@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 S 0 , cellobiose-grown cells generated twice as much protein and had 50%-higher specific H 2 generation rates than maltose-grown cultures. Addition of S 0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H 2 to H 2 S. In contrast, the presence of S 0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H 2 to H 2 S production, with 2.5 times more H 2 than H 2 S 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 S 0 -independent manner. Most differentially transcribed open reading frames (ORFs) responding to S 0 in cellobiose-grown cells also responded to S 0 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 S 0 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 S 0 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={The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one alpha protein (PF1571) and two beta proteins (beta1-PF1404 and beta2-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 degrees C) showed that the beta1 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 beta1 protein relative to beta2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the beta2/beta1 ratio varied linearly with temperature from 3.8, when assembled at 80 degrees C, to 0.9 at 105 degrees C. The recombinant alpha+beta1+beta2 CP assembled at 105 degrees C was more thermostable than either the alpha+beta1+beta2 version assembled at 90 degrees C or the alpha+beta2 version assembled at either 90 degrees C or 105 degrees C, based on melting temperature and the biocatalytic inactivation rate at 115 degrees C. The recombinant CP assembled at 105 degrees C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90 degrees C assembly (measured at 95 degrees C). Combination of the alpha and beta1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the beta1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that beta 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 H 2 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}, 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. 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. analysis of variance carboxymethylcellulose phosphotransferase system carbon catabolite repression Saccharolytic microorganisms employ a range of proteins to hydrolyze, transport, and utilize complex carbohydrates that serve as carbon and energy sources (1de Vos W.M. Kengen S.W.M. Voorhorst W.G.B. van der Oost J. Extremophiles. 1998; 2: 201-205Crossref PubMed Scopus (38) Google Scholar). In some cases, these proteins are very specific to particular carbohydrates, whereas in other situations they mediate the processing of a broader range of glycosides. For simple sugars, such as glucose, binding and transport proteins alone mediate substrate entry into specific intracellular anabolic and catabolic pathways (2Galperin M.Y. Noll K.M. Romano A.H. Appl. Environ. Microbiol. 1996; 62: 2915-2918PubMed Google Scholar). However, for complex carbohydrates, a series of glycoside hydrolases must first process the polysaccharide so that its backbone and side chain glycosidic linkages are hydrolyzed to the extent needed for binding, transport, and intracellular utilization. How specific organisms develop the capacity to utilize complex carbohydrates is not known, but this probably involves evolutionary pressures in addition to acquisition of this genetic potential through horizontal gene transfer events. In any case, a microorganism's capacity to utilize carbohydrates presumably reflects the availability of such substrates in its habitat. Therefore, insights into the repertoire of carbohydrate-active proteins in a given organism and how the expression of these proteins is regulated would reveal much about particular metabolic features in addition to how it interacts within a given ecosystem. Thermotoga maritima is an obligately anaerobic, heterotrophic, hyperthermophilic bacterium originally isolated from geothermal features associated with Vulcano Island, Italy (3Huber R. Langworthy T.A. Konig H. Thomm M. Woese C.R. Sleytr U.B. Stetter K.O. Arch. Microbiol. 1986; 144: 324-333Crossref Scopus (623) Google Scholar). Its capacity to utilize a wide range of simple and complex carbohydrates was confirmed by the inventory of glycoside hydrolases encoded in its genome (4Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. Fraser C.M. et al.Nature. 1999; 399: 323-329Crossref PubMed Scopus (1206) Google Scholar). In fact, the T. maritima genome, despite its relatively small size, encodes the largest number of glycoside hydrolases of any bacterial or archaeal genome sequenced to date (see Fig. 1). From growth experiments and characterization of specific glycoside hydrolases (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar), T. maritima is known to metabolize both polysaccharides and simple sugars, including carboxymethylcellulose, barley glucan, starch, galactomannan (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar), xylan (6Bronnenmeier K. Kern A. Liebl W. Staudenbauer W.L. Appl. Environ. Microbiol. 1995; 61: 1399-1407Crossref PubMed Google Scholar), pectin, 1L. D. Kluskens, personal communication. 1L. D. Kluskens, personal communication. mannose, xylose, and glucose (2Galperin M.Y. Noll K.M. Romano A.H. Appl. Environ. Microbiol. 1996; 62: 2915-2918PubMed Google Scholar). In some cases, the proteins involved in the processing, transport, and utilization of these glycosides can be inferred from their apparent organization into operons in the T. maritimagenome sequence (4Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. Fraser C.M. et al.Nature. 1999; 399: 323-329Crossref PubMed Scopus (1206) Google Scholar), whereas in other cases such classification is not clear. Regulation of genes encoding specific carbohydrate-active proteins in T. maritima has only been studied to a limited extent thus far (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar, 7Nguyen T.N. Borges K.M. Romano A.H. Noll K.M. FEMS Microbiol. Lett. 2001; 195: 79-83Crossref PubMed Google Scholar), and the coordinated regulation of related genes involved in polysaccharide utilization has not been examined. Here, a targeted cDNA microarray, based on carbohydrate-active proteins from T. maritima, was used in conjunction with mixed model analysis (8Jin W. Riley R.M. Wolfinger R.D. White K.P. Passador-Gurgel G. Gibson G. Nat. Genet. 2001; 29: 389-395Crossref PubMed Scopus (522) Google Scholar, 9Wolfinger R.D. Gibson G. Wolfinger E.D. Bennett L. Hamadeh H. Bushel P. Afshari C. Paules R.S. J. Comput. Biol. 2001; 8: 625-637Crossref PubMed Scopus (856) Google Scholar) to explore issues related to saccharide utilization by this organism. Despite the fact thatT. maritima has been phylogenetically characterized as a primitive microorganism from an evolutionary perspective (10Achenbach-Richter L. Gupta R. Stetter K.O. Woese C.R. Syst. Appl. Microbiol. 1987; 9: 34-39Crossref PubMed Scopus (180) Google Scholar), results here support that it has versatile and discriminating mechanisms for regulating and effecting complex carbohydrate utilization. The relative importance of evolutionary processes and horizontal gene transfer (4Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. Fraser C.M. et al.Nature. 1999; 399: 323-329Crossref PubMed Scopus (1206) Google Scholar) in developing its carbohydrate utilization capacity is not known, butT. maritima's ability to respond to various substrates in its growth environment underlies its ubiquity in global geothermal settings (11Nesbo C.L. Nelson K.E. Doolittle W.F. J. Bacteriol. 2002; 184: 4475-4488Crossref PubMed Scopus (55) Google Scholar). Open reading frames (total of 269) of known and putative genes related to sugar processing and other related metabolic functions were identified through BLAST (12Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69678) Google Scholar) comparisons of protein sequences from the T. maritima MSB8 genome available on the World Wide Web at www.tigr.org/ tigrscripts/CMR2/GenomePage3.spl?database=btm. DNA primers were designed with similar annealing temperatures and minimal hairpin formation using Vector NTI 7.0 (Informax, Bethesda, MD). The selected probes were PCR-amplified in a PTC-100 Thermocycler (MJ Research, Inc., Waltham, MA) using Taq polymerase (Roche Molecular Biochemicals) and T. maritima genomic DNA, isolated as described previously (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar). The integrity and concentration of the PCR products were verified on 1% agarose gels. PCR products were purified to 100 ng/μl using 96-well QIAquick PCR purification kits (Qiagen, Valencia, CA), resuspended in 50% Me2SO, and printed onto CMT-GAPS aminosilane-coated microscope slides (Corning Glass) using a 417 Arrayer (Affymetrix, Santa Clara, CA) in the North Carolina State University Genome Research Laboratory (Raleigh, NC). Eight replicates of each gene fragment were printed onto each slide. The DNA was then attached to the slides by UV cross-linking using a GS GeneLinker UV Chamber (Bio-Rad) set at 250 mJ and baked at 75 °C for 2 h. Growth ofT. maritima MSB8 cultures in artificial sea water was followed using optical density measurements and epifluorescence microscopic cell density enumeration, as described previously (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar). Growth substrates glucose, mannose, xylose, β-xylan (birchwood), laminarin (Laminaria digitata), and starch (potato) were obtained from Sigma. Galactomannan (carob), glucomannan (konjac), carboxymethylcellulose, and β-glucan (barley) were obtained from Megazyme (Wicklow, Ireland). Growth substrates were prepared as described previously (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar) and included in the medium at a final concentration of 0.25% (w/v). Substrate purities as provided by the manufacturers varied from 95 to 99%. To ensure minimum carryover between substrates, cells were grown for at least 10 passes on each carbon source using a 0.5% (v/v) starting innoculum before obtaining the growth curves. Specific growth rates on mono- and polysaccharide substrates were determined from the slopes of semilog plots of exponential cell growth versus time. Isolation of total RNA from T. maritima was performed on cells that were grown until early- to mid-exponential phase on the various growth substrates, as described in detail previously (5Chhabra S.R. Shockley K.R. Ward D.E. Kelly R.M. Appl. Environ. Microbiol. 2002; 68: 545-554Crossref PubMed Scopus (91) Google Scholar). First-strand cDNA was prepared from T. maritima total RNA using Stratascript (Stratagene, La Jolla, CA) and random hexamer primers (Invitrogen) by the incorporation of 5-[3-aminoallyl]-2′-deoxyuridine-5′-triphosphate (Sigma) as described elsewhere (13Hasseman J. TIGR Microarray Protocols. 2001; (http://www.tigr.org/tdb/microarray/protocolsTIGR.shtml)Google Scholar). The slides were scanned using a Scanarray 4000 scanner (GSI Lumonics and Billerica) in the North Carolina State University Genome Research Laboratory. Signal intensity data were obtained using Quantarray (GSI Lumonics). A loop design was constructed (see Fig. 2) to ensure reciprocal labeling for all 10 different experimental conditions. Replication of treatments, arrays, dyes, and cDNA spots allowed the use of analysis of variance (ANOVA)2 models for data analysis. ANOVAs are especially appropriate for loop designs in which a large number of conditions are compared with one another, eliminating uninteresting reference samples and allowing for the collection of more information on experimental conditions (14Kerr M.K. Churchill G.A. Genet. Res. 2001; 77: 123-128Crossref PubMed Scopus (465) Google Scholar). Mixed ANOVA models, in which some effects are considered fixed and others are considered random, have been used to re-examine published microarray data sets (9Wolfinger R.D. Gibson G. Wolfinger E.D. Bennett L. Hamadeh H. Bushel P. Afshari C. Paules R.S. J. Comput. Biol. 2001; 8: 625-637Crossref PubMed Scopus (856) Google Scholar) and examine the effects of sex, genotype, and age on transcription inDrosophila melanogaster (8Jin W. Riley R.M. Wolfinger R.D. White K.P. Passador-Gurgel G. Gibson G. Nat. Genet. 2001; 29: 389-395Crossref PubMed Scopus (522) Google Scholar). Using existing SAS procedures and customized Perl code, an automated data import system was developed to merge Quantarray intensity measurements, coordinate files generated by the array printer, and corresponding T. maritima locus numbers in a SAS data set (SAS Institute, Cary, NC). The data import system was verified through independent calculations in Excel (Microsoft, Seattle, WA). A linear normalization ANOVA model (9Wolfinger R.D. Gibson G. Wolfinger E.D. Bennett L. Hamadeh H. Bushel P. Afshari C. Paules R.S. J. Comput. Biol. 2001; 8: 625-637Crossref PubMed Scopus (856) Google Scholar) of log base 2 intensities was used to estimate global variation in the form of fixed (dye, treatment) and random (array, pin within array, pin spot within array) effects and random error using the following model: log2(y ijklmn) =m + Dj + T k +A i + A i(P1) +A i(S m P l) + εijklmn. The estimated effects calculated from this model were used to predict an expected intensity for each value, and then a residual was calculated as the difference between a replicate's observed and predicted intensity and then used as data to capture variation attributable to gene-specific effects after accounting for global variation. Gene-specific ANOVA models were then used to partition variation into gene-specific treatment effects, dye effects, and the same hierarchy of random effects described previously. Specifically, the model r ijklmn =m+ D i + T k +A i + A i(P1) +A i(S m P1) + εijklmn was fit separately to the residuals for each gene, and the resulting parameter estimates and S.E. values were then used for statistical inference. Volcano plots were used to visualize interesting contrasts or comparisons between two treatments or two groups of treatments (9Wolfinger R.D. Gibson G. Wolfinger E.D. Bennett L. Hamadeh H. Bushel P. Afshari C. Paules R.S. J. Comput. Biol. 2001; 8: 625-637Crossref PubMed Scopus (856) Google Scholar). A Bonferroni correction was utilized to adjust for the expected increase in false positives due to multiple comparisons (9Wolfinger R.D. Gibson G. Wolfinger E.D. Bennett L. Hamadeh H. Bushel P. Afshari C. Paules R.S. J. Comput. Biol. 2001; 8: 625-637Crossref PubMed Scopus (856) Google Scholar). Genes meeting the Bonferroni significance criteria were selected for further study, ensuring that genes with inconsistent fold changes would be eliminated from further analysis. Two complementary approaches were utilized to cluster data from T. maritima growth on 10 saccharides. To visualize the relative expression levels of all genes withina treatment, hierarchical clustering was performed on least squares means calculated from the linear models for each sugar (Fig. 3). To visualize the expression pattern of each single gene acrosstreatments, the least squares mean estimates were standardized using the mean and S.D. of the 10 least squares means estimates for a given gene. Each of the 10 least squares means estimates were standardized accordingly with the formula Y i = (X i − μ)/ς, where Y i = the standardized least squares means variable, μ = ΣX i/n, and ς = (Σ(X i − μ)2) 12. The standardized variable was then utilized for clustering (Fig. 3). For complete information on signal intensity, significance of expression changes, -fold changes, pairwise volcano plots, and hierarchical clustering for all of the genes included on the array, see the Supplemental Material. A targeted cDNA microarray for T. maritima was constructed that included 269 known and putative genes or about 15% of the total open reading frames in the T. maritima genome. This included the known set of genes related to glycoside utilization and modification (65 genes), proteolysis (40 genes), stress response, and proteolytic fermentation. Genes related to sugar transport (21 genes) or transcriptional regulation (69 genes) and 66 other genes of interest were also included. Genes apparently related to glycoside utilization and modification in T. maritima include 41 glycoside hydrolases, 17 glycosyl transferases, 6 carbohydrate esters, and 1 polysaccharide lyase. The corresponding encoded proteins have been classified into several families, based on amino acid sequence homology (15Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1179) Google Scholar) (available on the World Wide Web at afmb.cnrs-mrs.fr/CAZY). There are over 130T. maritima proteins with sufficient BLAST homology to be classified into transcriptional regulatory or signal transduction COG categories (16Tatusov R.L. Natale D.A. Garkavtsev I.V. Tatusova T.A. Shankavaram U.T. Rao B.S. Kiryutin B. Galperin M.Y. Fedorova N.D. Koonin E.V. Nucleic Acids Res. 2001; 29: 22-28Crossref PubMed Scopus (1539) Google Scholar). These regulatory proteins have been assigned to families based on sequence homology; however, different proteins in the same families may have different DNA and substrate-binding specificities (17Mirny L.A. Gelfand M.S. J. Mol. Biol. 2002; 321: 7-20Crossref PubMed Scopus (116) Google Scholar). Also, proteins placed in different families may share the same name because of their regulon composition, as in the case of the Escherichia coli and Bacillus subtilis xylR protein (18Song S. Park C. J. Bacteriol. 1997; 179: 7025-7032Crossref PubMed Scopus (120) Google Scholar, 19Kreuzer P. Gartner D. Allmansberger R. Hillen W. J. Bacteriol. 1989; 171: 3840-3845Crossref PubMed Google Scholar). Of the 69 transcription/transduction genes on the array, six share similarity with the ROK (receptor, open reading frame,kinase) family of transcriptional regulators, which include glucokinases, B. subtilis XylR, and E. coli NagC (COG1940) (20Titgemeyer F. J. Cell. Biochem. 1993; 51: 69-74Crossref PubMed Scopus (19) Google Scholar). Six members of the PurR/LacI superfamily (COG1609) were included (21Mirny L.A. Gelfand M.S. Nucleic Acids Res. 2002; 30: 1704-1711Crossref PubMed Scopus (58) Google Scholar) along with the T. maritima IclR transcriptional regulator, whose structure was recently solved (22Zhang R.G. Kim Y. Skarina T. Beasley S. Laskowski R. Arrowsmith C. Edwards A. Joachimiak A. Savchenko A. J. Biol. Chem. 2002; 277: 19183-19190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Several pairs of sensor histidine kinases and response regulators of putative two-component regulatory systems were included, as were regulators from the MarR (23Cohen S.P. Hachler H. Levy S.B. J. Bacteriol. 1993; 175: 1484-1492Crossref PubMed Scopus (274) Google Scholar), AraC (24Martin R.G. Rosner J.L. Curr. Opin. Microbiol. 2001; 4: 132-137Crossref PubMed Scopus (182) Google Scholar), TroR (25Hardham J.M. Stamm L.V. Porcella S.F. Frye J.G. Barnes N.Y. Howell J.K. Mueller S.L. Radolf J.D. Weinstock G.M. Norris S.J. Gene (Amst.). 1997; 197: 47-64Crossref PubMed Scopus (62) Google Scholar), LytR (26Nikolskaya A.N. Galperin M.Y. Nucleic Acids Res. 2002; 30: 2453-2459Crossref PubMed Scopus (147) Google Scholar), ArsR (27Diorio C. Cai J. Marmor J. Shinder R. DuBow M.S. J. Bacteriol. 1995; 177: 2050-2056Crossref PubMed Google Scholar), and CspC (28Phadtare S. Alsina J. Inouye M. Curr. Opin. Microbiol. 1999; 2: 175-180Crossref PubMed Scopus (271) Google Scholar) families. The T. maritima genome contains ∼120 genes involved in oligopeptide/sugar transport. In the targeted microarray used here, 21 genes related to sugar transport were included on the basis of their proximity to the genes involved in glycoside utilization. This targeted microarray was used to examine the differential response of T. maritima grown on a range of mono- and polysaccharides at its optimal growth temperature of 80 °C. Growth conditions were analyzed based on an incomplete loop design (Fig.2). Treatments in the loop design were balanced with respect to dyes so that treatment effects were not confounded with dye effects. T. maritima cultures were grown on a variety of saccharides, including the monosaccharides glucose, mannose, and xylose. The polysaccharides investigated differed in backbone sugar type (glucose, mannose, and xylose), backbone linkage type (β-1,3; β-1,4; or α-1,4), and side chain residue type (galactose, glucuronic acid, or glucose) (see TableI). Included in these were a mixed backbone (konjac glucomannan: glucose/mannose) and a mixed linkage (barley glucan: β-1,4/1,3) polysaccharide. Final cell densities were in the range of 108 to 109cells/ml in all cases. Doubling times (min) for galactomannan (carob), β-glucan (barley), laminarin (L. digitata), β-xylan (birchwood), starch (potato), glucomannan (konjac), and carboxymethylcellulose were estimated to be 85, 72, 143, 61, 117, 74, and 78, respectively. On the monosaccharides, the doubling times (min) were 162, 253, and 188, for glucose, mannose, and xylose, respectively. Under identical conditions, the average doubling time for growth on monosaccharides (201 min) was observed to be substantially higher than that on the corresponding polysaccharide substrates (90 min).Table ICarbon sources used in this studyPoly/monosaccharideSourceBackbone structureSide chainMassDaGlucoseNAaNA, not available.Glc180MannoseNAMan180XyloseNAXylbXyl, xylose.150GalactomannanCarob(Man β1→4 Man)nGal (α1→6)NAGlucomannanKonjac(Glc β1→4 Man)n100,000Carboxymethyl celluloseNA(Glc β1→4 Glc)n90,000β-1,3/1,4-GlucanBarley(Glc β1→3,4 Glc)n250,000LaminarinL. digitata(Glc β1→3 Glc)n5,000StarchPotato(Glc α1→4 Glc)nGlc (α→16)nNAβ-XylanBirchwood(Xyl β1→4 Xyl)nGlr (α1→6)cGlr, glucuronic acid.NAa NA, not available.b Xyl, xylose.c Glr, glucuronic acid. Open table in a new tab Two hierarchical clusters are shown in Fig.3 to summarize the expression patterns of 269 T. maritima genes during growth on 10 saccharides. The first cluster is based on least squares means and compares the normalized expression levels of all genes within each treatment condition. The second cluster is based on standardized least squares means for a single gene across all 10 treatments to show the effect of different treatments on the relative expression of a particular gene. The hierarchical clustering based on standardized least squares means revealed many cases of apparent co-regulation of genes within potential operons (29McGuire A.M. Hughes J.D. Church G.M. Genome Res. 2000; 10: 744-757Crossref PubMed Scopus (160) Google Scholar). Several sets of spatially distant gene strings were observed to cluster with similar expression profiles, suggesting the presence of regulons in the T. maritima genome. Representative clusters are displayed in Fig.4. Overall expression levels of a number of genes remained consistently high or low regardless of the growth condition. These included constitutively expressed genes like TM0017 (pyruvate ferredoxin oxidoreductase) and TM0688 (glyceraldehyde-3-phosphate dehydrogenase) (30Blamey J.M. Adams M.W. Biochemistry. 1994; 33: 1000-1007Crossref PubMed Scopus (92) Google Scholar) as well as genes related to proteolytic activity. Both sets of genes with the corresponding known or putative functions are displayed in Fig. 5. Individual genes with high overall expression levels on only a single carbon source are indicated in Table II. Least squares means for all genes included in this study for all growth conditions are shown in Supplemental Table IV, along with the corresponding standardized values in Supplemental Table V. Below, gene regulation patterns within each functional category are examined for each monosaccharide and corresponding polysaccharide growth substrate.Figure 4Substrate-dependent regulation. Sample Clusters constructed using standardized least squares means. Known or putative functions as reported in the genome sequence are indicated.View Large Image Figure ViewerDownload (PPT)Figure 4Substrate-dependent regulation. Sample Clusters constructed using standardized least squares means. Known or putative functions as reported in the genome sequence are indicated.View Large Image Figure ViewerDownload (PPT)Figure 5Genes with overall high or low expression levels for all growth substrates. Clusters constructed using least squares means. Known or putative functions as reported in the genome sequence are indicated.View Large Image Figure ViewerDownload (PPT)Table IIGenes with high overall expression levels (log2R ≥ 0.6) on indicated growth substrateGrowth substrateLocusFunctionCarboxymethylcelluloseTM0963Oligoendopeptidase, putativeMannoseTM1755Phosphate butyryltransferaseTM1754Butyrate kinase, putativeTM1756Branched chain fatty acid kinase, putativeLaminarinTM0024LaminarinaseTM0032Transcriptional regulator, XylR-relatedStarchTM1835Cyclomaltodextrinase, putativeTM1840α-AmylaseTM1845PullulanaseXylanTM0055α-GlucuronidaseTM0065Transcriptional regulator, IclR familyXyloseTM0949Transcriptional regulator, LacI family Open table in a new tab Backbone- and linkage-specific gene regulation was observed in the case of endoglycoside hydrolase genes for growth on α- and β-specific glucans. Growth on carboxymethylcellulose (CMC) (see cluster 4.1), a β-1,4-linked glucose polymer, induced genes encoding extracellular endoglucanases TM1525 (cel12B) and TM0305 (cel74), as well as the intracellular endoglucanase TM1524 (cel12A) and the intracellular cellobiosyl phosphorylase, TM1848. Examination of cluster I (Fig. 3) reveals that expression levels of cel74 were substantially lower than those ofcel12A on glucan polysaccharides. Although the presence of a β-1,4-glucosidase gene (bglA) (accession number CAA52276) in T. maritima MSB8 has been reported (31Liebl W. Methods Enzymol. 2001; 330: 290-300Crossref PubMed Scopus (16) Google Scholar), the corresponding protein sequence does not show homology to deduced sequences identified in the T. maritima MSB8 genome (4Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart}, 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 (ChiA BD ), 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 (ChiB BD [11 kDa]), followed by a catalytic domain (ChiB cat [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 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} }