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Characterisation of the soluble domain of nitrite reductase from Neisseria meningitidis strains

Abstract number: 1733_305

Stefanelli P., Colotti G., Neri A., Salucci M.L., Miccoli R., Di Leandro L., Ippoliti R.

Objectives: The objective of the study was to characterise the molecular and biochemical properties of the soluble domain of the AniA protein of N. meningitidis serogroup B MC58, and thereafter evaluate genomic conservation and protein expression in a panel of N. meningitidis clinical isolates.

Methods: The soluble domain of the aniA gene of N. meningitidis MC58 was cloned and expressed in E. coli following standard procedures. Nitrite consumption was evaluated by the Griess method at different time points. For the pH dependence, the experiments were performed using different buffers 20 mM acetate buffer in the range 4.5–5.5, 20 mM Hepes buffer in the range 5.5–6.6, and 20 mM phosphate buffer in the range 6.5–7.5. Direct Nitrogen Oxide production was recorded by a specific electrode (WPI, UK). A total of 11 N. meningitidis strains, 7 disease-associated and 4 carriers, were cultured with 5% CO2 at 37°C on Thayer-Martin plates. The aniA sequences were analyzed with the BLAST programme. The antisera was prepared with 1.5 mg of purified protein to immunise New Zealand rabbits, Charles River and used to perform western-blot analysis.

Results: The biochemical results show an ordered mechanism for the catalysis, since the binding of nitrite needed to induce electron transfer from the type I to the type II Cu-site. Furthermore sAniA shows a dependence of the catalytic activity upon acidification. Sequence analysis of the coding region of the gene in 11 N. meningitidis strains showed that, notwithstanding a high degree of gene sequence similarity among them, two gene variants were found due to insertions or deletions. In 1 disease associated strain a 9-residue insertion was located close to the catalytic site. No amplification was obtained in 2 carried strains due to the presence of a gene for a transposase protein. The AniA was expressed in all the strains, except for two with no amplification reaction.

Conclusion: These preliminary results describe the basic biochemical properties of the sAniA from N. meningitidis MC58, in particular its pH dependence. The conservation of the gene among the majority of the examined strains suggests that preservation of its integrity is important for bacterial survival. However, further investigation will be performed to understand how ani A gene variations in the two carrier strains could affect the enzymatic activity of the protein.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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