A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem - PubMed (original) (raw)

A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem

L V Hooper et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Little is known about how members of the indigenous microflora interact with their mammalian hosts to establish mutually beneficial relationships. We have used a gnotobiotic mouse model to show that Bacteroides thetaiotaomicron, a component of the intestinal microflora of mice and humans, uses a repressor, FucR, as a molecular sensor of L-fucose availability. FucR coordinates expression of an operon encoding enzymes in the L-fucose metabolic pathway with expression of another locus that regulates production of fucosylated glycans in intestinal enterocytes. Genetic and biochemical studies indicate that FucR does this by using fucose as an inducer at one locus and as a corepressor at the other locus. Coordinating this commensal's immediate nutritional requirements with production of a host-derived energy source is consistent with its need to enter and persist within a competitive ecosystem.

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Figures

Figure 1

Figure 1

B. thetaiotaomicron

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-fucose-utilization gene cluster. (A) ORFs and the site of Tn4351 insertion. The bacterial

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-fucose metabolic pathway is shown. The product of an ORF terminating 177 bp upstream of fucR shows homology to the 30S ribosomal protein S16 from several species. Another ORF beginning 187 bp downstream of fucP shares homology with several RNA polymerase σ factors. The GenBank accession number for this 10671-bp locus is AF137263. (B) Amino acid sequence of FucR. The underlined region encompasses a helix–turn–helix DNA binding region conserved in GntR family members.

Figure 2

Figure 2

FucR functions as a repressor of the fucRIAK operon. (A) Transcription reporter assay. Strains BT7 (wild-type background) and BT11(_fucR_∷pGERM background) contain an integrated chromosomal copy of GUS fused to 422 bp of sequence that is normally located directly upstream of the fucR start codon. Note that integration produces a duplication of the 422-bp element, with one copy linked to GUS and the other copy remaining at its endogenous location. GUS mRNA was detected by RNase protection, and signals were compared with those obtained by using a probe to 30S ribosomal protein S16 (rp16) mRNA. The results are representative of three independent experiments. (B) FucR expression. Strains were grown to mid-logarithmic phase on defined medium, with or without

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-fucose. Soluble cellular proteins (10 μg per lane) were separated by SDS/PAGE. Western blots were prepared and probed with a rabbit antiserum generated against purified FucR. (C) Assays of cellular

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-fucose isomerase activity from mid-logarithmic phase cultures of isogenic strains. (D) Evidence that fucA and fucK are expressed cotranscriptionally with fucRI. The shuttle vector pLYL01 containing fucRIAK rescues the ability of the _fucI_∷pGERM strain to grow on defined medium containing

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-fucose, whereas fucRI does not. (E)

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-Fucose permease activity in cells recovered from mid-logarithmic phase cultures of wild-type and mutant strains.

Figure 3

Figure 3

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-Fucose binds to FucR and causes it to dissociate from the fucose pathway promoter. (A) Gel mobility-shift assay. A 32P-labeled DNA fragment, corresponding to the 283 bp of sequence 5′ to the initiator Met codon of fucR, was incubated with 30 ng of purified FucR and 1 mM of

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-fucose,

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-fuculose,

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-fuculose-1-phosphate,

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-glucose, or

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-rhamnose. (B) Determination of

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-fucose binding affinity by ligand-dependent quenching of FucR’s intrinsic fluorescence spectrum (see Materials and Methods).

Figure 4

Figure 4

Regulation of Fucα1,2Gal-containing glycan production in the ileum of germ-free NMRI mice. (A) Representative whole-mount preparations of ileum obtained 10 days after inoculation of the indicated strain and stained with peroxidase-conjugated UEA-I (brown). Villi appear as finger-like projections. (B). Model for the coordinate regulation of bacterial fucose metabolism with host fucosylated glycan production.

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-fucose acts through FucR both as the inducer of transcription of fucRIAK and as a corepressor of transcription at csp, which is directly or indirectly responsible for production of a bacterial signal that results in increased production of host fucosylated glycoconjugates.

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