@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{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{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{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} }
 @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={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, PhiAT3, 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{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={ABSTRACT 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 φ31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of φ31 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 φ31 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{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{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{durmaz_klaenhammer_1995, title={A starter culture rotation strategy incorporating paired restriction/modification and abortive infection bacteriophage defenses in a single Lactococcus lactis strain}, volume={61}, number={4}, journal={Applied and Environmental Microbiology}, author={Durmaz, E. and Klaenhammer, T. R.}, year={1995}, pages={1266} }
 @article{durmaz_higgins_klaenhammer_1992, title={MOLECULAR CHARACTERIZATION OF A 2ND ABORTIVE PHAGE RESISTANCE GENE PRESENT IN LACTOCOCCUS-LACTIS SUBSP LACTIS ME2}, volume={174}, ISSN={["0021-9193"]}, DOI={10.1128/jb.174.22.7463-7469.1992}, abstractNote={The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.}, number={22}, journal={JOURNAL OF BACTERIOLOGY}, author={DURMAZ, E and HIGGINS, DL and KLAENHAMMER, TR}, year={1992}, month={Nov}, pages={7463–7469} }
 @article{durmaz_klaenhammer_1991, title={A fourth mechanism for bacteriophage resistance in Lactococcus lactis subsp. lactis ME2}, volume={74}, journal={Journal of Dairy Science}, author={Durmaz, E. and Klaenhammer, T. R.}, year={1991}, pages={120} }