Orientations of the Bacteroides fragilis Capsular Polysaccharide Biosynthesis Locus Promoters during Symbiosis and Infection

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

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Orientations of the Bacteroides fragilis Capsular Polysaccharide Biosynthesis Locus Promoters during Symbiosis and Infection

Journal of Bacteriology ◽

10.1128/jb.00555-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5832-5836 ◽

Cited By ~ 13

Author(s):

Erin B. Troy ◽

Vincent J. Carey ◽

Dennis L. Kasper ◽

Laurie E. Comstock

Keyword(s):

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

Polysaccharide Biosynthesis

ABSTRACT Orientations of the seven invertible polysaccharide biosynthesis locus promoters of B acteroides fragilis were determined from bacteria grown in vitro, from feces of monoassociated and complex colonized mice, and from B. fragilis-induced murine abscesses. Bacteria grown in vivo have greater variability in orientation of polysaccharide locus promoters than culture-grown organisms.

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Genetic Diversity of the Capsular Polysaccharide C Biosynthesis Region of Bacteroides fragilis

Infection and Immunity ◽

10.1128/iai.68.11.6182-6188.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6182-6188 ◽

Cited By ~ 12

Author(s):

Laurie E. Comstock ◽

Annalisa Pantosti ◽

Dennis L. Kasper

Keyword(s):

Genetic Diversity ◽

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

Hybridization Analysis ◽

Clinical Isolates ◽

Pcr Analysis ◽

Capsular Polysaccharides ◽

Genetic Approach ◽

Polysaccharide Biosynthesis ◽

Putative Aminotransferase

ABSTRACT A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus ofBacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. OnlycfiA + B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs ofwcgC (N-acetylmannosamine dehydrogenase),wcfF (putative dehydrogenase), and wcgP(putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.

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Interstrain Variation of the Polysaccharide B Biosynthesis Locus of Bacteroides fragilis: Characterization of the Region from Strain 638R

Journal of Bacteriology ◽

10.1128/jb.181.19.6192-6196.1999 ◽

1999 ◽

Vol 181 (19) ◽

pp. 6192-6196 ◽

Cited By ~ 13

Author(s):

Laurie E. Comstock ◽

Michael J. Coyne ◽

Arthur O. Tzianabos ◽

Dennis L. Kasper

Keyword(s):

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

Polysaccharide Biosynthesis

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.

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Bacteroides fragilis Synthesizes a DNA Invertase Affecting both a Local and a Distant Region

Journal of Bacteriology ◽

10.1128/jb.01362-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 2119-2124 ◽

Cited By ~ 12

Author(s):

Hazeline Roche-Hakansson ◽

Maria Chatzidaki-Livanis ◽

Michael J. Coyne ◽

Laurie E. Comstock

Keyword(s):

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

The Other ◽

Site Specific ◽

Polysaccharide Biosynthesis ◽

Its Gene ◽

Distant Region ◽

Conserved Tyrosine

ABSTRACT The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of Bacteroides fragilis was characterized. Its gene, tsr19, is adjacent to mpi, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of B. fragilis, Tsr19 brings about inversion of two DNA regions, one local and one located distantly.

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Analysis of a Capsular Polysaccharide Biosynthesis Locus of Bacteroides fragilis

Infection and Immunity ◽

10.1128/iai.67.7.3525-3532.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3525-3532 ◽

Cited By ~ 40

Author(s):

Laurie E. Comstock ◽

Michael J. Coyne ◽

Arthur O. Tzianabos ◽

Annalisa Pantosti ◽

Andrew B. Onderdonk ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Bacteroides Fragilis ◽

Open Reading Frames ◽

Capsular Polysaccharides ◽

Nucleotide Sugar ◽

Polysaccharide Biosynthesis ◽

Abdominal Abscesses ◽

Intra Abdominal Infection ◽

Reading Frames ◽

Nucleotide Sugar Biosynthesis

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 toO-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.

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