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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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PCR-RFLP analysis of Brucella LPS genes manAO-Ag, manBO-Ag, wbkD, wbkF, wboA and wa**. Panel A. Lanes: 1, molecular size markers; 2, manAO-Ag from B. melitensis 16 M uncut; 3, manAO-Ag from B. melitensis 16 M cut by AvaII; 4, manAO-Ag from B. neotomae cut by AvaII; 5, wbkF from B. melitensis 16 M uncut; 6, wbkF from B. melitensis 16 M cut by AluI; 7, wbkF from B. melitensis bv2 cut by AluI; 8, wbkF from B. abortus bv2 cut by AluI; 9, wbkF2* from B. melitensis 16 M uncut; 10, wbkF2* from B. canis uncut; 11, wbkF2* from B. melitensis 16 M cut by EcoRV; 12, wbkF2* from B. canis cut by EcoRV; 13, wboA from B. melitensis 16 M uncut; 14, wboA from B. melitensis 16 M cut by AluI; 15, wboA from B. abortus cut by AluI; 16, wa** from B. melitensis 16 M uncut; 17, wa** from B. melitensis 16 M cut by AvaII; 18, wa** from B. suis bv2 cut by AvaII; 19, wa** from B. melitensis 16 M cut by HinfI; 20, wa** from B. ovis cut by HinfI. Panel B. Lanes: 1, molecular size markers; 2, manBO-Ag from B. melitensis 16 M uncut; 3, manBO-Ag from B. pinnipedialis uncut; 4, manBO-Ag from B. melitensis 16 M cut by Sau3A; 5, manBO-Ag from B. melitensis bv2 cut by Sau3A; 6, manBO-Ag from B. abortus cut by Sau3A; 7, manBO-Ag from B. suis cut by Sau3A; 8, manBO-Ag from B. suis bv2 cut by Sau3A; 9, manBO-Ag from B. ovis cut by Sau3A; 10, manBO-Ag from B. pinnipedialis cut by Sau3A; 11, manBO-Agfrom B. ceti cut by Sau3A; 12, manBO-Agfrom B. melitensis 16 M cut by EcoRV; 13, manBO-Ag from B. abortus cut by EcoRV. Panel C. Lanes: 1, molecular size markers; 2, wbkD from B. melitensis 16 M uncut; 3, wbkD from B. abortus uncut; 4, wbkD from B. canis uncut; 5, wbkD from B. melitensis 16 M cut by Sau3A; 6, wbkD from B. abortus cut by Sau3A; 7, wbkD from B. canis cut by Sau3A.
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Figure 3: PCR-RFLP analysis of Brucella LPS genes manAO-Ag, manBO-Ag, wbkD, wbkF, wboA and wa**. Panel A. Lanes: 1, molecular size markers; 2, manAO-Ag from B. melitensis 16 M uncut; 3, manAO-Ag from B. melitensis 16 M cut by AvaII; 4, manAO-Ag from B. neotomae cut by AvaII; 5, wbkF from B. melitensis 16 M uncut; 6, wbkF from B. melitensis 16 M cut by AluI; 7, wbkF from B. melitensis bv2 cut by AluI; 8, wbkF from B. abortus bv2 cut by AluI; 9, wbkF2* from B. melitensis 16 M uncut; 10, wbkF2* from B. canis uncut; 11, wbkF2* from B. melitensis 16 M cut by EcoRV; 12, wbkF2* from B. canis cut by EcoRV; 13, wboA from B. melitensis 16 M uncut; 14, wboA from B. melitensis 16 M cut by AluI; 15, wboA from B. abortus cut by AluI; 16, wa** from B. melitensis 16 M uncut; 17, wa** from B. melitensis 16 M cut by AvaII; 18, wa** from B. suis bv2 cut by AvaII; 19, wa** from B. melitensis 16 M cut by HinfI; 20, wa** from B. ovis cut by HinfI. Panel B. Lanes: 1, molecular size markers; 2, manBO-Ag from B. melitensis 16 M uncut; 3, manBO-Ag from B. pinnipedialis uncut; 4, manBO-Ag from B. melitensis 16 M cut by Sau3A; 5, manBO-Ag from B. melitensis bv2 cut by Sau3A; 6, manBO-Ag from B. abortus cut by Sau3A; 7, manBO-Ag from B. suis cut by Sau3A; 8, manBO-Ag from B. suis bv2 cut by Sau3A; 9, manBO-Ag from B. ovis cut by Sau3A; 10, manBO-Ag from B. pinnipedialis cut by Sau3A; 11, manBO-Agfrom B. ceti cut by Sau3A; 12, manBO-Agfrom B. melitensis 16 M cut by EcoRV; 13, manBO-Ag from B. abortus cut by EcoRV. Panel C. Lanes: 1, molecular size markers; 2, wbkD from B. melitensis 16 M uncut; 3, wbkD from B. abortus uncut; 4, wbkD from B. canis uncut; 5, wbkD from B. melitensis 16 M cut by Sau3A; 6, wbkD from B. abortus cut by Sau3A; 7, wbkD from B. canis cut by Sau3A.

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)

PCR-RFLP analysis of Brucella LPS genes manAO-Ag, manBO-Ag, wbkD, wbkF, wboA and wa**. Panel A. Lanes: 1, molecular size markers; 2, manAO-Ag from B. melitensis 16 M uncut; 3, manAO-Ag from B. melitensis 16 M cut by AvaII; 4, manAO-Ag from B. neotomae cut by AvaII; 5, wbkF from B. melitensis 16 M uncut; 6, wbkF from B. melitensis 16 M cut by AluI; 7, wbkF from B. melitensis bv2 cut by AluI; 8, wbkF from B. abortus bv2 cut by AluI; 9, wbkF2* from B. melitensis 16 M uncut; 10, wbkF2* from B. canis uncut; 11, wbkF2* from B. melitensis 16 M cut by EcoRV; 12, wbkF2* from B. canis cut by EcoRV; 13, wboA from B. melitensis 16 M uncut; 14, wboA from B. melitensis 16 M cut by AluI; 15, wboA from B. abortus cut by AluI; 16, wa** from B. melitensis 16 M uncut; 17, wa** from B. melitensis 16 M cut by AvaII; 18, wa** from B. suis bv2 cut by AvaII; 19, wa** from B. melitensis 16 M cut by HinfI; 20, wa** from B. ovis cut by HinfI. Panel B. Lanes: 1, molecular size markers; 2, manBO-Ag from B. melitensis 16 M uncut; 3, manBO-Ag from B. pinnipedialis uncut; 4, manBO-Ag from B. melitensis 16 M cut by Sau3A; 5, manBO-Ag from B. melitensis bv2 cut by Sau3A; 6, manBO-Ag from B. abortus cut by Sau3A; 7, manBO-Ag from B. suis cut by Sau3A; 8, manBO-Ag from B. suis bv2 cut by Sau3A; 9, manBO-Ag from B. ovis cut by Sau3A; 10, manBO-Ag from B. pinnipedialis cut by Sau3A; 11, manBO-Agfrom B. ceti cut by Sau3A; 12, manBO-Agfrom B. melitensis 16 M cut by EcoRV; 13, manBO-Ag from B. abortus cut by EcoRV. Panel C. Lanes: 1, molecular size markers; 2, wbkD from B. melitensis 16 M uncut; 3, wbkD from B. abortus uncut; 4, wbkD from B. canis uncut; 5, wbkD from B. melitensis 16 M cut by Sau3A; 6, wbkD from B. abortus cut by Sau3A; 7, wbkD from B. canis cut by Sau3A.
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Figure 3: PCR-RFLP analysis of Brucella LPS genes manAO-Ag, manBO-Ag, wbkD, wbkF, wboA and wa**. Panel A. Lanes: 1, molecular size markers; 2, manAO-Ag from B. melitensis 16 M uncut; 3, manAO-Ag from B. melitensis 16 M cut by AvaII; 4, manAO-Ag from B. neotomae cut by AvaII; 5, wbkF from B. melitensis 16 M uncut; 6, wbkF from B. melitensis 16 M cut by AluI; 7, wbkF from B. melitensis bv2 cut by AluI; 8, wbkF from B. abortus bv2 cut by AluI; 9, wbkF2* from B. melitensis 16 M uncut; 10, wbkF2* from B. canis uncut; 11, wbkF2* from B. melitensis 16 M cut by EcoRV; 12, wbkF2* from B. canis cut by EcoRV; 13, wboA from B. melitensis 16 M uncut; 14, wboA from B. melitensis 16 M cut by AluI; 15, wboA from B. abortus cut by AluI; 16, wa** from B. melitensis 16 M uncut; 17, wa** from B. melitensis 16 M cut by AvaII; 18, wa** from B. suis bv2 cut by AvaII; 19, wa** from B. melitensis 16 M cut by HinfI; 20, wa** from B. ovis cut by HinfI. Panel B. Lanes: 1, molecular size markers; 2, manBO-Ag from B. melitensis 16 M uncut; 3, manBO-Ag from B. pinnipedialis uncut; 4, manBO-Ag from B. melitensis 16 M cut by Sau3A; 5, manBO-Ag from B. melitensis bv2 cut by Sau3A; 6, manBO-Ag from B. abortus cut by Sau3A; 7, manBO-Ag from B. suis cut by Sau3A; 8, manBO-Ag from B. suis bv2 cut by Sau3A; 9, manBO-Ag from B. ovis cut by Sau3A; 10, manBO-Ag from B. pinnipedialis cut by Sau3A; 11, manBO-Agfrom B. ceti cut by Sau3A; 12, manBO-Agfrom B. melitensis 16 M cut by EcoRV; 13, manBO-Ag from B. abortus cut by EcoRV. Panel C. Lanes: 1, molecular size markers; 2, wbkD from B. melitensis 16 M uncut; 3, wbkD from B. abortus uncut; 4, wbkD from B. canis uncut; 5, wbkD from B. melitensis 16 M cut by Sau3A; 6, wbkD from B. abortus cut by Sau3A; 7, wbkD from B. canis cut by Sau3A.
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