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Hyperthermophilic Thermotoga species differ with respect to specific carbohydrate transporters and glycoside hydrolases.

ABSTRACT: Four hyperthermophilic members of the bacterial genus Thermotoga (T. maritima, T. neapolitana, T. petrophila, and Thermotoga sp. strain RQ2) share a core genome of 1,470 open reading frames (ORFs), or about 75% of their genomes. Nonetheless, each species exhibited certain distinguishing features during growth on simple and complex carbohydrates that correlated with genomic inventories of specific ABC sugar transporters and glycoside hydrolases. These differences were consistent with transcriptomic analysis based on a multispecies cDNA microarray. Growth on a mixture of six pentoses and hexoses showed no significant utilization of galactose or mannose by any of the four species. T. maritima and T. neapolitana exhibited similar monosaccharide utilization profiles, with a strong preference for glucose and xylose over fructose and arabinose. Thermotoga sp. strain RQ2 also used glucose and xylose, but was the only species to utilize fructose to any extent, consistent with a phosphotransferase system (PTS) specific for this sugar encoded in its genome. T. petrophila used glucose to a significantly lesser extent than the other species. In fact, the XylR regulon was triggered by growth on glucose for T. petrophila, which was attributed to the absence of a glucose transporter (XylE2F2K2), otherwise present in the other Thermotoga species. This suggested that T. petrophila acquires glucose through the XylE1F1K1 transporter, which primarily serves to transport xylose in the other three Thermotoga species. The results here show that subtle differences exist among the hyperthermophilic Thermotogales with respect to carbohydrate utilization, which supports their designation as separate species.

SUBMITTER: Frock AD

PROVIDER: S-EPMC3298158 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Hyperthermophilic Thermotoga species differ with respect to specific carbohydrate transporters and glycoside hydrolases.

Frock Andrew D AD Gray Steven R SR Kelly Robert M RM

Applied and environmental microbiology 20120113 6

Four hyperthermophilic members of the bacterial genus Thermotoga (T. maritima, T. neapolitana, T. petrophila, and Thermotoga sp. strain RQ2) share a core genome of 1,470 open reading frames (ORFs), or about 75% of their genomes. Nonetheless, each species exhibited certain distinguishing features during growth on simple and complex carbohydrates that correlated with genomic inventories of specific ABC sugar transporters and glycoside hydrolases. These differences were consistent with transcriptom ...[more]

PMID: 22247137

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Project description: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.

Project description:Arabinoxylan hydrolysates (AXH) are the hydrolyzed products of the major components of the dietary fiber arabinoxylan. AXH include diverse oligosaccharides varying in xylose polymerization and side residue modifications with arabinose at the O-2 and/or O-3 position of the xylose unit. Previous studies have reported that AXH exhibit prebiotic properties on gut bifidobacteria; moreover, several adult-associated bifidobacterial species (e.g., Bifidobacterium adolescentis and Bifidobacterium longum subsp. longum) are known to utilize AXH. In this study, we tried to elucidate the molecular mechanisms of AXH utilization by Bifidobacterium pseudocatenulatum, which is a common bifidobacterial species found in adult feces. We performed transcriptomic analysis of B. pseudocatenulatum YIT 4072T, which identified three upregulated gene clusters during AXH utilization. The gene clusters encoded three sets of ATP-binding cassette (ABC) transporters and five enzymes belonging to glycoside hydrolase family 43 (GH43). By characterizing the recombinant proteins, we found that three solute-binding proteins of ABC transporters showed either broad or narrow specificity, two arabinofuranosidases hydrolyzed either single- or double-decorated arabinoxylooligosaccharides, and three xylosidases exhibited functionally identical activity. These data collectively suggest that the transporters and glycoside hydrolases, encoded in the three gene clusters, work together to utilize AXH of different sizes and with different side residue modifications. Thus, our study sheds light on the overall picture of how these proteins collaborate for the utilization of AXH in B. pseudocatenulatum and may explain the predominance of this symbiont species in the adult human gut.IMPORTANCE Bifidobacteria commonly reside in the human intestine and possess abundant genes involved in carbohydrate utilization. Arabinoxylan hydrolysates (AXH) are hydrolyzed products of arabinoxylan, one of the most abundant dietary fibers, and they include xylooligosaccharides and those decorated with arabinofuranosyl residues. The molecular mechanism by which B. pseudocatenulatum, a common bifidobacterial species found in adult feces, utilizes structurally and compositionally variable AXH has yet to be extensively investigated. In this study, we identified three gene clusters (encoding five GH43 enzymes and three solute-binding proteins of ABC transporters) that were upregulated in B. pseudocatenulatum YIT 4072T during AXH utilization. By investigating their substrate specificities, we revealed how these proteins are involved in the uptake and degradation of AXH. These molecular insights may provide a better understanding of how resident bifidobacteria colonize the colon.

Dataset Information

Hyperthermophilic Thermotoga species differ with respect to specific carbohydrate transporters and glycoside hydrolases.

Publications

Hyperthermophilic Thermotoga species differ with respect to specific carbohydrate transporters and glycoside hydrolases.

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