Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R

Abstract

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

FIG. 1

Comparison of the PS B1 and PS B2 biosynthesis regions of B. fragilis NCTC 9343 and 638R. The direction of transcription of each ORF (boxes) is designated with an arrow. The ORFs that do not demonstrate homology with genes involved in polysaccharide biosynthesis are shaded. The area between the diagonal lines indicates where the two chromosomes diverge. Two stem-loop regions that may serve to terminate transcription in the 638R region are indicated, as are the locations of primers used to amplify wcgC and wcgD for complementation studies. The average G+C content of each ORF of the 638R region is indicated by a black bar. Cosmid clones and a PCR product used as sequencing templates are shown.

FIG. 2

Western immunoblot analysis of E. coli AB1133 and ECA mutants 21546 and 21566 containing B. fragilis wcg DNA in trans. Bacterial cultures were grown to an optical density at 600 nm of 0.8, the bacteria were pelleted by centrifugation and lysed with 1× loading buffer, and volumes of bacteria equivalent to 40 μl of culture were added to each well of the sodium dodecyl sulfate–12% polyacrylamide gel, transferred to a nylon membrane, and probed with MAb 898 (ECA specific). Lanes: 1, AB1133; 2, 21546 (pUC19); 3, 21546(pWCGC); 4, 21546(pWCGD); 5, 21546(pWCGCD); 6, 21566(pUC19); 7, 21566(pWCGC); 8, 21566(pWCGD); 9, 21566(pWCGCD).