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DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers.

Zygmunt MS, Blasco JM, Letesson JJ, Cloeckaert A, Moriyón I - BMC Microbiol. (2009)

Bottom Line: Although most genes were highly conserved, species- and biovar-specific restriction patterns were found.Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization.The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly, France. mzygmunt@tours.inra.fr

ABSTRACT

Background: The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide.

Results: The polymorphism of O-polysaccharide genes wbkE, manA(O-Ag), manB(O-Ag), manC(O-Ag), wbkF and wbkD) and wbo (wboA and wboB), and core genes manB(core) and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manB(O-Ag) carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism.

Conclusion: The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

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Related in: MedlinePlus

Restriction maps of the core- and O-polysaccharide genes with the restriction enzymes used. For each gene, restriction map A corresponds to that deduced from the nucleotide sequence of B. melitensis 16 M. Only differences compared to the nucleotide sequences of B. melitensis 16 M are indicated in restriction maps B, C, D, E, F and G. The restriction patterns A, B, C, D, E, F and G are further indicated in Table 1 for each gene and for each Brucella strain studied. Additional sites and their most probable location according to restriction patterns are indicated by the restriction name (e.g. Hf) and by the position name and an asterisk.
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Figure 2: Restriction maps of the core- and O-polysaccharide genes with the restriction enzymes used. For each gene, restriction map A corresponds to that deduced from the nucleotide sequence of B. melitensis 16 M. Only differences compared to the nucleotide sequences of B. melitensis 16 M are indicated in restriction maps B, C, D, E, F and G. The restriction patterns A, B, C, D, E, F and G are further indicated in Table 1 for each gene and for each Brucella strain studied. Additional sites and their most probable location according to restriction patterns are indicated by the restriction name (e.g. Hf) and by the position name and an asterisk.

Mentions: B. neotomae had a distinct manAO-Ag restriction pattern consisting of an additional AvaII site (Figures 2 and 3, Table 1). Moreover, in silico analysis showed a specific profile for B. ovis consisting of a nucleotide substitution (GAA to GGA) at position 497 which modified the ManA C-terminal sequence at amino acid 165 (not shown). Also, a single nucleotide deletion (CAAT to CA-T) was detected at position 738; this frame shift leads to a change in amino acid sequence after position 246. Nucleotide sequence of PCR products from several strains confirmed the deletion in manAO-Ag as characteristic of B. ovis (not shown).


DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers.

Zygmunt MS, Blasco JM, Letesson JJ, Cloeckaert A, Moriyón I - BMC Microbiol. (2009)

Restriction maps of the core- and O-polysaccharide genes with the restriction enzymes used. For each gene, restriction map A corresponds to that deduced from the nucleotide sequence of B. melitensis 16 M. Only differences compared to the nucleotide sequences of B. melitensis 16 M are indicated in restriction maps B, C, D, E, F and G. The restriction patterns A, B, C, D, E, F and G are further indicated in Table 1 for each gene and for each Brucella strain studied. Additional sites and their most probable location according to restriction patterns are indicated by the restriction name (e.g. Hf) and by the position name and an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698832&req=5

Figure 2: Restriction maps of the core- and O-polysaccharide genes with the restriction enzymes used. For each gene, restriction map A corresponds to that deduced from the nucleotide sequence of B. melitensis 16 M. Only differences compared to the nucleotide sequences of B. melitensis 16 M are indicated in restriction maps B, C, D, E, F and G. The restriction patterns A, B, C, D, E, F and G are further indicated in Table 1 for each gene and for each Brucella strain studied. Additional sites and their most probable location according to restriction patterns are indicated by the restriction name (e.g. Hf) and by the position name and an asterisk.
Mentions: B. neotomae had a distinct manAO-Ag restriction pattern consisting of an additional AvaII site (Figures 2 and 3, Table 1). Moreover, in silico analysis showed a specific profile for B. ovis consisting of a nucleotide substitution (GAA to GGA) at position 497 which modified the ManA C-terminal sequence at amino acid 165 (not shown). Also, a single nucleotide deletion (CAAT to CA-T) was detected at position 738; this frame shift leads to a change in amino acid sequence after position 246. Nucleotide sequence of PCR products from several strains confirmed the deletion in manAO-Ag as characteristic of B. ovis (not shown).

Bottom Line: Although most genes were highly conserved, species- and biovar-specific restriction patterns were found.Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization.The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly, France. mzygmunt@tours.inra.fr

ABSTRACT

Background: The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide.

Results: The polymorphism of O-polysaccharide genes wbkE, manA(O-Ag), manB(O-Ag), manC(O-Ag), wbkF and wbkD) and wbo (wboA and wboB), and core genes manB(core) and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manB(O-Ag) carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism.

Conclusion: The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

Show MeSH
Related in: MedlinePlus