermann_russell_2008, title={Lactobacillus acidophilus nucleic acid sequences encoding protease homologues and uses therefore}, volume={7,455,992}, number={2008 Nov. 25}, author={Klaenhammer, T. R. and Altermann, E. and Russell, W. M.}, year={2008} }
 @misc{klaenhammer_azcarate-peril_altermann_2008, title={Lactobacillus acidophilus nucleic acids and uses thereof}, volume={7,468,182}, number={2008 Dec. 23}, author={Klaenhammer, T. and Azcarate-Peril, A. and Altermann, E.}, year={2008} }
 @article{konstantinov_smidt_vos_bruijns_singh_valence_molle_lortal_altermann_klaenhammer_et al._2008, title={S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions}, volume={105}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0810305105}, abstractNote={Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFalpha, and IL-1beta were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA-DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.}, number={49}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Konstantinov, Sergey R. and Smidt, Hauke and Vos, Willem M. and Bruijns, Sven C. M. and Singh, Satwinder Kaur and Valence, Florence and Molle, Daniel and Lortal, Sylvie and Altermann, Eric and Klaenhammer, Todd R. and et al.}, year={2008}, month={Dec}, pages={19474–19479} }
 @article{mohamadzadeh_klaenhammer_2008, title={Specific Lactobacillus species differentially activate Toll-like receptors and downstream signals in dendritic cells}, volume={7}, ISSN={["1744-8395"]}, DOI={10.1586/14760584.7.8.1155}, abstractNote={Background: Dendritic cells (DCs) regulate mucosal T-cell immunity and encounter several distinct bacteria of the gut flora, including lactobacilli. Gram-positive lactobacilli have been suggested to play an important role in exerting adjuvanticity effects on innate immune cells at mucosal sites. Aims & methods: In the present report, we studied the effects of specific Lactobacillus species on human monocyte derived DCs. Results: We show that lactobacilli activate DCs by differentially inducing the expression of Toll-like receptors and bioactive IL-12 in Lactobacillus-treated DCs. Further, these specific Lactobacillus spp. did not activate the phosphorylation of p38 MAPK, which might be a downstream effect of the remarkable capacity of lactobacilli to induce IL-12 in DCs that skew T cells significantly toward an IFN-γ-secreting Th1 response. Conclusion: These results highlight an important role of specific Lactobacillus spp. as adjuvants in triggering DC function, which in turn may determine the immunological outcome in an environment wherein innate cells reside.}, number={8}, journal={EXPERT REVIEW OF VACCINES}, author={Mohamadzadeh, Mansour and Klaenhammer, Todd R.}, year={2008}, month={Oct}, pages={1155–1164} }
 @misc{mohamadzadeh_duong_hoover_klaenhammer_2008, title={Targeting mucosal dendritic cells with microbial antigens from probiotic lactic acid bacteria}, volume={7}, ISSN={["1744-8395"]}, DOI={10.1586/14760584.7.2.163}, abstractNote={The use of vaccines against infectious microbes has been critical to the advancement of medicine. Vaccine strategies combined with, or without, adjuvants have been established to eradicate various bacterial and viral pathogens. A new generation of vaccines is being developed using specific strains of Gram-positive, lactic acid bacteria and, notably, some probiotic lactobacilli. These bacteria have been safely consumed by humans for centuries in fermented foods. Thus, they can be orally administered, are well tolerated by recipients and could be easily and economically provided to large populations. In this overview, we focus on mucosal immunity and how its cellular component(s), particularly dendritic cells, can be specifically targeted to deliver immunogenic subunits, such as the protective antigen from Bacillus anthracis (the causative agent of anthrax). An antigen-specific immune response can be elicited using specific strains of Lactobacillus acidophilus expressing the protective antigen. A mucosal, dendritic cell-targeted approach increases the bioavailability of an immunogen of interest when delivered orally by L. acidophilus. This provides an efficiently elegant natural strategy and serves a dual function as an immune-stimulating adjuvant in vivo.}, number={2}, journal={EXPERT REVIEW OF VACCINES}, author={Mohamadzadeh, Mansour and Duong, Tri and Hoover, Timothy and Klaenhammer, Todd R.}, year={2008}, month={Mar}, pages={163–174} }
 @misc{durmaz_klaenhammer_2007, title={AbiZ phage resistance gene}, volume={7,169,911}, number={2007 Jan. 30}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Durmaz, E. and Klaenhammer, T. R.}, year={2007} }
 @article{durmaz_klaenhammer_2007, title={Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis}, volume={189}, ISSN={["1098-5530"]}, DOI={10.1128/JB.00904-06}, abstractNote={The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.}, number={4}, journal={JOURNAL OF BACTERIOLOGY}, author={Durmaz, Evelyn and Klaenhammer, Todd R.}, year={2007}, month={Feb}, pages={1417–1425} }
 @article{carroll_andrus_bruno-barcena_klaenhammer_hassan_threadgill_2007, title={Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase using the interleukin 10-deficient mouse model of colitis}, volume={293}, ISSN={["1522-1547"]}, url={http://europepmc.org/abstract/med/17640978}, DOI={10.1152/ajpgi.00132.2007}, abstractNote={Emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of inflammatory bowel disease (IBD). Although intestinal epithelial cells produce the ROS-neutralizing enzyme superoxide dismutase (SOD), the protein and activity levels of copper/zinc (Cu/Zn) and manganese (Mn) SOD are perturbed in inflamed tissues of IBD patients. Thus we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in interleukin (IL) 10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13–15 IL-10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for 4 wk. Colonic tissue was collected and inflammation was histologically scored. The presence of innate immune cells was investigated by immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least 3 days. L. gasseri producing MnSOD significantly reduced inflammation in IL-10-deficient mice compared with untreated controls ( P < 0.05), whereas the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. Transcripts of antioxidant genes were equivalent in colonic tissues obtained from control and probiotic-treated IL-10-deficient mice. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity that reduces the severity of colitis in the IL-10-deficient mouse.}, number={4}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY}, author={Carroll, Ian M. and Andrus, Jason M. and Bruno-Barcena, Jose M. and Klaenhammer, Todd R. and Hassan, Hosni M. and Threadgill, Deborah S.}, year={2007}, month={Oct}, pages={G729–G738} }
 @article{pfeiler_azcarate-peril_klaenhammer_2007, title={Characterization of a novel bile-inducible operon encoding a two-component regulatory system in Lactobacillus acidophilus}, volume={189}, ISSN={["1098-5530"]}, DOI={10.1128/JB.00337-07}, abstractNote={Lactobacillus acidophilus NCFM is an industrially important strain used extensively as a probiotic culture. Tolerance of the presence of bile is an attribute important to microbial survival in the intestinal tract. A whole-genome microarray was employed to examine the effects of bile on the global transcriptional profile of this strain, with the intention of elucidating genes contributing to bile tolerance. Genes involved in carbohydrate metabolism were generally induced, while genes involved in other aspects of cellular growth were mostly repressed. A 7-kb eight-gene operon encoding a two-component regulatory system (2CRS), a transporter, an oxidoreductase, and four hypothetical proteins was significantly upregulated in the presence of bile. Deletion mutations were constructed in six genes of the operon. Transcriptional analysis of the 2CRS mutants showed that mutation of the histidine protein kinase (HPK) had no effect on the induction of the operon, whereas the mutated response regulator (RR) showed enhanced induction when the cells were exposed to bile. These results indicate that the 2CRS plays a role in bile tolerance and that the operon it resides in is negatively controlled by the RR. Mutations in the transporter, the HPK, the RR, and a hypothetical protein each resulted in loss of tolerance of bile. Mutations in genes encoding another hypothetical protein and a putative oxidoreductase resulted in significant increases in bile tolerance. This functional analysis showed that the operon encoded proteins involved in both bile tolerance and bile sensitivity.}, number={13}, journal={JOURNAL OF BACTERIOLOGY}, author={Pfeiler, Erika A. and Azcarate-Peril, M. Andrea and Klaenhammer, Todd R.}, year={2007}, month={Jul}, pages={4624–4634} }
 @article{dobson_sanozky-dawes_klaenhammer_2007, title={Identification of an operon and inducing peptide involved in the production of lactacin B by Lactobacillus acidophilus}, volume={103}, ISSN={["1365-2672"]}, DOI={10.1111/j.1365-2672.2007.03417.x}, abstractNote={Aim: To determine if a 9·5-kb region on the Lactobacillus acidophilus NCFM genome, encoded the genetic determinants for regulation and production of lactacin B, a class II bacteriocin. Methods: Transcriptional analysis was used to identify a 9·5-kb polycistronic region suspected of encoding the lab operon. The 12 putative open reading frames (LBA1803–LBA1791) were organized into three clusters: a production and regulation cluster encoding a putative two-component signal transduction system; an export cluster encoding a putative ABC transporter and a final cluster composed of three unknown proteins. Seven genes were typical of bacteriocins, encoding small, cationic peptides, each with an N-terminal double-glycine leader motif. Inactivation of a predicted ABC transporter completely abolished bacteriocin activity. When cloned and expressed together, LBA1803–LBA1800 resulted in markedly higher levels of lactacin B activity. The four peptides were chemically synthesized but exhibited no bacteriocin activity, alone or in combination. Only LBA1800 induced lactacin B production in broth cultures. Conclusions: Lactacin B production is encoded within the 9·5-kb lab operon of 12 genes that are transcribed in a single transcript. LBA1800 is an inducing peptide of bacteriocin production. Significance and Impact of the Study: A three-component regulatory system common to class II bacteriocins regulates the production of this bacteriocin by Lact. acidophilus.}, number={5}, journal={JOURNAL OF APPLIED MICROBIOLOGY}, author={Dobson, A. E. and Sanozky-Dawes, R. B. and Klaenhammer, T. R.}, year={2007}, month={Nov}, pages={1766–1778} }
 @article{klaenhammer_azcarate-peril_altermann_barrangou_2007, title={Influence of the dairy environment on gene expression and substrate(1-3)}, volume={137}, ISSN={["0022-3166"]}, DOI={10.1093/jn/137.3.748s}, abstractNote={Lactic acid bacteria (LAB) are widely used for the industrial production of fermented dairy products and form a group of related low-GC-content gram-positive bacteria. The major species used in dairy manufacturing are Lactobacillus, Lactococcus, Streptococcus, and Leuconostoc. Traditionally most are applied as starter cultures for dairy fermentations or used as probiotic cultures, delivered in dairy vehicles. The appearance of the genomes of Lactococcus lactis, Bidifobacterium longum, Lactobacillus plantarum, L. johnsonii, L. acidophilus, 2 strains of Streptococcus thermophilus, and pending completion of many draft genomic sequences, is now promoting in-depth investigation into the comparative genetic content of LAB. Moreover, whole-genome transcriptional arrays are quickly revealing critical genes/operons that are coordinately expressed and the impact of environmental factors on expression of multiple gene sets. Comparative genomics between multiple genomes is providing insights into genes that are important in metabolic, physiological, and functional roles for different LAB in the environments they inhabit, ranging from the gastrointestinal tract to milk and acidified dairy products.}, number={3}, journal={JOURNAL OF NUTRITION}, author={Klaenhammer, Todd R. and Azcarate-Peril, M. Andrea and Altermann, Eric and Barrangou, Rodolphe}, year={2007}, month={Mar}, pages={748S–750S} }
 @article{sturino_klaenhammer_2007, title={Inhibition of bacteriophage replication in Streptococcus thermophilus by subunit poisoning of primase}, volume={153}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.2007/007567-0}, abstractNote={Invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutations within the putative primase of Streptococcus thermophilus phage κ3. PCR products containing the desired mutation(s) within putative ATPase/helicase and/or oligomerization domains of the κ3-encoded primase gene were cloned into a high-copy-number vector and expressed in S. thermophilus NCK1125. The majority of the plasmid constructs failed to alter phage sensitivity; however, four of the constructs conferred strong phage resistance upon the host. Expression of the K238(A/T) and RR340-341AA mutant proteins in trans suppressed the function of the native phage primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing and centre of infection formation by more than 9 and 3.5 logs, respectively. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and sensitivity of the host, indicating that translation was required to confer phage resistance. Introduction of an E437A mutation in a putative oligomerization domain located downstream of the transdominant K238T mutation also completely suppressed phage resistance. This study appears to represent the first use of transdominant proteins to inhibit phages that are disruptive to cultures used in industrial fermentations.}, journal={MICROBIOLOGY-SGM}, author={Sturino, Joseph M. and Klaenhammer, Todd R.}, year={2007}, month={Oct}, pages={3295–3302} }
 @article{callanan_russell_klaenhammer_2007, title={Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis}, volume={389}, ISSN={["0378-1119"]}, DOI={10.1016/j.gene.2006.10.022}, abstractNote={The Lactobacillus gasseri ADH beta-glucuronidase gene, gusA, was cloned previously and found to exhibit excellent activity in acidic pH ranges, with maximal activity at pH 5.0. In contrast, activity was limited in neutral pH ranges of 6-7. In an effort to improve the activity of the reporter enzyme in neutral pH ranges, the gusA gene was cloned into the broad host range vector, pGK12, and subjected to random mutagenesis by passage through Epicurian coli mutator strain XL1-Red. Two mutant alleles, gusA2 and gusA3, were recovered that produced beta-glucuronidase with increased activity in neutral pH ranges. One of these, gusA3, was significantly more active in the pH range of 4-8 in both Escherichia coli and L. gasseri. Sequence analysis of gusA2 and gusA3 revealed single base pair changes that resulted in D524G and D573A substitutions, respectively. The modified GusA3 enzyme has expanded potential for use as a reporter enzyme in expression hosts that are not acidophilic, as well as lactic acid bacteria and other microorganisms that grow in acidifying environments.}, number={2}, journal={GENE}, author={Callanan, Michael J. and Russell, William M. and Klaenhammer, Todd R.}, year={2007}, month={Mar}, pages={122–127} }
 @misc{pfeiler_klaenhammer_2007, title={The genomics of lactic acid bacteria}, volume={15}, ISSN={["1878-4380"]}, DOI={10.1016/j.tim.2007.09.010}, abstractNote={The lactic acid bacteria (LAB) are one of the most industrially important groups of bacteria. These organisms are used in a variety of ways, including food production, health improvement and production of macromolecules, enzymes and metabolites. The genome sequencing of 20 LAB provides an expanded view of their genetic and metabolic capacities and enables researchers to perform functional and comparative genomic studies. This review highlights some of the findings from these analyses in the context of the numerous roles the LAB play.}, number={12}, journal={TRENDS IN MICROBIOLOGY}, author={Pfeiler, Erika A. and Klaenhammer, Todd R.}, year={2007}, month={Dec}, pages={546–553} }
 @article{engelbrektson_korzenik_sanders_clement_leyer_klaenhammer_kitts_2006, title={Analysis of treatment effects on the microbial ecology of the human intestine}, volume={57}, ISSN={["0168-6496"]}, DOI={10.1111/j.1574-6941.2006.00112.x}, abstractNote={A large number of studies have investigated gastrointestinal microbiota and changes in the gastrointestinal community. However, a concern in these studies is how best to assess changes in gastrointestinal community structure. This paper presents two different human trials where the fecal terminal restriction fragment length polymorphism data sets were analyzed to search for treatment effects. Principle components analysis and cluster analysis based on grouped data are compared with analysis of data by subject using distance coefficients. Comparison with baseline within an individual before grouping by treatment provided a clearer indication of treatment effects than did an evaluation of data grouped before analysis. In addition, a large within-subject sample size and multiple baseline samples are necessary to accurately analyze treatment effects.}, number={2}, journal={FEMS MICROBIOLOGY ECOLOGY}, author={Engelbrektson, Anna L. and Korzenik, Joshua R. and Sanders, Mary Ellen and Clement, Brian G. and Leyer, Gregory and Klaenhammer, Todd R. and Kitts, Christopher L.}, year={2006}, month={Aug}, pages={239–250} }
 @article{duong_barrangou_russell_klaenhammer_2006, title={Characterization of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM}, volume={72}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.72.2.1218-1225.2006}, abstractNote={ABSTRACT Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations or product formulations. The compatible solute trehalose has been widely reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, particularly freezing and dehydration. Analysis of the Lactobacillus acidophilus NCFM genome revealed a putative trehalose utilization locus consisting of a transcriptional regulator, treR ; a trehalose phosphoenolpyruvate transferase system (PTS) transporter, treB ; and a trehalose-6-phosphate hydrolase, treC . The objective of this study was to characterize the tre locus in L. acidophilus and determine whether or not intracellular uptake of trehalose contributes to cryoprotection. Cells subjected to repeated freezing and thawing cycles were monitored for survival in the presence of various concentrations of trehalose. At 20% trehalose a 2-log increase in survival was observed. The trehalose PTS transporter and trehalose hydrolase were disrupted by targeted plasmid insertions. The resulting mutants were unable to grow on trehalose, indicating that both trehalose transport into the cell via a PTS and hydrolysis via a trehalose-6-phosphate hydrolase were necessary for trehalose fermentation. Trehalose uptake was found to be significantly reduced in the transporter mutant but unaffected in the hydrolase mutant. Additionally, the cryoprotective effect of trehalose was reduced in these mutants, suggesting that intracellular transport and hydrolysis contribute significantly to cryoprotection.}, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Duong, T and Barrangou, R and Russell, WM and Klaenhammer, TR}, year={2006}, month={Feb}, pages={1218–1225} }
 @article{ventura_canchaya_bernini_altermann_barrangou_mcgrath_claesson_li_leahy_walker_et al._2006, title={Comparative genomics and transcriptional analysis of prophages identified in the Genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei}, volume={72}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.72.5.3130-3146.2006}, abstractNote={ABSTRACT Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae , respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed-field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli.}, number={5}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Ventura, Marco and Canchaya, Carlos and Bernini, Valentina and Altermann, Eric and Barrangou, Rodolphe and McGrath, Stephen and Claesson, Marcus J. and Li, Yin and Leahy, Sinead and Walker, Carey D. and et al.}, year={2006}, month={May}, pages={3130–3146} }
 @article{makarova_slesarev_wolf_sorokin_mirkin_koonin_pavlov_pavlova_karamychev_polouchine_et al._2006, title={Comparative genomics of the lactic acid bacteria}, volume={103}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0607117103}, abstractNote={Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.}, number={42}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Makarova, K. and Slesarev, A. and Wolf, Y. and Sorokin, A. and Mirkin, B. and Koonin, E. and Pavlov, A. and Pavlova, N. and Karamychev, V. and Polouchine, N. and et al.}, year={2006}, month={Oct}, pages={15611–15616} }
 @misc{sturino_klaenhammer_2006, title={Engineered bacteriophage-defence systems in bioprocessing}, volume={4}, ISSN={["1740-1534"]}, DOI={10.1038/nrmicro1393}, number={5}, journal={NATURE REVIEWS MICROBIOLOGY}, author={Sturino, JM and Klaenhammer, TR}, year={2006}, month={May}, pages={395–404} }
 @article{barrangou_azcarate-peril_duong_conners_kelly_klaenhammer_2006, title={Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays}, volume={103}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0511287103}, abstractNote={The transport and catabolic machinery involved in carbohydrate utilization by Lactobacillus acidophilus was characterized genetically by using whole-genome cDNA microarrays. Global transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose, and fructooligosaccharides. Hybridizations were carried out by using a round-robin design, and microarray data were analyzed with a two-stage mixed model ANOVA. Differentially expressed genes were visualized by hierarchical clustering, volcano plots, and contour plots. Overall, only 63 genes (3% of the genome) showed a >4-fold induction. Specifically, transporters of the phospho enol pyruvate:sugar transferase system were identified for uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette transporters were identified for uptake of raffinose and fructooligosaccharides. A member of the LacS subfamily of galactoside-pentose hexuronide translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of monosaccharides, disaccharides, and polysaccharides into substrates of glycolysis were also found, including enzymatic machinery of the Leloir pathway. The transcriptome appeared to be regulated by carbon catabolite repression. Although substrate-specific carbohydrate transporters and hydrolases were regulated at the transcriptional level, genes encoding regulatory proteins CcpA, Hpr, HprK/P, and EI were consistently highly expressed. Genes central to glycolysis were among the most highly expressed in the genome. Collectively, microarray data revealed that coordinated and regulated transcription of genes involved in sugar uptake and metabolism is based on the specific carbohydrate provided. L. acidophilus 's adaptability to environmental conditions likely contributes to its competitive ability for limited carbohydrate sources available in the human gastrointestinal tract.}, number={10}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Barrangou, R and Azcarate-Peril, MA and Duong, T and Conners, SB and Kelly, RM and Klaenhammer, TR}, year={2006}, month={Mar}, pages={3816–3821} }
 @article{azcarate-peril_bruno-barcena_hassan_klaenhammer_2006, title={Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus}, volume={72}, ISSN={["1098-5336"]}, url={http://europepmc.org/abstract/med/16517636}, DOI={10.1128/AEM.72.3.1891-1899.2006}, abstractNote={ABSTRACT Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene ( frc ) and an oxalyl coenzyme A decarboxylase gene ( oxc ) was identified in the genome of the probiotic bacterium Lactobacillus acidophilus . Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequisite for frc and oxc transcription. As a consequence, oxalate-dependent induction of these genes occurred only in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome information was available, other lactic acid bacteria were screened for frc and oxc genes. With the exception of Lactobacillus gasseri and Bifidobacterium lactis , none of the other strains harbored genes for oxalate utilization.}, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Azcarate-Peril, MA and Bruno-Barcena, JM and Hassan, HM and Klaenhammer, TR}, year={2006}, month={Mar}, pages={1891–1899} }
 @article{altermann_russell_azcarate-peril_barrangou_buck_mcauliffe_souther_dobson_duong_callanan_et al._2005, title={Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM}, volume={102}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0409188102}, abstractNote={Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.}, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Altermann, E and Russell, WM and Azcarate-Peril, MA and Barrangou, R and Buck, BL and McAuliffe, O and Souther, N and Dobson, A and Duong, T and Callanan, M and et al.}, year={2005}, month={Mar}, pages={3906–3912} }
 @article{buck_altermann_svingerud_klaenhammer_2005, title={Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM}, volume={71}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.71.12.8344-8351.2005}, abstractNote={ABSTRACT Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.}, number={12}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Buck, BL and Altermann, E and Svingerud, T and Klaenhammer, TR}, year={2005}, month={Dec}, pages={8344–8351} }
 @article{mcauliffe_cano_klaenhammer_2005, title={Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM}, volume={71}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.71.8.4925-4929.2005}, abstractNote={ABSTRACT Two genes, bshA and bshB , encoding bile salt hydrolase enzymes (EC 3.5.1.24) were identified in the genome sequence of Lactobacillus acidophilus NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate specificities for these two enzymes.}, number={8}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={McAuliffe, O and Cano, RJ and Klaenhammer, TR}, year={2005}, month={Aug}, pages={4925–4929} }
 @article{klaenhammer_peril_barrangou_duong_altermann_2005, title={Genomic Perspectives on Probiotic Lactic Acid Bacteria}, volume={24}, ISBN={1342-1441}, DOI={10.12938/bifidus.24.31}, abstractNote={The lactic acid bacteria are Gram-positive fermentative microorganisms known primarily for their roles as starter cultures and probiotics. The food industry represents one of the largest manufacturing industries in the world and recent trends are rapidly expanding the use of probiotic cultures within functional foods. Understanding and control of lactic acid bacteria is now being revolutionized by genomic sciences and the appearance of the complete genome sequences for Bifidobacterium longum, Lactobacillus johnsonii, Lactobacillus plantarum, and draft sequences for Lactobacillus gasseri and Lactobacillus casei. This explosion of DNA sequence information, accompanied by the development of bioinformatic tools for nucleic acid and protein analysis, now allows rapid characterization of the lactic acid bacteria for their genomic content and expression profiles across the entire genome. Comparative genomics has already revealed important similarities and differences in strains, species, and genera and will likely identify key genetic features responsible for the beneficial properties ascribed to probiotic lactic acid bacteria. Practical genomics for the lactic acid bacteria promises to establish the genetic landscape, correlate genotypes with desirable phenotypes, establish genetic criteria for strain selection, improve culture stability by stress preconditioning, provide opportunities for metabolic engineering, and uncover a mechanistic basis for the beneficial activities of probiotics when delivered in various foods. This presentation will examine the genomic content of probiotic Lactobacillus cultures, compared to those lactic acid bacterial genomes that have appeared recently. In addition, expression profiling by whole genome microarrays will be used to illustrate how environmental conditions encountered during biomanufacturing, fermentation, and the gastrointestinal tract can impact gene expression and culture functionality.}, number={2}, journal={Bioscience and Microflora}, publisher={BMFH Press}, author={Klaenhammer, Todd R. and Peril, Andrea Azcarate and Barrangou, Rodolphe and Duong, Tri and Altermann, Eric}, year={2005}, pages={31–33} }
 @misc{klaenhammer_barrangou_buck_azcarate-peril_altermann_2005, title={Genomic features of lactic acid bacteria effecting bioprocessing and health}, volume={29}, ISSN={["1574-6976"]}, DOI={10.1016/j.femsre.2005.04.007}, abstractNote={The lactic acid bacteria are a functionally related group of organisms known primarily for their bioprocessing roles in food and beverages. More recently, selected members of the lactic acid bacteria have been implicated in a number of probiotic roles that impact general health and well-being. Genomic analyses of multiple members of the lactic acid bacteria, at the genus, species, and strain level, have now elucidated many genetic features that direct their fermentative and probiotic roles. This information is providing an important platform for understanding core mechanisms that control and regulate bacterial growth, survival, signaling, and fermentative processes and, in some cases, potentially underlying probiotic activities within complex microbial and host ecosystems.}, number={3}, journal={FEMS MICROBIOLOGY REVIEWS}, publisher={Wiley-Blackwell}, author={Klaenhammer, TR and Barrangou, R and Buck, BL and Azcarate-Peril, MA and Altermann, E}, year={2005}, month={Aug}, pages={393–409} }
 @article{mohamadzadeh_olson_kalina_ruthel_demmin_warfield_bavari_klaenhammer_2005, title={Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization}, volume={102}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0500098102}, abstractNote={Professional antigen-presenting dendritic cells (DCs) are critical in regulating T cell immune responses at both systemic and mucosal sites. Many Lactobacillus species are normal members of the human gut microflora and most are regarded as safe when administered as probiotics. Because DCs can naturally or therapeutically encounter lactobacilli, we investigated the effects of several well defined strains, representing three species of Lactobacillus on human myeloid DCs (MDCs) and found that they modulated the phenotype and functions of human MDCs. Lactobacillus -exposed MDCs up-regulated HLA-DR, CD83, CD40, CD80, and CD86 and secreted high levels of IL-12 and IL-18, but not IL-10. IL-12 was sustained in MDCs exposed to all three Lactobacillus species in the presence of LPS from Escherichia coli , whereas LPS-induced IL-10 was greatly inhibited. MDCs activated with lactobacilli clearly skewed CD4 + and CD8 + T cells to T helper 1 and Tc1 polarization, as evidenced by secretion of IFN-γ, but not IL-4 or IL-13. These results emphasize a potentially important role for lactobacilli in modulating immunological functions of DCs and suggest that certain strains could be particularly advantageous as vaccine adjuvants, by promoting DCs to regulate T cell responses toward T helper 1 and Tc1 pathways.}, number={8}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Mohamadzadeh, M and Olson, S and Kalina, WV and Ruthel, G and Demmin, GL and Warfield, KL and Bavari, S and Klaenhammer, TR}, year={2005}, month={Feb}, pages={2880–2885} }
 @article{bruno-barcena_azcarate-peril_klaenhammer_hassan_2005, title={Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri}, volume={246}, ISSN={["1574-6968"]}, url={http://europepmc.org/abstract/med/15869967}, DOI={10.1016/j.femsle.2005.03.044}, abstractNote={A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-β-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361–4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 °C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.}, number={1}, journal={FEMS MICROBIOLOGY LETTERS}, author={Bruno-Barcena, JM and Azcarate-Peril, MA and Klaenhammer, TR and Hassan, HM}, year={2005}, month={May}, pages={91–101} }
 @article{azcarate-peril_mcauliffe_altermann_lick_russell_klaenhammer_2005, title={Microarray analysis of a two-component regulatory system involved in acid resistance and proteolytic activity in Lactobacillus acidophilus}, volume={71}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.71.10.5794-5804.2005}, abstractNote={ABSTRACT Two-component regulatory systems are one primary mechanism for environmental sensing and signal transduction. Annotation of the complete genome sequence of the probiotic bacterium Lactobacillus acidophilus NCFM revealed nine two-component regulatory systems. In this study, the histidine protein kinase of a two-component regulatory system (LBA1524HPK-LBA1525RR), similar to the acid-related system lis RK from Listeria monocytogenes (P. D. Cotter et al., J. Bacteriol. 181:6840-6843, 1999), was insertionally inactivated. A whole-genome microarray containing 97.4% of the annotated genes of L. acidophilus was used to compare genome-wide patterns of transcription at various pHs between the control and the histidine protein kinase mutant. The expression pattern of approximately 80 genes was affected by the LBA1524HPK mutation. Putative LBA1525RR target loci included two oligopeptide-transport systems present in the L. acidophilus genome, other components of the proteolytic system, and a LuxS homolog, suspected of participating in synthesis of the AI-2 signaling compound. The mutant exhibited lower tolerance to acid and ethanol in logarithmic-phase cells and poor acidification rates in milk. Supplementation of milk with Casamino Acids essentially restored the acid-producing ability of the mutant, providing additional evidence for a role of this two component system in regulating proteolytic activity in L. acidophilus .}, number={10}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Azcarate-Peril, MA and McAuliffe, O and Altermann, E and Lick, S and Russell, WM and Klaenhammer, TR}, year={2005}, month={Oct}, pages={5794–5804} }
 @misc{rastall_gibson_gill_guarner_klaenhammer_pot_reid_rowland_sanders_2005, title={Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: An overview of enabling science and potential applications}, volume={52}, ISSN={["1574-6941"]}, DOI={10.1016/j.femsec.2005.01.003}, abstractNote={The application of probiotics and prebiotics to the manipulation of the microbial ecology of the human colon has recently seen many scientific advances. The sequencing of probiotic genomes is providing a wealth of new information on the biology of these microorganisms. In addition, we are learning more about the interactions of probiotics with human cells and with pathogenic bacteria. An alternative means of modulating the colonic microbial community is by the use of prebiotic oligosaccharides. Increasing knowledge of the metabolism of prebiotics by probiotics is allowing us to consider specifically targeting such dietary intervention tools at specific population groups and specific disease states.}, number={2}, journal={FEMS MICROBIOLOGY ECOLOGY}, author={Rastall, RA and Gibson, GR and Gill, HS and Guarner, F and Klaenhammer, TR and Pot, B and Reid, G and Rowland, IR and Sanders, ME}, year={2005}, month={Apr}, pages={145–152} }
 @article{altermann_klaenhammer_2005, title={PathwayVoyager: pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database}, volume={6}, journal={BMC Genomics}, author={Altermann, E. and Klaenhammer, T. R.}, year={2005} }
 @article{lu_altermann_breidt_predki_fleming_klaenhammer_2005, title={Sequence analysis of the Lactobacillus plantarum bacteriophage Phi JL-1}, volume={348}, ISSN={["1879-0038"]}, DOI={10.1016/j.gene.2004.12.052}, abstractNote={The complete genomic sequence of a Lactobacillus plantarum virulent phage PhiJL-1 was determined. The phage possesses a linear, double-stranded, DNA genome consisting of 36,677 bp with a G+C content of 39.36%. A total of 52 possible open reading frames (ORFs) were identified. According to N-terminal amino acid sequencing and bioinformatic analyses, proven or putative functions were assigned to 21 ORFs (41%), including 5 structural protein genes. The PhiJL-1 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication, DNA packaging, head and tail morphogenesis, and lysis. This type of modular genomic organization was similar to several other phages infecting lactic acid bacteria. The structural gene maps revealed that the order of the head and tail genes is highly conserved among the genomes of several Siphoviridae phages, allowing the assignment of probable functions to certain uncharacterized ORFs from phage PhiJL-1 and other Siphoviridae phages.}, journal={GENE}, author={Lu, Z and Altermann, E and Breidt, F and Predki, P and Fleming, HP and Klaenhammer, TR}, year={2005}, month={Mar}, pages={45–54} }
 @misc{sturino_klaenhammer_2004, title={Antisense RNA expression strategies effective against Streptococcus thermophilus bacteriophages}, volume={6,686,192}, number={2004 Feb. 3}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Sturino, J. M. and Klaenhammer, T. R.}, year={2004} }
 @article{sturino_klaenhammer_2004, title={Antisense RNA targeting of primase interferes with bacteriophage replication in Streptococcus thermophilus}, volume={70}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.70.3.1735-1743.2004}, abstractNote={ABSTRACT The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene ( pri3.1 ) from S. thermophilus phage κ3 provided significant protection from κ3 and two other Sfi21-type phages. Expression of pri3.10-AS , an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of κ3 to 3 × 10 −3 and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 × 10 −3 . The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5′ region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.}, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Sturino, JM and Klaenhammer, TR}, year={2004}, month={Mar}, pages={1735–1743} }
 @misc{sturino_klaenhammer_2004, title={Bacteriophage defense systems and strategies for lactic acid bacteria}, volume={56}, ISBN={["0-12-002658-9"]}, ISSN={["0065-2164"]}, DOI={10.1016/S0065-2164(04)56011-2}, abstractNote={This chapter describes the complex relationship that exists between strains of S. thermophilus and their bacteriophages. In particular, this chapter highlights the various defense strategies and systems that have been developed to curb the propagation and evolution of lytic phages. Point mutation and recombination are the two great engines of phage evolution. Their short generation time and large burst sizes can act to accelerate the rate at which mutant phages may overcome a given defense. The plasticity of phage genomes is critical to their rapid evolution. Phages are a leading cause of failure in industrial fermentations. Further, as the demand for fermented food products made with strains of S. thermophilus has increased, so has the incidence and severity of phage attacks against these thermophilic starter strains. With the expansion of fermentation and bioprocessing systems reliant on lactic acid bacteria (LAB), disruption by bacteriophages remains a serious concern. Together, these persistent pressures necessitate the continued development of starter cultures with enhanced phage resistance properties.}, journal={ADVANCES IN APPLIED MICROBIOLOGY, VOL 56}, author={Sturino, JM and Klaenhammer, TR}, year={2004}, pages={331-+} }
 @article{majhenic_venema_allison_matijasic_rogelj_klaenhammer_2004, title={DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B, two bacteriocins produced by Lactobacillus gasseri LF221}, volume={63}, ISSN={["1432-0614"]}, DOI={10.1007/s00253-003-1424-2}, abstractNote={Lactobacillus gasseri LF221, an isolate from the feces of a child, produces two bacteriocins. Standard procedures for molecular techniques were used to locate, clone and sequence the fragments of LF221 chromosomal DNA carrying the acidocin LF221 A and B structural genes, respectively. Sequencing analysis revealed the gene of acidocin LF221 A to be an open reading frame encoding a protein composed of 69 amino acids, including a 16-amino-acid N-terminal extension. The acidocin LF221 B gene was found to encode a 65-amino-acid bacteriocin precursor with a 17-amino-acid N-terminal leader peptide. DNA homology searches showed similarities of acidocin LF221 A to brochocin B, lactococcin N and thermophilin B, whereas acidocin LF221 B exhibited some homology to lactacin F and was virtually identical to gassericin X. The peptides encoded by orfA1 and orfB3 showed characteristics of class II bacteriocins and are suspected to be the complementary peptides of acidocin A and B, respectively. orfA3 and orfB5 are proposed to encode putative immunity proteins for the acidocins. Acidocin LF221 A and acidocin LF221 B are predicted to be members of the two-component class II bacteriocins, where acidocin LF221 A appears to be a novel bacteriocin. L. gasseri LF221 is being developed as a potential probiotic strain and a food/feed preservative. Detailed characterization of its acidocins is an important piece of background information useful in applying the strain into human or animal consumption. The genetic information on both acidocins also enables tracking of the LF221 strain in mixed populations and complex environments.}, number={6}, journal={APPLIED MICROBIOLOGY AND BIOTECHNOLOGY}, author={Majhenic, AC and Venema, K and Allison, GE and Matijasic, BB and Rogelj, I and Klaenhammer, TR}, year={2004}, month={Feb}, pages={705–714} }
 @article{yildirim_lin_hitchins_jaykus_altermann_klaenhammer_kathariou_2004, title={Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods}, volume={70}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.70.7.4158-4164.2004}, abstractNote={ABSTRACT Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.}, number={7}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Yildirim, S and Lin, W and Hitchins, AD and Jaykus, LA and Altermann, E and Klaenhammer, TR and Kathariou, S}, year={2004}, month={Jul}, pages={4158–4164} }
 @article{yildirim_lin_hitchins_jaykus_altermann_klaenhammer_kathariou_2004, title={Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods (vol 70, pg 4158, 2004)}, volume={70}, ISSN={["0099-2240"]}, DOI={10.1128/aem.70.12.7581.2004}, abstractNote={Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis. Food contamination by Listeria monocytogenes has been implicated in numerous outbreaks and sporadic cases of human illness. Most commonly implicated in listeriosis are highly processed, ready-to-eat (RTE) foods that are kept refrigerated for various periods of time. At risk for listeriosis are people in the extremes of age, pregnant women and their fetuses, cancer patients, and others experiencing immunosuppression (13, 24, 35, 38). Listeriosis can have severe symptoms (septicemia, meningitis, and stillbirths) and a high mortality rate (20 to 30%). Hence, regulations exist in numerous nations concerning the density (e.g., 1 CFU/25 g) of cells of the etiologic agent permissible in RTE foods. Such regulations are based on the hypothesis that any L. monocytogenes strain that can be detected in RTE foods has the potential to pose serious hazards to human health. The potential hazard posed by listerial contamination of RTE foods can be influenced by the number of cells at the point of consumption, which would depend on conditions of storage, type of food matrix and its impact on growth, presence of competing microflora and antimicrobial agents, etc. In addition, the strain type of L. monocytogenes involved may be of importance. It is likely, based on studies with other bacterial pathogens, that some strains and strain clusters (clonal groups) within the species might be more pathogenic than others. Speculations have been formulated that only a fraction of the strains of L. monocytogenes found in foods may be capable of causing human illness (20). There is indeed evidence that the repertoire of strains ca}, number={12}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Yildirim, S and Lin, W and Hitchins, AD and Jaykus, LA and Altermann, E and Klaenhammer, TR and Kathariou, S}, year={2004}, month={Dec}, pages={7581–7581} }
 @article{bruno-barcena_andrus_libby_klaenhammer_hassan_2004, title={Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity}, volume={70}, ISSN={["1098-5336"]}, url={http://europepmc.org/abstract/med/15294805}, DOI={10.1128/AEM.70.8.4702-4710.2004}, abstractNote={ABSTRACT In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD ( sodA ) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89 , Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene ( sodA ) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri , L. johnsonii , L. acidophilus , and L. reuteri , respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O 2 ·− and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.}, number={8}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Bruno-Barcena, JM and Andrus, JM and Libby, SL and Klaenhammer, TR and Hassan, HM}, year={2004}, month={Aug}, pages={4702–4710} }
 @article{evans_swaminathan_graves_altermann_klaenhammer_fink_kernodle_kathariou_2004, title={Genetic markers unique to Listeria monocytogenes serotype 4b differentiate epidemic clone II (hot dog outbreak strains) from other lineages}, volume={70}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.70.4.2383-2390.2004}, abstractNote={ABSTRACT A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA , which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.}, number={4}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Evans, MR and Swaminathan, B and Graves, LM and Altermann, E and Klaenhammer, TR and Fink, RC and Kernodle, S and Kathariou, S}, year={2004}, month={Apr}, pages={2383–2390} }
 @article{azcarate-peril_altermann_hoover-fitzula_cano_klaenhammer_2004, title={Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance}, volume={70}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.70.9.5315-5322.2004}, abstractNote={ABSTRACT Amino acid decarboxylation-antiporter reactions are one of the most important systems for maintaining intracellular pH between physiological limits under acid stress. We analyzed the Lactobacillus acidophilus NCFM complete genome sequence and selected four open reading frames with similarities to genes involved with decarboxylation reactions involved in acid tolerance in several microorganisms. Putative genes encoding an ornithine decarboxylase, an amino acid permease, a glutamate γ-aminobutyrate antiporter, and a transcriptional regulator were disrupted by insertional inactivation. The ability of L. acidophilus to survive low-pH conditions, such as those encountered in the stomach or fermented dairy foods, was investigated and compared to the abilities of early- and late-stationary-phase cells of the mutants by challenging them with a variety of acidic conditions. All of the integrants were more sensitive to low pH than the parental strain. Interestingly, each integrant also exhibited an adaptive acid response during logarithmic growth, indicating that multiple mechanisms are present and orchestrated in L. acidophilus in response to acid challenge.}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Azcarate-Peril, MA and Altermann, E and Hoover-Fitzula, RL and Cano, RJ and Klaenhammer, TR}, year={2004}, month={Sep}, pages={5315–5322} }
 @article{altermann_buck_cano_klaenhammer_2004, title={Identification and phenotypic characterization of the cell-division protein CdpA}, volume={342}, ISSN={["1879-0038"]}, DOI={10.1016/j.gene.2004.08.004}, abstractNote={Analysis of the automated computer annotation of the early draft phase genome of Lactobacillus acidophilus NCFM revealed the previously discovered S-layer gene slpA and an additional partial ORF with weak similarities to S-layer proteins. The entire gene was sequenced to reveal a 1799-bp gene coding for 599 amino acids with a calculated molecular mass of 64.8 kDa. No transcription or translation signals could be determined in close proximity to the 5′-region. However, a strong putative terminator with a free energy of −16.84 kcal/mol was identified directly downstream of the gene. A PSI-Blast analysis showed similarities to members of S-layer proteins, cell-wall associated proteinases and hexosyl-transferases. Calculation of an unrooted phylogenetic tree with other examples of S-layer proteins and proteinases placed the deduced protein separately from both groups. A derivative of L. acidophilus NCFM was constructed by targeted integration into the gene. SDS-PAGE analysis of non-covalently linked proteins of the cell wall of the mutant, compared to the wild type, revealed the loss of a cell-surface protein. Phenotypic analyses of the mutant revealed significant changes in cell morphology, altered responses to various environmental stresses, and lowered cell adhesion. Based on the in silico and functional analyses, we ascertained that this protein plays a role in cell-wall processing during the growth and cell–cell separation and designated the gene as cell-division protein, cdpA.}, number={1}, journal={GENE}, author={Altermann, E and Buck, BL and Cano, R and Klaenhammer, TR}, year={2004}, month={Nov}, pages={189–197} }
 @misc{barrangou_klaenhammer_altermann_2004, title={Lactobacillus acidophilus nucleic acids encoding fructo-oligosaccharide utilization compounds and uses thereof}, volume={7,407,787}, number={2004 Jun 22}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Barrangou, R. and Klaenhammer, T. R. and Altermann, E.}, year={2004} }
 @article{pridmore_berger_desiere_vilanova_barretto_pittet_zwahlen_rouvet_altermann_barrangou_et al._2004, title={The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533}, volume={101}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0307327101}, abstractNote={Lactobacillus johnsonii NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their “probiotic” activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation. To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of L. johnsonii NCC 533. Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients. Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT). Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected. In silico genome comparisons with the >95% complete genome sequence of the closely related Lactobacillus gasseri revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons. Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem.}, number={8}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Pridmore, RD and Berger, B and Desiere, F and Vilanova, D and Barretto, C and Pittet, AC and Zwahlen, MC and Rouvet, M and Altermann, E and Barrangou, R and et al.}, year={2004}, month={Feb}, pages={2512–2517} }
 @article{roberts_belfort_bestor_bhagwat_bickle_bitinaite_blumenthal_degtyarev_dryden_dybvig_et al._2003, title={A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes}, volume={31}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkg274}, abstractNote={A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.}, number={7}, journal={NUCLEIC ACIDS RESEARCH}, author={Roberts, RJ and Belfort, M and Bestor, T and Bhagwat, AS and Bickle, TA and Bitinaite, J and Blumenthal, RM and Degtyarev, SK and Dryden, DTF and Dybvig, K and et al.}, year={2003}, month={Apr}, pages={1805–1812} }
 @article{ventura_canchaya_meylan_klaenhammer_zink_2003, title={Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification}, volume={69}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.69.11.6908-6922.2003}, abstractNote={ABSTRACT We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli . In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus .}, number={11}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Ventura, M and Canchaya, C and Meylan, V and Klaenhammer, TR and Zink, R}, year={2003}, month={Nov}, pages={6908–6922} }
 @article{barrangou_altermann_hutkins_cano_klaenhammer_2003, title={Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus}, volume={100}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1332765100}, abstractNote={Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter-operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract.}, number={15}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Barrangou, R and Altermann, E and Hutkins, R and Cano, R and Klaenhammer, TR}, year={2003}, month={Jul}, pages={8957–8962} }
 @article{lu_breidt_fleming_altermann_klaenhammer_2003, title={Isolation and characterization of a Lactobacillus plantarum bacteriophage, Phi JL-1, from a cucumber fermentation}, volume={84}, ISSN={["1879-3460"]}, DOI={10.1016/S0168-1605(03)00111-9}, abstractNote={A virulent Lactobacillus plantarum bacteriophage, PhiJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of PhiJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that PhiJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl(2)) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of PhiJL-1 at pH 6.5 were estimated to be 2.7 min at 70 degrees C and 0.2 min at 80 degrees C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage PhiJL-1 may be important for the development of controlled vegetable fermentations.}, number={2}, journal={INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY}, author={Lu, Z and Breidt, F and Fleming, HP and Altermann, E and Klaenhammer, TR}, year={2003}, month={Jul}, pages={225–235} }
 @article{andrus_bowen_klaenhammer_hassan_2003, title={Molecular characterization and functional analysis of the manganese-containing superoxide dismutase gene (sodA) from Streptococcus thermophilus AO54}, volume={420}, ISSN={["1096-0384"]}, DOI={10.1016/j.abb.2003.09.007}, abstractNote={This report describes the isolation, sequencing, and functional analysis of the sodA gene, encoding Mn-superoxide dismutase, from Streptococcus thermophilus AO54. The gene was found to encode a 201 amino acid polypeptide with 88 and 83% identity to SodA from Streptococcus mutans and Streptococcus agalacticae, respectively. Primer extension analysis revealed a transcriptional start site 27 nucleotides upstream of initiation codon. The gene was expressed in Escherichia coli and was able to rescue the growth of a sodAsodB mutant in a minimal-medium containing 10(-6)M paraquat. A sodA mutant of S. thermophilus was constructed and found to be more sensitive to aerobic growth than its parent strain. Supplementing the medium with MnCl(2) improved the growth of the mutant, only under microaerophilic conditions. The results suggest that sodA is essential for the aerobic growth of S. thermophilus. In the absence of functional SodA, manganese ions may provide partial protection against oxygen toxicity.}, number={1}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, author={Andrus, JM and Bowen, SW and Klaenhammer, TR and Hassan, HM}, year={2003}, month={Dec}, pages={103–113} }
 @misc{russell_klaenhammer_2003, title={Polynucleotide encoding a Lactobacillus gasseri beta-glucuronidase polypeptide}, volume={6,664,097}, number={2003 Dec. 16}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Russell, W. M. and Klaenhammer, T. R.}, year={2003} }
 @article{barrangou_yoon_breidt_fleming_klaenhammer_2002, title={Characterization of six Leuconostoc fallax bacteriophages isolated from an industrial sauerkraut fermentation}, volume={68}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.68.11.5452-5458.2002}, abstractNote={ABSTRACT Six bacteriophages active against Leuconostoc fallax strains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the species L. fallax and had different host ranges among the strains of this species tested. Morphologically, three of the phages were assigned to the family Siphoviridae , and the three others were assigned to the family Myoviridae . Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes. Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct. These results revealed for the first time the existence of bacteriophages that are active against L. fallax and confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation. Since a variety of L. fallax strains have been shown to be present in sauerkraut fermentation, bacteriophages active against L. fallax are likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation.}, number={11}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Barrangou, R and Yoon, SS and Breidt, F and Fleming, HP and Klaenhammer, TR}, year={2002}, month={Nov}, pages={5452–5458} }
 @article{klaenhammer_altermann_arigoni_bolotin_breidt_broadbent_cano_chaillou_deutscher_gasson_et al._2002, title={Discovering lactic acid bacteria by genomics}, volume={82}, DOI={10.1007/978-94-017-2029-8_3}, abstractNote={This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, environmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram-positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.}, number={1-4}, journal={Antonie Van Leeuwenhoek}, author={Klaenhammer, T. and Altermann, E. and Arigoni, F. and Bolotin, A. and Breidt, F. and Broadbent, J. and Cano, R. and Chaillou, S. and Deutscher, J. and Gasson, M. and et al.}, year={2002}, pages={29–58} }
 @article{sturino_klaenhammer_2002, title={Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages}, volume={68}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.68.2.588-596.2002}, abstractNote={ABSTRACT Antisense RNA complementary to a putative helicase gene ( hel3.1 ) of a cos -type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos -type S. thermophilus bacteriophages and one pac -type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage κ3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the κ3-derived phage-encoded resistance (PER) plasmid pTRK690:: ori3.1 , the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10 −7 and 10 −8 , beyond the resistance conferred by the PER element alone (less than 10 −6 ). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.}, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Sturino, JM and Klaenhammer, TR}, year={2002}, month={Feb}, pages={588–596} }
 @article{mermelstein_fennema_batt_goff_griffiths_hoover_hsieh_juneja_kroger_lund_et al._2002, title={Food research trends - 2003 and beyond}, volume={56}, number={12}, journal={Food Technology}, author={Mermelstein, N. H. and Fennema, O. R. and Batt, C. A. and Goff, H. D. and Griffiths, M. W. and Hoover, D. G. and Hsieh, F. H. and Juneja, V. K. and Kroger, M. and Lund, D. B. and et al.}, year={2002}, pages={30-} }
 @article{barrangou_yoon_breidt_fleming_klaenhammer_2002, title={Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation}, volume={68}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.68.6.2877-2884.2002}, abstractNote={ABSTRACT Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides . Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides . The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides , a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.}, number={6}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Barrangou, R and Yoon, SS and Breidt, F and Fleming, HP and Klaenhammer, TR}, year={2002}, month={Jun}, pages={2877–2884} }
 @article{yoon_barrangou-poueys_breidt_klaenhammer_fleming_2002, title={Isolation and characterization of bacteriophages from fermenting sauerkraut}, volume={68}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.68.2.973-976.2002}, abstractNote={ABSTRACT This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.}, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Yoon, SS and Barrangou-Poueys, R and Breidt, F and Klaenhammer, TR and Fleming, HP}, year={2002}, month={Feb}, pages={973–976} }
 @article{durmaz_madsen_israelsen_klaenhammer_2002, title={Lactococcus lactis lytic bacteriophages of the p335 group are inhibited by overexpression of a truncated CI repressor}, volume={184}, ISSN={["0021-9193"]}, DOI={10.1128/JB.184.23.6532-6543.2002}, abstractNote={ABSTRACT Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage φ31 encodes a genetic switch region of c I and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative c I repressor gene of phage φ31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length c I gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the c I gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage φ31 was inhibited to an efficiency of plaquing (EOP) of 10 −6 in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the φ31 EOP to <10 −7 . Phage φ31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the φ31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.}, number={23}, journal={JOURNAL OF BACTERIOLOGY}, author={Durmaz, E and Madsen, SA and Israelsen, H and Klaenhammer, TR}, year={2002}, month={Dec}, pages={6532–6543} }
 @article{tuler_callanan_klaenhammer_2002, title={Overexpression of peptidases in Lactococcus and evaluation of their release from leaky cells}, volume={85}, ISSN={["1525-3198"]}, DOI={10.3168/jds.S0022-0302(02)74326-9}, abstractNote={Walker and Klaenhammer (2001) developed a novel expression system in Lactococcus lactis that facilitated the release of beta-galactosidase (117 kDa monomer) without the need for secretion or export signals. The system is based on the controlled expression of integrated prophage holin and lysin cassettes via a lactococcal bacteriophage phi31 transcriptional activator (Tac31A) that resides on a high-copy plasmid. Approximately 85% of beta-galactosidase activity was detected in the supernatant of leaky lactococci without evidence of hindered growth, cell lysis, or membrane damage. The objective of this study was to determine if intracellular peptidases were externalized from leaky lactococci. Five L. lactis peptidases (PepA, PepC, PepN, PepO and PepXP) and two Lactobacillus helveticus peptidases (PepN and PepO) were cloned and overexpressed on two high-copy vectors. The lactococcal peptidases were also cloned into the high-copy vector that contained the Tac31A transcriptional activator to determine if they were externalized from the leaky prophage-containing L. lactis subsp. lactis strain NCK203. Two of the lactococcal peptidases (PepA and PepO) required an additional strong promoter (Lactobacillus paracasei P144) and optimized assay conditions to detect enzyme activity. Results showed different levels of enzymatic overexpression associated with the cellular fraction (2 to 250-fold increases in activity) and negligible amounts of activity present within the supernatant fraction (0 to 6% of total peptidase activity). The lactococcal phage-based protein release mechanism did not facilitate the externalization of the lactococcal peptidases investigated in this study.}, number={10}, journal={JOURNAL OF DAIRY SCIENCE}, author={Tuler, TR and Callanan, MJ and Klaenhammer, TR}, year={2002}, month={Oct}, pages={2438–2450} }
 @misc{kullen_klaenhammer_2001, title={Acid-inducible promoters for gene expression}, volume={6,242,194}, number={2001 June 5}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Kullen, M. J. and Klaenhammer, T. R.}, year={2001} }
 @article{madsen_mills_djordjevic_israelsen_klaenhammer_2001, title={Analysis of the genetic switch and replication region of a P335-type bacteriophage with an obligate lytic lifestyle on Lactococcus lactis}, volume={67}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.67.3.1128-1139.2001}, abstractNote={The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of phi31 10(6)-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.}, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Madsen, SM and Mills, D and Djordjevic, G and Israelsen, H and Klaenhammer, TR}, year={2001}, month={Mar}, pages={1128–1139} }
 @article{russell_klaenhammer_2001, title={Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination}, volume={67}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.67.9.4361-4364.2001}, abstractNote={ABSTRACT An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri . The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35–50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding β-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding β-glucuronidase.}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Russell, WM and Klaenhammer, TR}, year={2001}, month={Sep}, pages={4361–4364} }
 @article{russell_klaenhammer_2001, title={Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH}, volume={67}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.67.3.1253-1261.2001}, abstractNote={The gusA gene, encoding a new beta-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a beta-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored beta-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to beta-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a beta-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified beta-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.}, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Russell, WM and Klaenhammer, TR}, year={2001}, month={Mar}, pages={1253–1261} }
 @article{walker_klaenhammer_2001, title={Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways}, volume={67}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.67.1.251-259.2001}, abstractNote={ABSTRACT A novel system that leaks β-galactosidase (β-gal) without a requirement for secretion or export signals was developed in Lactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage φ31 is an 888-bp fragment (P 15A10 ) encoding the transcriptional activator. When a high-copy-number P 15A10 :: lacZ.st fusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant β-gal activity were detected in the supernatant (approximately 85% of the total β-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P 15A10 fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of β-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage φ31 late promoter, into the host genome of MG1363 yielded a similar “leaky” phenotype, indicating that holin and lysin might play a critical role in the release of β-gal into the medium. In addition to β-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.}, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Walker, SA and Klaenhammer, TR}, year={2001}, month={Jan}, pages={251–259} }
 @misc{sanders_klaenhammer_2001, title={The scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic}, volume={84}, ISSN={["0022-0302"]}, DOI={10.3168/jds.S0022-0302(01)74481-5}, abstractNote={Lactobacillus acidophilus NCFM is a probiotic strain available in conventional foods (milk, yogurt, and toddler formula) and dietary supplements. Its commercial availability in the United States since the mid-1970s is predicated on its safety, its amenability to commercial manipulation, and its biochemical and physiological attributes presumed to be important to human probiotic functionality. The strain has been characterized in vitro, in animal studies, and in humans. NCFM is the progenitor of the strain being used for complete chromosome sequencing and therefore will be a cornerstone strain for understanding the relationship between genetics and probiotic functionality. Both phenotypic and genotypic techniques have verified its taxonomic status as a type A1 L. acidophilus strain. It adheres to Caco-2 and mucus-secreting HT-29 cell culture systems, produces antimicrobial compounds, and is amenable to genetic manipulation and directed DNA introduction. NCFM survives gastrointestinal tract transit in both healthy and diseased populations. NCFM inhibits aberrant crypt formation in mutagenized rats, indicative of activity that could decrease the risk of colon cancer. A blend of probiotic strains containing NCFM decreased the incidence of pediatric diarrhea. NCFM led to a significant decrease in levels of toxic amines in the blood of dialysis patients with small bowel bacterial overgrowth. At adequate daily feeding levels, NCFM may facilitate lactose digestion in lactose-intolerant subjects. Further validation of the probiotic properties of NCFM in humans and clarification of its mechanisms of probiotic action are needed to better understand the role this strain might play in promoting human health.}, number={2}, journal={JOURNAL OF DAIRY SCIENCE}, author={Sanders, ME and Klaenhammer, TR}, year={2001}, month={Feb}, pages={319–331} }
 @article{walker_klaenhammer_2000, title={An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage}, volume={66}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.66.1.310-319.2000}, abstractNote={ABSTRACT The coding regions of six putative open reading frames (ORFs) identified near the phage φ31 late promoter and the right cohesive end ( cos ) of lactococcal bacteriophage φ31 were used to develop antisense constructs to inhibit the proliferation of phage φ31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T T7 ) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage φ31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage φ31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage φ31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T T7 resulted in a threefold decrease in the EOP of phage φ31 (EOP = 0.11 ± 0.03 [mean ± standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage φ31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis , even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.}, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Walker, SA and Klaenhammer, TR}, year={2000}, month={Jan}, pages={310–319} }
 @article{durmaz_klaenhammer_2000, title={Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages}, volume={66}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.66.3.895-903.2000}, abstractNote={ABSTRACT Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi + ) with phage φ31. Hin dIII restriction maps of the variants (φ31.1, φ31.2, φ31.7, and φ31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage φ31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, φ31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the φ31.1 DNA, which replaced the φ31 origin. Three separate Hin dIII fragments, accounting for most of the recombinant region of φ31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.}, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Durmaz, E and Klaenhammer, TR}, year={2000}, month={Mar}, pages={895–903} }
 @article{kullen_klaenhammer_2000, title={Genetic modification of intestinal lactobacilli and bifidobacteria}, volume={2}, ISBN={1467-3037}, DOI={10.21775/cimb.002.041}, abstractNote={Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of man and animals. There is a substantial and growing body of evidence that these microbes provide benefits to the host in which they reside. Understanding the roles of these two groups of bacteria in the intestine continues to be a significant challenge. To this end, genetic characterisation and manipulation of intestinal lactobacilli and bifidobacteria is essential to define their contributions to the intestinal microflora, and to potentially exploit any beneficial or unique properties. This review will describe the tools and strategies currently available for the genetic manipulation of lactobacilli and bifidobacteria. Additionally, the ramifications and opportunities that may arise as a result of the genetic manipulation of probiotic lactobacilli and bifidobacteria will be addressed.}, number={2}, journal={Current Issues in Molecular Biology}, author={Kullen, M. J. and Klaenhammer, T. R.}, year={2000}, pages={41} }
 @article{klaenhammer_2000, title={Probiotic bacteria: Today and tomorrow}, volume={130}, ISSN={["0022-3166"]}, DOI={10.1093/jn/130.2.415s}, abstractNote={This paper provides an overview of the key issues raised during this symposium. Probiotic cultures have been associated historically with cultured milks and dairy products, from which there is substantial evidence for positive effects on human health and general well-being.}, number={2}, journal={JOURNAL OF NUTRITION}, author={Klaenhammer, TR}, year={2000}, month={Feb}, pages={415S–416S} }
 @article{kullen_sanozky-dawes_crowell_klaenhammer_2000, title={Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex}, volume={89}, ISSN={["1364-5072"]}, DOI={10.1046/j.1365-2672.2000.01146.x}, abstractNote={The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.}, number={3}, journal={JOURNAL OF APPLIED MICROBIOLOGY}, author={Kullen, MJ and Sanozky-Dawes, RB and Crowell, DC and Klaenhammer, TR}, year={2000}, month={Sep}, pages={511–516} }
 @article{mccormick_klaenhammer_stiles_1999, title={Colicin V can be produced by lactic acid bacteria}, volume={29}, ISSN={["0266-8254"]}, DOI={10.1046/j.1365-2672.1999.00571.x}, abstractNote={Colicin V is a small, proteinaceous bacterial toxin, produced by many strains of Escherichia coli and other members of the Enterobacteriaceae, that fits the definition of class II bacteriocins of Gram-positive bacteria. Export of colicin V is dependent on specific ABC (ATP-binding cassette) secretion proteins which recognize a double-glycine-type leader peptide on the immature colicin V bacteriocin. Replacement of the colicin V leader peptide by a signal peptide from the signal sequence-dependent bacteriocin divergicin A allowed expression of colicin V in lactic acid bacteria. This system may serve as a model for the heterologous expression of other small bacteriocins active against Gram-negative bacteria and other antibacterial peptides from lactic acid bacteria.}, number={1}, journal={LETTERS IN APPLIED MICROBIOLOGY}, author={McCormick, JK and Klaenhammer, TR and Stiles, ME}, year={1999}, month={Jul}, pages={37–41} }
 @article{kullen_klaenhammer_1999, title={Identification of the pH-inducible, proton-translocating F1F0- ATPase (atpBEFHAGDC) operon of Lactobacillus acidophilus by differential display: gene structure, cloning and characterization}, volume={33}, DOI={10.1046/j.1365-2958.1999.01557.x}, abstractNote={The influence of low pH on inducible gene expression in Lactobacillus acidophilus was investigated by the use of differential display. Logarithmic phase cultures were exposed to pH 3.5 for various intervals, and RNA was isolated and reverse transcribed. The resultant cDNAs were subjected to PCR and the products were resolved by electrophoresis. Several cDNA products were induced after exposure to pH 3.5. One of these products, a 0.7 kb fragment, showed sequence similarity to bacterial atpBEF genes of the atp operon, whose genes encode the various subunits of the F1F0-ATPase. With the 0.7 kb differential display product as a probe, hybridizations with total RNA from untreated and acid-treated L. acidophilus verified the acid inducibility of this operon. The increase in atp mRNA induced by low pH was accompanied by an increase in the activity of the enzyme in membrane extracts. The full-length atp operon was sequenced, and its genes were in the order of atpBEFHAGDC, coding for the a, c, b, delta, alpha, gamma, beta and epsilon subunits respectively. The operon contained no i gene, but was preceded by a 122 bp intergenic space, which contained putative extended -10 and -35 promoter regions. Primer extension analysis of RNA from cultures that were shifted from pH 5.6 to pH 3. 5, and held for 0, 30 or 45 min, revealed that the transcriptional start site did not change position as a function of culture pH or time after exposure to pH 3.5. The primary structure and genetic organization indicated that the H+-ATPase of L. acidophilus is a typical F1F0-type ATPase. The similarity to streptococcal ATPases and the acid inducibility of this operon suggest that it may function in the ATP-dependent extrusion of protons and maintenance of cytoplasmic pH. Finally, the use of differential display RT-PCR was an effective approach to identify genes in L. acidophilus induced by an environmental stimulus.}, number={6}, journal={Molecular Microbiology}, author={Kullen, M. J. and Klaenhammer, T. R.}, year={1999}, pages={1152–1161} }
 @article{klaenhammer_kullen_1999, title={Selection and design of probiotics}, volume={50}, ISSN={["0168-1605"]}, DOI={10.1016/S0168-1605(99)00076-8}, abstractNote={Over the past 5 years the probiotic field has exploded with a number of new cultures, each purported to elicit a variety of benefits. Lists of functional characteristics and benefits, in vivo, are now commonplace to any presentation on probiotics. Scientifically established health claims remain among the highest priorities to companies who seek to establish solid health benefits that will promote their particular probiotic. The scientific community faces a greater challenge and must objectively seek cause and effect relationships for many potential and currently investigated probiotic species and strain combinations. Rational selection and design of probiotics remains an important challenge and will require a platform of basic information about the physiology and genetics of candidate strains relevant to their intestinal roles, functional activities, and interactions with other resident microflora. In this context, genetic characterization of probiotic cultures is essential to unequivocally define their contributions to the intestinal microbiota and ultimately identify the genotypes that control any unique and beneficial properties. Strain selection and differentiation, based on the genetic complement and programming of a candidate probiotic, then becomes feasible. Looking ahead, it will be vital to the development of this exploding field to correlate important characteristics in probiotics with known genotypes and regulatory controls that are likely to affect functionality and beneficial outcomes, in vivo.}, number={1-2}, journal={INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY}, author={Klaenhammer, TR and Kullen, MJ}, year={1999}, month={Sep}, pages={45–57} }
 @article{walker_girgis_klaenhammer_1999, title={The groESL chaperone operon of Lactobacillus johnsonii}, volume={65}, number={7}, journal={Applied and Environmental Microbiology}, author={Walker, D. C. and Girgis, H. S. and Klaenhammer, T. R.}, year={1999}, pages={3033–3041} }
 @article{dinsmore_dj o'sullivan_klaenhammer_1998, title={A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.}, volume={212}, ISSN={["0378-1119"]}, DOI={10.1016/S0378-1119(98)00132-2}, abstractNote={The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, φ31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against φ31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.}, number={1}, journal={GENE}, author={Dinsmore, PK and DJ O'Sullivan and Klaenhammer, TR}, year={1998}, month={May}, pages={5–11} }
 @misc{klaenhammer_conkling_o'sullivan_djordjevic_walker_taylor_1998, title={Bacteriophage-triggered cell suicide systems and fermentation methods employing the same}, volume={5,792,625}, number={1998 Aug. 11}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Conkling, M. A. and O'Sullivan, D. and Djordjevic, G. and Walker, S. A. and Taylor, C. G.}, year={1998} }
 @article{walker_dombroski_klaenhammer_1998, title={Common elements regulating gene expression in temperate and lytic bacteriophages of Lactococcus species}, volume={64}, number={3}, journal={Applied and Environmental Microbiology}, author={Walker, S. A. and Dombroski, C. S. and Klaenhammer, T. R.}, year={1998}, pages={1147–1152} }
 @article{dj o'sullivan_klaenhammer_1998, title={Control of expression of LlaI restriction in Lactococcus lactis}, volume={27}, ISSN={["1365-2958"]}, DOI={10.1046/j.1365-2958.1998.00748.x}, abstractNote={The plasmid encoded LlaI R/M system from Lactococcus lactis ssp. lactis consists of a bidomain methylase, with close evolutionary ties to type IIS methylases, and a trisubunit restriction complex. Both the methylase and restriction subunits are encoded on a polycistronic 6.9 kb operon. In this study, the 5' end of the llal 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llalM. Deletion of this promoter region abolished LlaI restriction in L. lactis. Analysis of the intervening sequence revealed a 72-amino-acid open reading frame, designated llalC, with a conserved ribosome binding site and helix-turn-helix domain. Overexpression of llalC in Escherichia coli with a T7 expression vector produced the predicted protein of 8.2 kDa. Mutation and in trans complementation analyses indicated that C-LlaI positively enhanced LlaI restriction activity in vivo. Northern analysis and transcriptional fusions of the llal promoter to a lacZ reporter gene indicated that C x LlaI did not enhance transcription of the llal operon. Databank searches with the deduced protein sequence for llalC revealed significant homologies to the E. coli Rop regulatory and mRNA stabilizer protein. Investigation of the effect of C x LlaI on enhancement of LlaI restriction in L. lactis revealed that growth at elevated temperatures (40 degrees C) completely abolished any enhancement of restriction activity. These data provide molecular evidence for a mechanism on how the expression of a restriction system in a prokaryote can be drastically reduced during elevated growth temperatures, by a small regulatory protein.}, number={5}, journal={MOLECULAR MICROBIOLOGY}, author={DJ O'Sullivan and Klaenhammer, TR}, year={1998}, month={Mar}, pages={1009–1020} }
 @article{klaenhammer_1998, title={Functional activities of Lactobacillus probiotics: Genetic mandate}, volume={8}, ISSN={["0958-6946"]}, DOI={10.1016/S0958-6946(98)00076-4}, abstractNote={The lactobacilli are important inhabitants of the intestinal tract of man and animals. Considerable evidence has implicated them in a number of potentially beneficial roles including immunostimulation, pathogen exclusion, production of bioactive materials, and general intestinal health. Opportunities now abound for incorporation of lactobacilli into functional foods, dietary adjuncts, and health-related products. Over the past decade, developments in the molecular taxonomy of lactobacilli have identified six distinct species within the group previously designated Lactobacillus acidophilus. It remains an important challenge to understand the intestinal roles, activities, and interactions of the individual species, as well as the group at large. In this context, genetic characterization and manipulation of lactobacilli are essential to unequivocally define their contributions to the intestinal microbiota and ultimately control their unique and potentially beneficial properties. This presentation will examine gene systems and regulatory controls of lactobacilli that may be important to their activity, stability, and gastrointestinal functionality. The genetic tools and strategies currently available for characterization and manipulation of lactobacilli will be discussed relative to future targets for probiotic applications. From this framework, new opportunities for the development of safe and effective probiotics will be presented.}, number={5-6}, journal={INTERNATIONAL DAIRY JOURNAL}, author={Klaenhammer, TR}, year={1998}, pages={497–505} }
 @misc{djordjevic_klaenhammer_1998, title={Inducible gene expression systems in Lactococcus lactis}, volume={9}, ISSN={["1073-6085"]}, DOI={10.1007/BF02760814}, number={2}, journal={MOLECULAR BIOTECHNOLOGY}, author={Djordjevic, GM and Klaenhammer, TR}, year={1998}, month={Apr}, pages={127–139} }
 @article{walker_klaenhammer_1998, title={Molecular characterization of a phage-inducible middle promoter and its transcriptional activator from the lactococcal bacteriophage phi 31}, volume={180}, number={4}, journal={Journal of Bacteriology}, author={Walker, S. A. and Klaenhammer, T. R.}, year={1998}, pages={921–931} }
 @misc{allison_klaenhammer_1998, title={Phage resistance mechanisms in lactic acid bacteria}, volume={8}, ISSN={["0958-6946"]}, DOI={10.1016/S0958-6946(98)00043-0}, abstractNote={Dairy fermentations involving Lactococcus lactis and more recently Streptococcus thermophilus, are commonly attacked by bacteriophages. Efforts to protect these dairy starter cultures have resulted in a significant body of knowledge about the bacterio-phages, their interactions with the host, and natural phage defense mechanisms that have evolved within bacteria operating under the most dynamic and devastating phage environment faced by industrial fermentations. This paper will overview this area and discuss the novel genetic approaches that are now being investigated in an effort to provide long term phage protection to dairy starter cultures that are used extensively in the industry.}, number={3}, journal={INTERNATIONAL DAIRY JOURNAL}, author={Allison, GE and Klaenhammer, TR}, year={1998}, month={Mar}, pages={207–226} }
 @article{klaenhammer_connolly_fitzgerald_stanton_ross_1998, title={Special issue - Functional foods: Designer foods for the future}, volume={8}, number={5-6}, journal={International Dairy Journal}, author={Klaenhammer, T. R. and Connolly, J. F. and Fitzgerald, R. J. and Stanton, C. and Ross, R. P.}, year={1998}, pages={vv} }
 @article{djordjevic_osullivan_walker_conkling_klaenhammer_1997, title={A triggered-suicide system designed as a defense against bacteriophages}, volume={179}, ISSN={["0021-9193"]}, DOI={10.1128/jb.179.21.6741-6748.1997}, abstractNote={A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.}, number={21}, journal={JOURNAL OF BACTERIOLOGY}, author={Djordjevic, GM and OSullivan, DJ and Walker, SA and Conkling, MA and Klaenhammer, TR}, year={1997}, month={Nov}, pages={6741–6748} }
 @article{djordjevic_klaenhammer_1997, title={Bacteriophage-triggered defense systems: Phage adaptation and design improvements}, volume={63}, number={11}, journal={Applied and Environmental Microbiology}, author={Djordjevic, G. M. and Klaenhammer, T. R.}, year={1997}, pages={4370–4376} }
 @misc{djordjevic_klaenhammer_1997, title={Genes and gene expression in Lactococcus bacteriophages}, volume={7}, ISSN={["0958-6946"]}, DOI={10.1016/S0958-6946(97)00060-5}, abstractNote={Lactococcus lactis is extensively used in the production of cheese and cultured dairy products in industrial fermentations worldwide. Bacteriophage infection of L. lactis imposes a constant threat to the fermentation industry and represents the major cause of fermentation failure. Numerous phage defense strategies have been developed over the years to protect industrial starter cultures, particularly L. lactis. Numerous genes from lactococcal bacteriophages have been cloned and characterized and mechanisms that regulate their expression elucidated. Complete genome sequences of several L. lactis bacteriophages have also been determined. This accumulation of genetic information on lactococcal bacteriophages has led to a better understanding of the phage life cycle, host interactions, relationships between lactococcal phages, and possible patterns of phage evolution. These advances in molecular biology of lactococcal bacteriophages will be discussed with a view towards the development of novel and more effective phage defenses.}, number={8-9}, journal={INTERNATIONAL DAIRY JOURNAL}, author={Djordjevic, GM and Klaenhammer, TR}, year={1997}, pages={489–508} }
 @misc{klaenhammer_moineau_1997, title={Method of eliminating genetic routes for bacteriophage evolution and products produced thereby}, volume={5,618,723}, number={1997 Apr. 8}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Moineau, S.}, year={1997} }
 @article{dinsmore_klaenhammer_1997, title={Molecular characterization of a genomic region in a Lactococcus bacteriophage that is involved in its sensitivity to the phage defense mechanism AbiA}, volume={179}, ISSN={["0021-9193"]}, DOI={10.1128/jb.179.9.2949-2957.1997}, abstractNote={A spontaneous mutant of the lactococcal phage phi31 that is insensitive to the phage defense mechanism AbiA was characterized in an effort to identify the phage factor(s) involved in sensitivity of phi31 to AbiA. A point mutation was localized in the genome of the AbiA-insensitive phage (phi31A) by heteroduplex analysis of a 9-kb region. The mutation (G to T) was within a 738-bp open reading frame (ORF245) and resulted in an arginine-to-leucine change in the predicted amino acid sequence of the protein. The mutant phi31A-ORF245 reduced the sensitivity of phi31 to AbiA when present in trans, indicating that the mutation in ORF245 is responsible for the AbiA insensitivity of phi31A. Transcription of ORF245 occurs early in the phage infection cycles of phi31 and phi31A and is unaffected by AbiA. Expansion of the phi31 sequence revealed ORF169 (immediately upstream of ORF245) and ORF71 (which ends 84 bp upstream of ORF169). Two inverted repeats lie within the 84-bp region between ORF71 and ORF169. Sequence analysis of an independently isolated AbiA-insensitive phage, phi31B, identified a mutation (G to A) in one of the inverted repeats. A 118-bp fragment from phi31, encompassing the 84-bp region between ORF71 and ORF169, eliminates AbiA activity against phi31 when present in trans, establishing a relationship between AbiA and this fragment. The study of this region of phage phi31 has identified an open reading frame (ORF245) and a 118-bp DNA fragment that interact with AbiA and are likely to be involved in the sensitivity of this phage to AbiA.}, number={9}, journal={JOURNAL OF BACTERIOLOGY}, author={Dinsmore, PK and Klaenhammer, TR}, year={1997}, month={May}, pages={2949–2957} }
 @misc{klaenhammer_sing_hill_1997, title={Phage defense rotation strategy}, volume={5,593,885}, number={1997 Jan. 14}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Sing, W. D. and Hill, C. J.}, year={1997} }
 @misc{hill_klaenhammer_1996, title={Bacteriophage resistant recombinant bacteria}, volume={5,538,864}, number={1996 Jul. 23}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Hill, C. J. and Klaenhammer, T. R.}, year={1996} }
 @misc{klaenhammer_moineau_1996, title={Method of eliminating genetic routes for bacteriophage evolution and products produced thereby}, volume={5,580,725}, number={1996 Dec. 3}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Moineau, S.}, year={1996} }
 @article{klaenhammer_1995, title={Genetics of intestinal lactobacilli}, volume={5}, ISSN={["1879-0143"]}, DOI={10.1016/0958-6946(95)00044-5}, abstractNote={The lactobacilli are important inhabitants of the intestinal tract of man and animals. Considerable evidence has implicated them in a number of potentially beneficial roles including immuno-stimulation, pathogen exclusion, and production of bioactive peptides and enzymes. Opportunities abound for incorporation of lactobacilli into functional foods, dietary adjuncts, and health-related products. Over the past decade, developments in the molecular taxonomy of lactobacilli have identified six distinct species within the group previously designated Lactobacillus acidophilus. It remains an important challenge to understand the intestinal roles and activities of the individual species and the group at large. In this context, genetic characterization and manipulation of lactobacilli is essential to define their contributions to the intestinal microbiota, and potentially exploit any beneficial or unique properties. This presentation will address the genetics of Lactobacillus species and generally describe the tools and strategies currently available for their manipulation. From this framework, future genetic targets and opportunities for the development of specialized Lactobacillus strains will be discussed.}, number={8}, journal={INTERNATIONAL DAIRY JOURNAL}, author={Klaenhammer, TR}, year={1995}, pages={1019–1058} }
 @article{klaenhammer_1993, title={Genetics of bacteriocins produced by lactic-acid bacteria}, volume={12}, DOI={10.1111/j.1574-6976.1993.tb00012.x}, abstractNote={Lactic acid bacteria produce a variety of bacteriocins that have recently come under detailed investigation. The biochemical and genetic characteristics of these antimicrobial proteins are reviewed and common elements are discussed between the different classes of bacteriocins produced by these Gram-positive bacteria.}, number={39085}, journal={FEMS Microbiology Reviews}, author={Klaenhammer, T. R.}, year={1993}, pages={39–86} }
 @misc{klaenhammer_sanozky_steenson_1992, title={PTR2030, a conjugal plasmid and derivatives thereof that confer phage resistance to group N streptococci}, volume={5,139,950}, number={1992 Aug. 18}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Sanozky, R. B. and Steenson, L. R.}, year={1992} }
 @article{klaenhammer_sing_1991, title={A novel rotation strategy using different phage defenses in a single-strain starter culture system}, volume={74}, journal={Journal of Dairy Science}, author={Klaenhammer, T. R. and Sing, W. D.}, year={1991}, pages={120} }
 @article{klaenhammer_1991, title={DEVELOPMENT OF BACTERIOPHAGE-RESISTANT STRAINS OF LACTIC-ACID BACTERIA}, volume={19}, ISSN={["0300-5127"]}, DOI={10.1042/bst0190675}, abstractNote={Conference Article| August 01 1991 Development of bacteriophage-resistant strains of lactic acid bacteria Todd R. Klaenhammer Todd R. Klaenhammer 1Department of Food Science, Southeast Center for Dairy Foods Research, North Carolina State University, Raleigh, NC 27695–7624, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (3): 675–681. https://doi.org/10.1042/bst0190675 Article history Received: April 26 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Todd R. Klaenhammer; Development of bacteriophage-resistant strains of lactic acid bacteria. Biochem Soc Trans 1 August 1991; 19 (3): 675–681. doi: https://doi.org/10.1042/bst0190675 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: IS, insertion sequence, LlaI and FokI, type II-A-methylase genes This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.}, number={3}, journal={BIOCHEMICAL SOCIETY TRANSACTIONS}, author={KLAENHAMMER, TR}, year={1991}, month={Aug}, pages={675–681} }
 @misc{klaenhammer_sanozky_steenson_1990, title={pTR2030, a conjugal plasmid and derivatives thereof that confer phage reisistance to group N streptococci}, volume={4,931,396}, number={1990 Jun. 5}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Sanozky, R. B. and Steenson, L. R.}, year={1990} }
 @article{klaenhammer_1989, title={Genetic characterization of multiple mechanisms of phage defense from a prototype phage-insensitive strain, Lactococcus lactis ME2}, volume={72}, DOI={10.3168/jds.S0022-0302(89)79505-9}, abstractNote={Abstract Lactococci used as starter cultures in dairy fermentations are highly susceptible to attack by bacteriophage. Genetic studies with Lactococcus lactis ME2, a prototype phage-insensitive strain, have identified plasmid-encoded defenses, which interfere with phage adsorption, restrict and modify phages, or abort lytic phage infection. Restriction and modification and abortion of phage infection were localized on two distinct self-transmissible plasmids, pTN20 and pTR2030, respectively, originating from L. lactis ME2. A comparison of the physical and genetic characteristics of these two conjugative plasmids is presented. Conjugation and cloning strategies employed to assemble these complementary mechanisms of phage resistance will be discussed. The collective expression of different defense systems provided a greater phage resistance to dairy lactococci. Starter cultures that are recalcitrant to phage attack can be constructed from existing strains through application of genetic technologies, which assemble complementary mechanisms of phage defense.}, number={12}, journal={Journal of Dairy Science}, author={Klaenhammer, T. R.}, year={1989}, pages={3429} }
 @misc{klaenhammer_sanozky-dawes_1989, title={pTN1060, a conjugal plasmid and derivatives thereof that confer phage resistance to group N streptococci}, volume={4,883,756}, number={1989 Nov. 28}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Sanozky-Dawes, R. B.}, year={1989} }
 @article{klaenhammer_1988, title={BACTERIOCINS OF LACTIC-ACID BACTERIA}, volume={70}, ISSN={["1638-6183"]}, DOI={10.1016/0300-9084(88)90206-4}, abstractNote={Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.}, number={3}, journal={BIOCHIMIE}, author={KLAENHAMMER, TR}, year={1988}, month={Mar}, pages={337–349} }
 @misc{daeschel_mcfeeters_fleming_klaenhammer_sanozky_1987, title={Lactic acid bacteria which do not decarboxylate malic acid and fermentation therewith}, volume={4,666,849}, number={1987 May 19}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Daeschel, M. A. and McFeeters, R. F. and Fleming, H. P and Klaenhammer, T. R. and Sanozky, R. B.}, year={1987} }
 @article{klaenhammer_1987, title={Plasmid-directed mechanisms for bacteriophage defense in lactic streptococci}, volume={46}, DOI={10.1111/j.1574-6968.1987.tb02468.x}, abstractNote={Genetic studies with lactic streptococci have identified a variety of plasmids coding for systems that interfere with phage adsorption, direct restriction and modification activities, and disrupt various stages in the phage lytic cycle. This review describes mechanisms of phage defense that are plasmid-directed in lactic streptococci, examines the physical and genetic properties of the plasmids involved, and discusses genetic strategies for construction of phage-insensitive starter cultures for dairy fermentations.}, number={3}, journal={FEMS Microbiology Reviews}, author={Klaenhammer, T. R.}, year={1987}, pages={313} }
 @article{joerger_klaenhammer_1986, title={CHARACTERIZATION AND PURIFICATION OF HELVETICIN-J AND EVIDENCE FOR A CHROMOSOMALLY DETERMINED BACTERIOCIN PRODUCED BY LACTOBACILLUS-HELVETICUS-481}, volume={167}, ISSN={["0021-9193"]}, DOI={10.1128/jb.167.2.439-446.1986}, abstractNote={Lactobacillus helveticus 481 produced an antimicrobial agent active against five closely related species. The sensitive indicators included L. helveticus 1846 and 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. The antimicrobial compound was active at neutral pH under aerobic or anaerobic conditions, was sensitive to proteolytic enzymes and heat (30 min at 100 degrees C), and demonstrated a bactericidal mode of action against sensitive indicators. These data confirmed that antimicrobial activity of L. helveticus 481 was mediated by a bacteriocin, designated helveticin J. Production of helveticin J was maximized in an anaerobic fermentor held at a constant pH of 5.5. Ultrafiltration experiments on culture supernatants containing the bacteriocin revealed that helveticin J was present as an aggregate with a molecular weight in excess of 300,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of helveticin J purified through Sephadex chromatography resolved a 37,000-dalton protein band with bacteriocin activity. L. helveticus 481 was shown to harbor a single 8-megadalton plasmid (pMJ1008). Isolates cured of pMJ1008 were phenotypically identical to plasmid-bearing cells in fermentation patterns, helveticin J activity, and immunity spectra. The data provided evidence for a chromosomal location of helveticin J and host immunity determinants.}, number={2}, journal={JOURNAL OF BACTERIOLOGY}, author={JOERGER, MC and KLAENHAMMER, TR}, year={1986}, month={Aug}, pages={439–446} }
 @article{barefoot_klaenhammer_1983, title={Detection and activity of lactacin-b, a bacteriocin produced by lactobacillus-acidophilus}, volume={45}, number={6}, journal={Applied and Environmental Microbiology}, author={Barefoot, S. F. and Klaenhammer, T. R.}, year={1983}, pages={1808–1815} }