scholarly journals Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1039-1049 ◽  
Author(s):  
Sheila Patrick ◽  
Simon Houston ◽  
Zubin Thacker ◽  
Garry W. Blakely
Keyword(s):  
Capsular Polysaccharide ◽  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Gene Clusters ◽  
Opportunistic Pathogen ◽  
Extracellular Polysaccharides ◽  
Phase Variable ◽  
Multiple Gene ◽  
Polysaccharide Biosynthesis ◽  
Group 1

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.

scholarly journals Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 4170-4183 ◽  
Author(s):  
Hung-Yu Shu ◽  
Chang-Phone Fung ◽  
Yen-Ming Liu ◽  
Keh-Ming Wu ◽  
Ying-Tsong Chen ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Sequence Data ◽  
Gene Clusters ◽  
Epidemiological Studies ◽  
Clinical Isolates ◽  
Polysaccharide Biosynthesis ◽  
Bacterial Surface ◽  
Polysaccharide Synthesis ◽  
Hospital Acquired

Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 5′ end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 3′ end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.

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

2010 ◽  
Vol 192 (21) ◽  
pp. 5832-5836 ◽  
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.

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

2000 ◽  
Vol 68 (11) ◽  
pp. 6182-6188 ◽  
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.

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

1999 ◽  
Vol 181 (19) ◽  
pp. 6192-6196 ◽  
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.

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

2006 ◽  
Vol 189 (5) ◽  
pp. 2119-2124 ◽  
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.

scholarly journals Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

2006 ◽  
Vol 188 (5) ◽  
pp. 1987-1998 ◽  
Author(s):  
Maria Chatzidaki-Livanis ◽  
Melissa K. Jones ◽  
Anita C. Wright
Keyword(s):  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Allelic Variation ◽  
Phase Variation ◽  
Phase Variable ◽  
Deletion Mutations ◽  
Underlying Illness ◽  
Dna Elements ◽  
Repetitive Dna Elements ◽  
Group 1

ABSTRACT Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show “phase variation” to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.

scholarly journals Analysis of a Capsular Polysaccharide Biosynthesis Locus of Bacteroides fragilis

1999 ◽  
Vol 67 (7) ◽  
pp. 3525-3532 ◽  
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.

scholarly journals A Novel Outer Membrane Protein, Wzi, Is Involved in Surface Assembly of the Escherichia coli K30 Group 1 Capsule

2003 ◽  
Vol 185 (19) ◽  
pp. 5882-5890 ◽  
Author(s):  
Andrea Rahn ◽  
Kostantinos Beis ◽  
James H. Naismith ◽  
Chris Whitfield
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Signal Sequence ◽  
Surface Group ◽  
Gene Clusters ◽  
Capsule Assembly ◽  
Free Material ◽  
Group 1

ABSTRACT Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a β-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.

scholarly journals A Family of Transcriptional Antitermination Factors Necessary for Synthesis of the Capsular Polysaccharides of Bacteroides fragilis

2009 ◽  
Vol 191 (23) ◽  
pp. 7288-7295 ◽  
Author(s):  
Maria Chatzidaki-Livanis ◽  
Michael J. Coyne ◽  
Laurie E. Comstock
Keyword(s):  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Small Region ◽  
Capsular Polysaccharides ◽  
Single Strain ◽  
Conserved Residues ◽  
Polysaccharide Biosynthesis ◽  
Transcriptional Termination ◽  
Amino Acid Segment ◽  
By Products

ABSTRACT A single strain of Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, designated PSA to PSH. These polysaccharides are synthesized by-products encoded by eight separate polysaccharide biosynthesis loci. The genetic architecture of each of these eight loci is similar, including the fact that the first gene of each locus is a paralog of the first gene of each of the other PS loci. These proteins are designated the UpxY family, where x is replaced by a to h, depending upon the polysaccharide locus from which it is produced. Mutational analysis of three separate upxY genes demonstrated that they are necessary and specific for transcription of their respective polysaccharide biosynthesis operon and that they function in trans. Transcriptional reporter constructs, reverse transcriptase PCR, and deletion analysis demonstrated that the UpxYs do not affect initiation of transcription, but rather prevent premature transcriptional termination within the 5′ untranslated region between the promoter and the upxY gene. The UpxYs have conserved motifs that are present in NusG and NusG-like proteins. Mutation of two conserved residues within the conserved KOW motif abrogated UpaY activity, further confirming that these proteins belong to the NusG-like (NusGSP) family. Alignment of highly similar UpxYs led to the identification of a small region of these proteins predicted to confer specificity for their respective loci. Construction of an upaY-upeY hybrid that produced a protein in which a 17-amino-acid segment of UpaY was changed to that of UpeY altered UpaY's specificity, as it was now able to function in transcriptional antitermination of the PSE biosynthesis operon.

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