@article{rudershausen_m. lee_lombardo_merrell_buckel_2019, title={Survival and Habitat of Yellow‐Phase American Eels in North Carolina Tidal Creeks}, volume={148}, ISSN={0002-8487 1548-8659}, url={http://dx.doi.org/10.1002/tafs.10190}, DOI={10.1002/tafs.10190}, abstractNote={AbstractWe estimated rates of survival as well as effects of habitat on catch rates of juvenile yellow‐phase American Eels Anguilla rostrata in southeastern U.S. tidal creeks. We trapped and marked eels with PIT tags at 24 fixed sites in eight North Carolina tidal creeks and then recaptured and resighted the tagged individuals to estimate apparent survival. Separate Cormack–Jolly–Seber (CJS) models were fitted to mark–recapture data (eight creeks) versus mark–resight data (four creeks) to estimate apparent survival. Median annual apparent survival (Φ) was higher when the CJS model was fitted to mark–resight data (Φ = 0.15) than to mark–recapture data (Φ = 0.013). Negative binomially distributed models were fitted to catch rates of both tagged and untagged eels to test for habitat, development, and seasonal effects. The presence/absence of culverts and season were meaningful covariates of catch rates; greater catches were found at sites possessing culverts and during the spring. Other habitat and development factors at the site, creek, and watershed levels were not important covariates of catch rates. Partitioning the sources of loss of yellow‐phase American Eels from these systems into mortality versus emigration would be useful future research in the southeastern U.S. coastal region. Further study into how culverts affect yellow‐phase American Eel habitation and movement in southeastern U.S. estuaries is also warranted.}, number={5}, journal={Transactions of the American Fisheries Society}, publisher={Wiley}, author={Rudershausen, Paul J. and M. Lee, Laura and Lombardo, Steven M. and Merrell, Jeffery H. and Buckel, Jeffrey A.}, year={2019}, month={Aug}, pages={978–990} }
 @article{lee_crosby_rubinstein_laemthong_bing_straub_adams_kelly_2020, title={The biology and biotechnology of the genus Caldicellulosiruptor: recent developments in 'Caldi World'}, volume={24}, ISSN={["1433-4909"]}, url={https://doi.org/10.1007/s00792-019-01116-5}, DOI={10.1007/s00792-019-01116-5}, abstractNote={Terrestrial hot springs near neutral pH harbor extremely thermophilic bacteria from the genus Caldicellulosiruptor, which utilize the carbohydrates of lignocellulose for growth. These bacteria are technologically important because they produce novel, multi-domain glycoside hydrolases that are prolific at deconstructing microcrystalline cellulose and hemicelluloses found in plant biomass. Among other interesting features, Caldicellulosiruptor species have successfully adapted to bind specifically to lignocellulosic substrates via surface layer homology (SLH) domains associated with glycoside hydrolases and unique binding proteins (tāpirins) present only in these bacteria. They also utilize a parallel pathway for conversion of glyceraldehyde-3-phosphate into 3-phosphoglycerate via a ferredoxin-dependent oxidoreductase that is conserved across the genus. Advances in the genetic tools for Caldicellulosiruptor bescii, including the development of a high-temperature kanamycin-resistance marker and xylose-inducible promoter, have opened the door for metabolic engineering applications and some progress along these lines has been reported. While several species of Caldicellulosiruptor can readily deconstruct lignocellulose, improvements in the amount of carbohydrate released and in the production of bio-based chemicals are required to successfully realize the biotechnological potential of these organisms.}, number={1}, journal={EXTREMOPHILES}, author={Lee, Laura L. and Crosby, James R. and Rubinstein, Gabriel M. and Laemthong, Tunyaboon and Bing, Ryan G. and Straub, Christopher T. and Adams, Michael W. W. and Kelly, Robert M.}, year={2020}, month={Jan}, pages={1–15} }
 @article{lee_hart_lunin_alahuhta_bomble_himmel_blumer-schuette_adams_kelly_2019, title={Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tapirins) from Extremely Thermophilic Caldicellulosiruptor Species}, volume={85}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.01983-18}, abstractNote={
            The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed.
            Caldicellulosiruptor
            species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called tāpirins, are involved in the way that
            Caldicellulosiruptor
            species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.
          }, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Lee, Laura L. and Hart, William S. and Lunin, Vladimir V. and Alahuhta, Markus and Bomble, Yannick J. and Himmel, Michael E. and Blumer-Schuette, Sara E. and Adams, Michael W. W. and Kelly, Robert M.}, year={2019}, month={Feb} }
 @article{conway_crosby_hren_southerland_lee_lunin_alahuhta_himmel_bomble_adams_et al._2018, title={Novel multidomain, multifunctional glycoside hydrolases from highly lignocellulolytic Caldicellulosiruptor species}, volume={64}, ISSN={["1547-5905"]}, DOI={10.1002/aic.16354}, abstractNote={Biological hydrolysis of microcrystalline cellulose is an uncommon feature in the microbial world, especially among bacteria and archaea growing optimally above 70°C (the so‐called extreme thermophiles). In fact, among this group only certain species in the genus Caldicellulosiruptor are capable of rapid and extensive cellulose degradation. Four novel multidomain glycoside hydrolases (GHs) from Caldicellulosiruptor morganii and Caldicellulosiruptor danielii were produced recombinantly in Caldicellulosiruptor bescii and characterized. These GHs are structurally organized with two or three catalytic domains flanking carbohydrate binding modules from Family 3. Collectively, these enzymes represent GH families 5, 9, 10, 12, 44, 48, and 74, and hydrolyze crystalline cellulose, glucan, xylan, and mannan, the primary carbohydrates in plant biomass. Degradation of microcrystalline cellulose by cocktails of GHs from three Caldicellulosiruptor species demonstrated that synergistic interactions enable mixtures of multiple enzymes to outperform single enzymes, suggesting a community mode of action for lignocellulose utilization in thermal environments. © 2018 American Institute of Chemical Engineers AIChE J, 64: 4218–4228, 2018}, number={12}, journal={AICHE JOURNAL}, author={Conway, Jonathan M. and Crosby, James R. and Hren, Andrew P. and Southerland, Robert T. and Lee, Laura L. and Lunin, Vladimir V. and Alahuhta, Petri and Himmel, Michael E. and Bomble, Yannick J. and Adams, Michael W. W. and et al.}, year={2018}, month={Dec}, pages={4218–4228} }
 @article{zurawski_khatibi_akinosho_straub_compton_conway_lee_ragauskas_davison_adams_et al._2017, title={Bioavailability of Carbohydrate Content in Natural and Transgenic Switchgrasses for the Extreme Thermophile Caldicellulosiruptor bescii}, volume={83}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00969-17}, abstractNote={ABSTRACT
          
            Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals.
            Caldicellulosiruptor bescii
            completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to
            C. bescii
            at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(−) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by
            C. bescii
            to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While
            C. bescii
            could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance.
          
          
            IMPORTANCE
            Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium
            Caldicellulosiruptor bescii
            was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.
          }, number={17}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Zurawski, Jeffrey V. and Khatibi, Piyum A. and Akinosho, Hannah O. and Straub, Christopher T. and Compton, Scott H. and Conway, Jonathan M. and Lee, Laura L. and Ragauskas, Arthur J. and Davison, Brian H. and Adams, Michael W. W. and et al.}, year={2017}, month={Sep} }
 @article{williams-rhaesa_poole_dinsmore_lipscomb_rubinstein_scott_conway_lee_khatibi_kelly_et al._2017, title={Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii}, volume={83}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00444-17}, abstractNote={ABSTRACT
          
            Caldicellulosiruptor bescii
            is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of
            C. bescii
            strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes
            pyrFA
            , and the second (MACB1018) is based on the targeted deletion of
            pyrE
            , making use of a kanamycin resistance marker. Importantly, an active insertion element, IS
            Cbe4
            , was discovered in
            C. bescii
            when it disrupted the gene for lactate dehydrogenase (
            ldh
            ) in strain JWCB018, constructed in the JWCB005 background. Additional instances of IS
            Cbe4
            movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of
            C. bescii
            from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and IS
            Cbe4
            elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in
            C. bescii
            . Furthermore, a facile approach for assessing genomic stability in
            C. bescii
            has been established.
          
          
            IMPORTANCE
            Caldicellulosiruptor bescii
            is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in
            C. bescii
            has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes (
            pyrFA
            ) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and IS
            Cbe4
            insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these IS
            Cbe4
            elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene
            pyrE
            have a stable genome structure, making them preferable for future metabolic engineering studies.
          }, number={14}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Williams-Rhaesa, Amanda M. and Poole, Farris L., II and Dinsmore, Jessica T. and Lipscomb, Gina L. and Rubinstein, Gabriel M. and Scott, Israel M. and Conway, Jonathan M. and Lee, Laura L. and Khatibi, Piyum A. and Kelly, Robert M. and et al.}, year={2017}, month={Jul} }
 @misc{counts_zeldes_lee_straub_adams_kelly_2017, title={Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms}, volume={9}, ISSN={["1939-005X"]}, DOI={10.1002/wsbm.1377}, abstractNote={The current upper thermal limit for life as we know it is approximately 120°C. Microorganisms that grow optimally at temperatures of 75°C and above are usually referred to as ‘extreme thermophiles’ and include both bacteria and archaea. For over a century, there has been great scientific curiosity in the basic tenets that support life in thermal biotopes on earth and potentially on other solar bodies. Extreme thermophiles can be aerobes, anaerobes, autotrophs, heterotrophs, or chemolithotrophs, and are found in diverse environments including shallow marine fissures, deep sea hydrothermal vents, terrestrial hot springs—basically, anywhere there is hot water. Initial efforts to study extreme thermophiles faced challenges with their isolation from difficult to access locales, problems with their cultivation in laboratories, and lack of molecular tools. Fortunately, because of their relatively small genomes, many extreme thermophiles were among the first organisms to be sequenced, thereby opening up the application of systems biology‐based methods to probe their unique physiological, metabolic and biotechnological features. The bacterial genera Caldicellulosiruptor, Thermotoga and Thermus, and the archaea belonging to the orders Thermococcales and Sulfolobales, are among the most studied extreme thermophiles to date. The recent emergence of genetic tools for many of these organisms provides the opportunity to move beyond basic discovery and manipulation to biotechnologically relevant applications of metabolic engineering. WIREs Syst Biol Med 2017, 9:e1377. doi: 10.1002/wsbm.1377This article is categorized under:

Biological Mechanisms > Metabolism
}, number={3}, journal={WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE}, author={Counts, James A. and Zeldes, Benjamin M. and Lee, Laura L. and Straub, Christopher T. and Adams, Michael W. W. and Kelly, Robert M.}, year={2017}, month={May} }
 @article{conway_pierce_le_harper_wright_tucker_zurawski_lee_blumer-schuette_kelly_2016, title={Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species}, volume={291}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.707810}, abstractNote={The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis encodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptor species genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique to C. kronotskyensis and contains SLH domains. Calkro_0111 was produced recombinantly in Escherichia coli as two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo- and exoglucanase activity on the β-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs in C. kronotskyensis, when produced in E. coli, was active on a variety of xylans and β-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptor and among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii. As such, the gene encoding Calkro_0402 was inserted into the C. bescii genome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-type C. bescii. The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptor species.}, number={13}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Conway, Jonathan M. and Pierce, William S. and Le, Jaycee H. and Harper, George W. and Wright, John H. and Tucker, Allyson L. and Zurawski, Jeffrey V. and Lee, Laura L. and Blumer-Schuette, Sara E. and Kelly, Robert M.}, year={2016}, month={Mar}, pages={6732–6747} }
 @article{zurawski_conway_lee_simpson_izquierdo_blumer-schuette_nookaew_adams_kelly_2015, title={Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization}, volume={81}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01622-15}, abstractNote={ABSTRACT
          
            Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus
            Caldicellulosiruptor
            with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO
            2
            , and H
            2
            .
            Caldicellulosiruptor bescii
            ,
            C. kronotskyensis
            , and
            C. saccharolyticus
            solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (
            Panicum virgatum
            ) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with
            C. saccharolyticus
            , not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of
            C. saccharolyticus
            lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in
            C. kronotskyensis
            and
            C. bescii
            during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across
            Caldicellulosiruptor
            species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation.
          }, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Zurawski, Jeffrey V. and Conway, Jonathan M. and Lee, Laura L. and Simpson, Hunter J. and Izquierdo, Javier A. and Blumer-Schuette, Sara and Nookaew, Intawat and Adams, Michael W. W. and Kelly, Robert M.}, year={2015}, month={Oct}, pages={7159–7170} }
 @article{blumer-schuette_alahuhta_conway_lee_zurawski_giannone_hettich_lunin_himmel_kelly_2015, title={Discrete and Structurally Unique Proteins (Tapirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose}, volume={290}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.641480}, abstractNote={Background: Lignocellulose-degrading microorganisms utilize binding modules associated with glycosidic enzymes to attach to polysaccharides. Results: Structurally unique, discrete proteins (tāpirins) bind to cellulose with a high affinity. Conclusion: Tāpirins represent a new class of proteins used by Caldicellulosiruptor species to attach to cellulose. Significance: The tāpirins establish a new paradigm for how cellulolytic bacteria adhere to cellulose. A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tāpirins,” origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.}, number={17}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Blumer-Schuette, Sara E. and Alahuhta, Markus and Conway, Jonathan M. and Lee, Laura L. and Zurawski, Jeffrey V. and Giannone, Richard J. and Hettich, Robert L. and Lunin, Vladimir V. and Himmel, Michael E. and Kelly, Robert M.}, year={2015}, month={Apr}, pages={10645–10656} }
 @article{conway_zurawski_lee_blumer-schuette_kelly, title={Lignocellulosic biomass deconstruction by the extremely thermophilic genus caldicellulosiruptor}, journal={Thermophilic Microorganisms}, author={Conway, J. M. and Zurawski, J. V. and Lee, L. L. and Blumer-Schuette, S. E. and Kelly, R. M.}, pages={91–119} }