@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={ABSTRACTComprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacteriumThermotoga maritimaon 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 thatT. maritimarelies 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 toT. maritimagenome 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 forT. 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{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{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_kelly_2002, title={Biochemical characterization of Thermotoga maritima endoglucanase Ce174 with and without a carbohydrate binding module (CBM)}, volume={531}, ISSN={["1873-3468"]}, DOI={10.1016/S0014-5793(02)03493-2}, abstractNote={The genome of the hyperthermophilic bacteriumThermotoga maritima(Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino‐terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide inEscherichia coliand found to be optimally active at pH 6, 90°C, with a melting temperature of approximately 105°C. Thecel74gene was previously found to be induced duringT. maritimagrowth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile,Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan‐based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.}, number={2}, journal={FEBS LETTERS}, author={Chhabra, SR and Kelly, RM}, year={2002}, month={Nov}, pages={375–380} } @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 10 9 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 ( M r , 76,900) and Cel5A ( M r , 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} } @article{parker_chhabra_lam_callen_duffaud_snead_short_mathur_kelly_2001, title={Galactomannanases man2 and man5 from Thermotoga species: Growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes}, volume={75}, ISSN={["0006-3592"]}, DOI={10.1002/bit.10020}, abstractNote={AbstractThe enzymatic hydrolysis of mannan‐based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding β‐mannosidase (man2) and β‐mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an Mr of approximately 92 kDa. The translated nucleotide sequences for the β‐mannanase genes (man5) encoded polypeptides with an Mr of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90°C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that β‐mannanase and β‐mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83°C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan‐degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 322–333, 2001.}, number={3}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Parker, KN and Chhabra, SR and Lam, D and Callen, W and Duffaud, GD and Snead, MA and Short, JM and Mathur, EJ and Kelly, RM}, year={2001}, month={Nov}, pages={322–333} } @article{chhabra_parker_lam_callen_snead_mathur_short_kelly_2001, title={beta-Mannanases from Thermotoga species}, volume={330}, journal={Hyperthermophilic enzymes. Part A}, publisher={San Diego, Calif.: Academic Press}, author={Chhabra, S. and Parker, K. N. and Lam, D. and Callen, W. and Snead, M. A. and Mathur, E. J. and Short, J. M. and Kelly, R. M.}, year={2001}, pages={224–238} } @article{parker_chhabra_lam_snead_mathur_kelly_2001, title={beta-Mannosidase from Thermotoga species}, volume={330}, journal={Hyperthermophilic enzymes. Part A}, publisher={San Diego, Calif.: Academic Press}, author={Parker, K. N. and Chhabra, S. and Lam, D. and Snead, M. A. and Mathur, E. J. and Kelly, R. M.}, year={2001}, pages={238–246} }