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.

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 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 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 Polysaccharide Biosynthesis Locus Required for Virulence of Bacteroides fragilis

2001 ◽  
Vol 69 (7) ◽  
pp. 4342-4350 ◽  
Author(s):  
Michael J. Coyne ◽  
Arthur O. Tzianabos ◽  
Benjamin C. Mallory ◽  
Vincent J. Carey ◽  
Dennis L. Kasper ◽  
...  
Keyword(s):  
Free Amino ◽  
Animal Experiments ◽  
Bacteroides Fragilis ◽  
Minor Component ◽  
Amino Sugar ◽  
Abscess Formation ◽  
Pcr Analysis ◽  
Capsular Polysaccharides ◽  
Polysaccharide Biosynthesis ◽  
Abdominal Abscesses

ABSTRACT Bacteroides fragilis, though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses. B. fragilis 9343 expresses at least three capsular polysaccharides—polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50 B. fragilis isolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature of B. fragilis.

scholarly journals Bacteroides fragilis strains express multiple capsular polysaccharides.

1993 ◽  
Vol 31 (7) ◽  
pp. 1850-1855 ◽  
Author(s):  
A Pantosti ◽  
A O Tzianabos ◽  
B G Reinap ◽  
A B Onderdonk ◽  
D L Kasper
Keyword(s):  
Bacteroides Fragilis ◽  
Capsular Polysaccharides

scholarly journals A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1795
Author(s):  
Markus B. Tomek ◽  
Bettina Janesch ◽  
Matthias L. Braun ◽  
Manfred Taschner ◽  
Rudolf Figl ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Host Colonization ◽  
Acceptor Specificity ◽  
Hexose Sugars ◽  
Full Structure ◽  
Bacteroidetes Phylum ◽  
General Protein

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Capsular Polysaccharides and Lipopolysaccharides from Two Strains of Bacteroides fragilis

1984 ◽  
Vol 6 (Supplement_1) ◽  
pp. S25-S29 ◽  
Author(s):  
Dennis L. Kasper ◽  
Alf A. Lindberg ◽  
Andrej Weintraub ◽  
Andrew B. Onderdonk ◽  
Jörgen Lönngren
Keyword(s):  
Bacteroides Fragilis ◽  
Capsular Polysaccharides

scholarly journals Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A ofBacillus subtilis

2000 ◽  
Vol 182 (24) ◽  
pp. 6975-6982 ◽  
Author(s):  
Janet K. Hatt ◽  
Philip Youngman
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Response Regulator ◽  
Gene Activation ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Specific Gene ◽  
Dna Binding Domain ◽  
Conserved Residues

ABSTRACT The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-formingBacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

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 The Oncogenic Potential of the Pax3-FKHR Fusion Protein Requires the Pax3 Homeodomain Recognition Helix but Not the Pax3 Paired-Box DNA Binding Domain

1999 ◽  
Vol 19 (1) ◽  
pp. 594-601 ◽  
Author(s):  
Paula Y. P. Lam ◽  
Jack E. Sublett ◽  
Andrew D. Hollenbach ◽  
Martine F. Roussel
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Chromosomal Translocation ◽  
Cellular Transformation ◽  
Small Region ◽  
Transactivation Domain ◽  
Recognition Helix

ABSTRACT The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third α-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

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