Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose-and pectin-containing biomass to acetate, CO 2 , and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocelluloseutilizing, biofuel-producing bacterium.Microbial hydrogen production from biomass has been recognized as an important source of renewable energy (15, 47). High-temperature microorganisms are well suited for production of biohydrogen from plant polysaccharides, as anaerobic fermentation is thermodynamically favored at elevated temperatures (17, 43). The extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903, a fermentative anaerobe initially isolated from wood in the flow of a thermal spring in New Zealand, first received attention because of its capacity to utilize cellulose at its optimal growth temperature, 70°C (37). Further work showed that C. saccharolyticus (i) can utilize a wide range of plant materials, including cellulose, hemicellulose, starch, and pectin, (ii) has a very high hydrogen yield (almost 4 mol of H 2 per mol of glucose) (14,20,48), and (iii) can ferment C 5 and C 6 sugars simultaneously. These features have led to the development of bioprocessing schemes based on C. saccharolyticus. For example, H 2 production is now being investigated using a two-step process in which H 2 and acetate are generated from biomass hydrolysates in one bioreactor and the acetate is fed to a second bioreactor and used by phototrophic organisms (Rhodobacter spp.) to produce additional H 2 in the presence of...

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 ␤-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 ␤-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative ␤-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, ␤-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 ␤-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.Genome sequence information for hyperthermophiles has provided significant insights into the metabolic features of these microorganisms (40). With respect to the physiology of growth of...

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.Information gleaned from genome sequence data indicates that heat shock response in hyperthermophilic archaea has several distinguishing features. For example, hyperthermophilic archaea lack Hsp70 (DnaK), Hsp40 (DnaJ), and GrpE, all of which are centrally important in the heat shock response of most known microorganisms (26). Also, the major chaperonin found thus far in thermophilic archaea, an Hsp60 homolog referred to as the thermosome, is more closely related to chaperonins associated with the eukaryotic cytosol (TriCC/CCT complex) than to the bacterial GroEL/ES system (19, 32). Energy-dependent proteolysis plays a major role during heat shock in bacteria in which genes encoding ATP-dependent proteases, such as lon, clp, and hfl, are linked to heat shock promoters (13). However, based on available genome sequence data, hyperthermophilic archaea lack the Clp and HflB (FtsH) family of proteins and have a different version of the Lon protease (43). Hyperthermophilic archaea, which typically have proteasomes, lack the eukaryotic ubiquitination pathway for selective protein degradation by the proteasome and, therefore, seem to modulate proteolysis at the protease level. Another interesting feature of hyperthermophilic archaeal heat shock response is the induced formation of unique compatible solutes that have been proposed to stabilize intracellular proteins against thermal denaturation (33). Whether compatible solutes reduce the need for protein turnover mechanisms is not known.The relative contributions to the collective response of chaperones, chaperonins, proteases, and compatible solutes during heat shock in hyperthermophilic archaea have yet to be examined. Here, the heat shock response of the hyperthermophilic archaeon Pyrococcus furiosus (9) was investigated by using Northern analyses in conjunction with a targeted cDNA microarray, based on genes encoding the thermosome, molecular chaperones, proteases, glycoside hydrolases, and other relevant cellular functions expected to be affected during thermal stress.Experimental approach and data analysis. Relevant open reading frames (ORFs) were located from the P. furiosus genome at NCBI (http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez /framik?dbϭgenome&gi ϭ 228) and BLAST searches of prokaryotic genomes found at The Institute for Genomic Research (www.TIGR.org). SCANPROSITE (http://expasy.cbr .nrc.ca/tools/scnpsit1.html) and PFAM HMM (Ͼhttp://pfam .wustl.edu/hmmsearch.shtml) search tools were used to verify the presence of putative catalytic domains. ORF fra...

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 ␤-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 ␤-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.

While surveying the genomes of hyperthermophilic and thermophilic Archaea for homologues of the flavoprotein disulfide reductases, many homologues with a high degree of identity to the branch of this family represented by glutathione reductase were found [1]. Most of the homologues appear to belong to the subfamily that depend on a redox-active single cysteine, analogous to the NADH oxidase and per-oxidase of Enterococcus and the coenzyme A disulfide reductase (CoADR; EC 1.8.1.14) of Staphylococcus Correspondence E. J. Crane III, We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H + fi 2CoA-SH + NAD(P) +). The P. horikoshii enzyme shows a k cat app of 7.2 s)1 with NADPH at 75 °C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal k cat s, while the K m for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephos-pho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH 2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furio-sus cells were assayed for small molecular mass thiols and found to contain 0.64 lmol CoAAEg dry weight)1 (corresponding to 210 lm CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents. Abbreviations CoADR, coenzyme A disulfide reductase (EC# 1.8.1.14); pfCoADR, P. furiosus coenzyme A disulfide reductase; phCoADR, P. horikoshii coenzyme A disulfide reductase; DTNB, 5,5¢ dithiobis(2-nitrobenzoic acid); EH 2 , two-electron reduced enzyme; EH 4 , four-electron reduced enzyme; HEPPS, N-(2-hydroxyethyl)piperazine-N¢-3-propanesulfonic acid; NOX, NADH oxidase; NPX, NADH peroxidase; TCA, trichloroacetic acid.