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New regulatory gene that contributes to control of Bacteroides thetaiotaomicron starch utilization genes.

ABSTRACT: Bacteroides thetaiotaomicron uses starch as a source of carbon and energy. Early steps in the pathway of starch utilization, such as starch binding and starch hydrolysis, are encoded by sus genes, which have been characterized previously. The sus structural genes are expressed only if cells are grown in medium containing maltose or higher oligomers of glucose. Regulation of the sus structural genes is mediated by SusR, an activator that is encoded by a gene located next to the sus structural genes. A strain with a disruption in susR cannot grow on starch but can still grow on maltose and maltotriose. A search for transposon-generated mutants that could not grow on maltose and maltotriose unexpectedly located a gene, designated malR, which regulates expression of an alpha-glucosidase not controlled by SusR. Although a disruption in susR did not affect expression of the malR controlled gene, a disruption in malR reduced expression of the sus structural genes. Thus, MalR appears to participate with SusR in regulation of the sus genes. Results of transcriptional fusion assays and reverse transcription-PCR experiments showed that malR is expressed constitutively. Moreover, multiple copies of malR provided on a plasmid (5 to 10 copies per cell) more than doubled the amount of alpha-glucosidase activity in cell extracts. Our results demonstrate that the starch utilization system of B. thetaiotaomicron is controlled on at least two levels by the regulatory proteins SusR and MalR.

SUBMITTER: Cho KH

PROVIDER: S-EPMC95569 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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New regulatory gene that contributes to control of Bacteroides thetaiotaomicron starch utilization genes.

Cho K H KH Cho D D Wang G R GR Salyers A A AA

Journal of bacteriology 20011201 24

Bacteroides thetaiotaomicron uses starch as a source of carbon and energy. Early steps in the pathway of starch utilization, such as starch binding and starch hydrolysis, are encoded by sus genes, which have been characterized previously. The sus structural genes are expressed only if cells are grown in medium containing maltose or higher oligomers of glucose. Regulation of the sus structural genes is mediated by SusR, an activator that is encoded by a gene located next to the sus structural gen ...[more]

PMID: 11717279

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Project description:Previously, we isolated two Tn4351-generated mutants of Bacteroides thetaiotaomicron (46-1 and CS3) that were unable to grow either on heparin or on chondroitin sulfate. This phenotype was unexpected, since the heparin and chondroitin sulfate utilization pathways had appeared from earlier studies to be independent of each other. Mutants 46-1 and CS3 were also of interest because both were unable to compete successfully with wild-type B. thetaiotaomicron in the intestinal tracts of germfree mice. Thus, both appeared to have a colonization defect. We have now cloned the chromosomal locus in which the transposon insertions in 46-1 and CS3 occurred. Southern blot analysis showed that the Tn4351 insertions in 46-1 and CS3 were about 100 bp apart. Using complementation and insertional mutagenesis, we localized the region affected by the 46-1 and CS3 insertions to within 2.5 kbp. This DNA segment was sequenced and found to contain a 401-codon open reading frame (ORF1) and the N-terminal segment of a second open reading frame (ORF2), which was downstream of ORF1 and transcribed in the same direction. The deduced amino acid sequence of ORF1 showed significant homology to that of a putative positive regulator of an arylsulfatase gene in Klebsiella aerogenes. ORF2 was at least 381 amino acids long and did not exhibit homology to any proteins in the data bases searched. Transposon insertions in both mutants 46-1 and CS3 disrupted ORF1. The results of insertional mutagenesis and complementation experiments indicated that ORF2 was not essential for growth on chondroitin sulfate or heparin. Thus, the chondroitin sulfate-negative and heparin-negative phenotypes of 46-1 and CS3 appear to be due to the interruption of a regulatory gene encoded by ORF1 and not to a polar effect of the insertions on a downstream gene(s). The gene encoding ORF1 has been designated chuR, for regulation of chondroitin sulfate and heparin utilization. Transcriptional fusion studies showed that the expression of chuR occurred at the same level under inducing and noninducing conditions, in contrast to the regulated expression of structural genes of the chondroitin sulfate utilization system. chuR was not autoregulated, nor was its expression affected by a mutation (46-4) that eliminated the expression of all chondroitin sulfate utilization genes but did not affect the utilization of heparin.

Project description:The utilization of simple sugars is widespread across all domains of life. In contrast, the breakdown of complex carbohydrates is restricted to a subset of organisms. A regulatory paradigm for integration of complex polysaccharide breakdown with simple sugar utilization was established in the mammalian gut symbiont Bacteroides thetaiotaomicron, whereby sensing of monomeric fructose regulates catabolism of both fructose and polymeric fructans. We now report that a different regulatory paradigm governs utilization of monomeric arabinose and the arabinose polymer arabinan. We establish that (i) arabinan utilization genes are controlled by a transcriptional activator that responds to arabinan and by a transcriptional repressor that responds to arabinose, (ii) arabinose utilization genes are regulated directly by the arabinose-responding repressor but indirectly by the arabinan-responding activator, and (iii) activation of both arabinan and arabinose utilization genes requires a pleiotropic transcriptional regulator necessary for survival in the mammalian gut. Genomic analysis predicts that this paradigm is broadly applicable to the breakdown of other polysaccharides in both B. thetaiotaomicron and other gut Bacteroides spp. The uncovered mechanism enables regulation of polysaccharide utilization genes in response to both the polysaccharide and its breakdown products.ImportanceBreakdown of complex polysaccharides derived from "dietary fiber" is achieved by the mammalian gut microbiota. This breakdown creates a critical nutrient source for both the microbiota and its mammalian host. Because the availability of individual polysaccharides fluctuates with variations in the host diet, members of the microbiota strictly control expression of polysaccharide utilization genes. Our findings define a regulatory architecture that controls the breakdown of a polysaccharide by a gut bacterium in response to three distinct signals. This architecture integrates perception of a complex polysaccharide and its monomeric constituent as well as feedback of central metabolism. Moreover, it is broadly applicable to several prominent members of the mammalian gut microbiota. The identified regulatory strategy may contribute to the abundance of gut Bacteroides, despite fluctuations in the host diet.

Dataset Information

New regulatory gene that contributes to control of Bacteroides thetaiotaomicron starch utilization genes.

Publications

New regulatory gene that contributes to control of Bacteroides thetaiotaomicron starch utilization genes.

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