@article{chou_shockley_conners_lewis_comfort_adams_kelly_2007, title={Impact of substrate glycoside linkage. and elemental sulfur on bioenergetics, of and hydrogen production by the hyperthermophilic Archaeon Pyrococcus furiosus}, volume={73}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00597-07}, abstractNote={ABSTRACT Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h−1 at 90°C. In the absence of S0, cellobiose-grown cells generated twice as much protein and had 50%-higher specific H2 generation rates than maltose-grown cultures. Addition of S0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H2 to H2S. In contrast, the presence of S0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H2 to H2S production, with 2.5 times more H2 than H2S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in α- or β-glucan uptake and processing were up-regulated in an S0-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S0 in cellobiose-grown cells also responded to S0 in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S0 was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S0 metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.}, number={21}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Chou, Chung-Jung and Shockley, Keith R. and Conners, Shannon B. and Lewis, Derrick L. and Comfort, Donald A. and Adams, Michael W. W. and Kelly, Robert M.}, year={2007}, month={Nov}, pages={6842–6853} }
 @article{madding_michel_shockley_conners_epting_johnson_kelly_2007, title={Role of the beta 1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus}, volume={189}, ISSN={["0021-9193"]}, DOI={10.1128/JB.01382-06}, abstractNote={ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.}, number={2}, journal={JOURNAL OF BACTERIOLOGY}, author={Madding, Lara S. and Michel, Joshua K. and Shockley, Keith R. and Conners, Shannon B. and Epting, Kevin L. and Johnson, Matthew R. and Kelly, Robert M.}, year={2007}, month={Jan}, pages={583–590} }
 @article{johnson_conners_montero_chou_shockley_kelly_2006, title={The Thermotoga maritima phenotype is impacted by syntrophic interaction with Methanococcus jannaschii in hyperthermophilic coculture}, volume={72}, ISSN={["0099-2240"]}, DOI={10.1128/aem.72.1.811-818.2006}, abstractNote={ABSTRACT Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium Thermotoga maritima when it was cocultured with the hyperthermophilic archaeon Methanococcus jannaschii. For the mid-log-to-early-stationary-phase transition of a T. maritima monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in T. maritima at this level (15.5% of the genome) for the same growth phase transition. Interspecies H2 transfer resulted in three- to fivefold-higher T. maritima cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding β-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of β-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where T. maritima coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.}, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Johnson, MR and Conners, SB and Montero, CI and Chou, CJ and Shockley, KR and Kelly, RM}, year={2006}, month={Jan}, pages={811–818} }
 @article{lee_shockley_schut_conners_montero_johnson_chou_bridger_wigner_brehm_et al._2006, title={Transcriptional and biochemical analysis of starch metabolism in the hyperthermophilic archaeon Pyrococcus furiosus}, volume={188}, ISSN={["1098-5530"]}, DOI={10.1128/JB.188.6.2115-2125.2006}, abstractNote={ABSTRACT Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these α-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-α-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of α-glucan substrates by P. furiosus.}, number={6}, journal={JOURNAL OF BACTERIOLOGY}, author={Lee, HS and Shockley, KR and Schut, GJ and Conners, SB and Montero, CI and Johnson, MR and Chou, CJ and Bridger, SL and Wigner, N and Brehm, SD and et al.}, year={2006}, month={Mar}, pages={2115–2125} }
 @article{conners_montero_comfort_shockley_johnson_chhabra_kelly_2005, title={An expression-driven approach to the prediction of carbohydrate transport and utilization regulons in the hyperthermophilic bacterium Thermotoga maritima}, volume={187}, ISSN={["1098-5530"]}, DOI={10.1128/JB.187.21.7267-7282.2005}, abstractNote={ABSTRACT Comprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixed-model analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T. maritima relies extensively on ABC transporters for carbohydrate uptake, many of which are likely controlled by local regulators responsive to either the transport substrate or a key metabolic degradation product. Roles in uptake of specific carbohydrates were suggested for members of the expanded Opp/Dpp family of ABC transporters. In this family, phylogenetic relationships among transport systems revealed patterns of possible duplication and divergence as a strategy for the evolution of new uptake capabilities. The presence of GC-rich hairpin sequences between substrate-binding proteins and other components of Opp/Dpp family transporters offers a possible explanation for differential regulation of transporter subunit genes. Numerous improvements to T. maritima genome annotations were proposed, including the identification of ABC transport systems originally annotated as oligopeptide transporters as candidate transporters for rhamnose, xylose, β-xylan, andβ -glucans and identification of genes likely to encode proteins missing from current annotations of the pentose phosphate pathway. Beyond the information obtained for T. maritima, the present study illustrates how expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable.}, number={21}, journal={JOURNAL OF BACTERIOLOGY}, author={Conners, SB and Montero, CI and Comfort, DA and Shockley, KR and Johnson, MR and Chhabra, SR and Kelly, RM}, year={2005}, month={Nov}, pages={7267–7282} }
 @article{shockley_scott_pysz_conners_johnson_montero_wolfinger_kelly_2005, title={Genorne-wide transcriptional variation within and between steady states for continuous growth of the hyperthermophile Thermotoga maritima}, volume={71}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.71.9.5572-5576.2005}, abstractNote={ABSTRACT Maltose-limited, continuous growth of the hyperthermophile Thermotoga maritima at different temperatures and dilution rates (80°C/0.25 h−1, 80°C/0.17 h−1, and 85°C/0.25 h−1) showed that transcriptome-wide variation in gene expression within mechanical steady states was minimal compared to that between steady states, supporting the efficacy of chemostat-based approaches for functional genomics studies.}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Shockley, KR and Scott, KL and Pysz, MA and Conners, SB and Johnson, MR and Montero, CI and Wolfinger, RD and Kelly, RM}, year={2005}, month={Sep}, pages={5572–5576} }
 @article{johnson_montero_conners_shockley_bridger_kelly_2005, title={Population density-dependent regulation of exopolysaccharide formation in the hyperthermophilic bacterium Thermotoga maritima}, volume={55}, ISSN={["1365-2958"]}, DOI={10.1111/j.1365-2958.2004.04419.x}, abstractNote={Co‐cultivation of the hyperthermophiles Thermotoga maritima and Methanococcus jannaschii resulted in fivefold higher T. maritima cell densities when compared with monoculture as well as concomitant formation of exopolysaccharide and flocculation of heterotroph‐methanogen cellular aggregates. Transcriptional analysis of T. maritima cells from these aggregates using a whole genome cDNA microarray revealed the induction of a putative exopolysaccharide synthesis pathway, regulated by intracellular levels of cyclic diguanosine 3′,5′‐(cyclic)phosphate (cyclic di‐GMP) and mediated by the action of several GGDEF proteins, including a putative diguanylate cyclase (TM1163) and a putative phosphodiesterase (TM1184). Transcriptional analysis also showed that TM0504, which encodes a polypeptide containing a motif common to known peptide‐signalling molecules in mesophilic bacteria, was strongly upregulated in the co‐culture. Indeed, when a synthetically produced peptide based on TM0504 was dosed into the culture at ecologically relevant levels, the production of exopolysaccharide was induced at significantly lower cell densities than was observed in cultures lacking added peptide. In addition to identifying a pathway for polysaccharide formation in T. maritima, these results point to the existence of peptide‐based quorum sensing in hyperthermophilic bacteria and indicate that cellular communication should be considered as a component of the microbial ecology within hydrothermal habitats.}, number={3}, journal={MOLECULAR MICROBIOLOGY}, author={Johnson, MR and Montero, CI and Conners, SB and Shockley, KR and Bridger, SL and Kelly, RM}, year={2005}, month={Feb}, pages={664–674} }
 @article{johnson_montero_conners_shockley_pysz_kelly_2004, title={Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms}, volume={32}, ISSN={["1470-8752"]}, DOI={10.1042/BST0320188}, abstractNote={Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures. Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats. Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.}, number={2004 Apr}, journal={BIOCHEMICAL SOCIETY TRANSACTIONS}, author={Johnson, MR and Montero, CI and Conners, SB and Shockley, KR and Pysz, MA and Kelly, RM}, year={2004}, month={Apr}, pages={188–192} }
 @misc{pysz_conners_montero_shockley_johnson_ward_kelly_2004, title={Transcriptional analysis of biofilm formation processes in the anaerobic, hyperthermophilic bacterium Thermotoga maritima}, volume={70}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.70.10.6098-6112.2004}, abstractNote={ABSTRACT Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several β-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased β-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.}, number={10}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Pysz, MA and Conners, SB and Montero, CI and Shockley, KR and Johnson, MR and Ward, DE and Kelly, RA}, year={2004}, month={Oct}, pages={6098–6112} }
 @article{pysz_ward_shockley_montero_conners_johnson_kelly_2004, title={Transcriptional analysis of dynamic heat-shock response by the hyperthermophilic bacterium Thermotoga maritima}, volume={8}, ISSN={["1433-4909"]}, DOI={10.1007/s00792-004-0379-2}, abstractNote={The thermal stress response of the hyperthermophilic bacterium Thermotoga maritima was characterized using a 407-open reading frame-targeted cDNA microarray. Transient gene expression was followed for 90 min, following a shift from 80 degrees C to 90 degrees C. While some aspects of mesophilic heat-shock response were conserved in T. maritima, genome content suggested differentiating features that were borne out by transcriptional analysis. Early induction of predicted heat-shock operons hrcA-grpE-dnaJ (TM0851-TM0850-TM0849), groES-groEL (TM0505-TM0506), and dnaK-sHSP (TM0373-TM0374) was consistent with conserved CIRCE elements upstream of hrcA and groES. Induction of the T. maritima rpoE/ sigW and rpoD/ sigA homologs suggests a mechanism for global heat-shock response in the absence of an identifiable ortholog to a major heat-shock sigma factor. In contrast to heat-shock response in Escherichia coli, the majority of genes encoding ATP-dependent proteases were downregulated, including clpP (TM0695), clpQ (TM0521), clpY (TM0522), lonA (TM1633), and lonB (TM1869). Notably, T. maritima showed indications of a late heat-shock response with the induction of a marR homolog (TM0816), several other putative transcriptional regulators (TM1023, TM1069), and two alpha-glucosidases (TM0434 and TM1068). Taken together, the results reported here indicate that, while T. maritima shares core elements of the bacterial heat-shock response with mesophiles, the thermal stress regulatory strategies of this organism differ significantly. However, it remains to be elucidated whether these differences are related to thermophilicity or phylogenetic placement.}, number={3}, journal={EXTREMOPHILES}, author={Pysz, MA and Ward, DE and Shockley, KR and Montero, CI and Conners, SB and Johnson, MR and Kelly, RM}, year={2004}, month={Jun}, pages={209–217} }
 @article{chhabra_shockley_conners_scott_wolfinger_kelly_2003, title={Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacterium Thermotoga maritima}, volume={278}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M211748200}, abstractNote={The hyperthermophilic bacteriumThermotoga maritima MSB8 was grown on a variety of carbohydrates to determine the influence of carbon and energy source on differential gene expression. Despite the fact that T. maritima has been phylogenetically characterized as a primitive microorganism from an evolutionary perspective, results here suggest that it has versatile and discriminating mechanisms for regulating and effecting complex carbohydrate utilization. Growth ofT. maritima on monosaccharides was found to be slower than growth on polysaccharides, although growth to cell densities of 108 to 109 cells/ml was observed on all carbohydrates tested. Differential expression of genes encoding carbohydrate-active proteins encoded in the T. maritimagenome was followed using a targeted cDNA microarray in conjunction with mixed model statistical analysis. Coordinated regulation of genes responding to specific carbohydrates was noted. Although glucose generally repressed expression of all glycoside hydrolase genes, other sugars induced or repressed these genes to varying extents. Expression profiles of most endo-acting glycoside hydrolase genes correlated well with their reported biochemical properties, although exo-acting glycoside hydrolase genes displayed less specific expression patterns. Genes encoding selected putative ABC sugar transporters were found to respond to specific carbohydrates, and in some cases putative oligopeptide transporter genes were also found to respond to specific sugar substrates. Several genes encoding putative transcriptional regulators were expressed during growth on specific sugars, thus suggesting functional assignments. The transcriptional response ofT. maritima to specific carbohydrate growth substrates indicated that sugar backbone- and linkage-specific regulatory networks are operational in this organism during the uptake and utilization of carbohydrate substrates. Furthermore, the wide ranging collection of such networks in T. maritima suggests that this organism is capable of adapting to a variety of growth environments containing carbohydrate growth substrates.}, number={9}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Chhabra, SR and Shockley, KR and Conners, SB and Scott, KL and Wolfinger, RD and Kelly, RM}, year={2003}, month={Feb}, pages={7540–7552} }
 @article{gao_bauer_shockley_pysz_kelly_2003, title={Growth of hyperthermophilic Archaeon Pyrococcus futiosus on chitin involves two family 18 chitinases}, volume={69}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.69.6.3119-3128.2003}, abstractNote={ABSTRACT Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiABD), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiBBD [11 kDa]), followed by a catalytic domain (ChiBcat [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.}, number={6}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Gao, J and Bauer, MW and Shockley, KR and Pysz, MA and Kelly, RM}, year={2003}, month={Jun}, pages={3119–3128} }
 @article{shockley_ward_chhabra_conners_montero_kelly_2003, title={Heat shock response by the hyperthermophilic archaeon Pyrococcus furiosus}, volume={69}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.69.4.2365-2371.2003}, abstractNote={ABSTRACT Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.}, number={4}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Shockley, KR and Ward, DE and Chhabra, SR and Conners, SB and Montero, CI and Kelly, RM}, year={2003}, month={Apr}, pages={2365–2371} }
 @article{chhabra_shockley_ward_kelly_2002, title={Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides}, volume={68}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.68.2.545-554.2002}, abstractNote={ABSTRACT The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley β-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 109 cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting β-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative β-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular, β-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (Mr, 76,900) and Cel5A (Mr, 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley β-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.}, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Chhabra, SR and Shockley, KR and Ward, DE and Kelly, RM}, year={2002}, month={Feb}, pages={545–554} }