Identification of the Mechanisms of Acetoin Production Change in Genetically Altered Vibrio cholerae Strains O1 Biovar El Tor

Abstract

Objective of the study was to determine the mechanisms of acetoin biosynthesis change in genetically altered El Tor V. cholerae strains in Voges-Proskauer test.Materials and methods. We used nine genetically altered V. cholerae O1 strains, biovar El Tor, imported in the territory of the Russian Federation and Ukraine between 1993–2011, and four typical strains isolated in 1970–1972. When assessing acetoin production in V. cholerae strains in Voges-Proskauer test, the strain V. cholerae 569B O1 serogroup, classical biovar served as negative control of the assay. Relative gene expression was studied using real-time RT-PCR. Protein model construction was performed by means of automated server SWISS – MODEL.Results and discussion. It has been demonstrated that diagnostically significant feature – VogesProskauer reaction, utilized for V. cholerae O1 biovar differentiation, is changed in all investigated genetically altered strains of cholera agent, isolated in different periods of the current seventh pandemic (66.7 % of the strains show weakly positive reaction, 33.3 % – negative one). The data obtained testify to the reduction or absence of acetoin production in the investigated strains. Analysis of four structural genes of als operon, as well as expression of regulatory genes alsR and аphA, responsible for acetoin biosynthesis, has revealed that changes in acetoin production in the genovariants stem from the deletion of a single nucleotide (T in the position 315) in the structural gene alsD, encoding acetolaktat decarboxylase, and also from high levels of negative acetoin biosynthesis regulator expression – аphA gene. Modeling of the spatial (3-D) structure of AlsD protein in the genovariant M1293 and the reference-strain N16961 has shown that AlsD protein of the genovariant is, indeed, considerably reduced. However, spontaneous decarboxylation is possible in the absence of acetolaktat decarboxylase, which phenotypically manifests itself in borderline positive Voges-Proskauer test.