title: Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides.
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 beta-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 beta-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative beta-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 beta-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 degrees C and an optimun temperature of 90 degrees C, compared to 90 and 80 degrees 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 beta-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.
authors.affiliation: Department of Chemical Engineering, North Carolina State University, Stinson Drive, Raleigh, NC 27695-7905, USA.
authors.forename: Swapnil R
authors.initials: SR
authors.lastname: Chhabra
authors.forename: Keith R
authors.initials: KR
authors.lastname: Shockley
authors.forename: Donald E
authors.initials: DE
authors.lastname: Ward
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D005936:Glucans / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
chemical_list: D003470:Culture Media; D005936:Glucans; D008351:Mannans; D011134:Polysaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases