Analysis of a capsular polysaccharide biosynthesis locus of Bacteroides fragilis

Abstract

A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar to O-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

FIG. 1

ORF map of a 24,454-bp region of the 9343 chromosome contained on cosmid pMJC2. Restriction sites within this region are shown for the following enzymes: PvuII (P), EcoRI (E), Asp718 (A), StuI (St), SmaI (Sm), SalI (Sl), MluI (M), and Sau3AI/BamHI junction with the pHC79 vector (Sa/B). ΔwcfD-L and ΔwcfF represent the regions that were deleted in the chromosomes of mutants 9343ΔwcfD-L and 9343ΔwcfF. The arrows indicate direction of transcription of each ORF. The ORFs that do not demonstrate homology with genes involved in polysaccharide biosynthesis (outside the wcf locus) are shaded. The lower diagram demonstrates the average G+C content for each ORF of the region. The dashed-line boxes represent a clustering of adjacent ORFs with similar G+C contents. A transcriptional terminator-like stem-loop region downstream of wcfL is indicated.

FIG. 2

Western immunoblot of cell lysates of B. fragilis 9343 and mutants probed with 9343ΔwcfD-L-adsorbed antiserum. The location of the 182-kDa marker is shown.