Identification of new genes of nodule bacteria sinorhizobium meliloti involved in the control of efficiency of symbiosis with alfalfa medicago sativa

Abstract

Background. Alfalfa root nodule bacteria (Sinorhizobium meliloti) are among the most active symbiotic N2-fixers. Their symbiotic efficiency (SE) defined as an ability to enhance the productivity of inoculated host plants is the polygenic trait controlled by a complicated system of genes, inactivation of which can result in either decrease or increase of SE. Analysis of previously identified eff-genes, whose mutations result in SE increase, revealed their location in different parts of genome (chromosome or megaplasmids) and demonstrated that these genes are not involved in operation of nitrogenase system. Mutations in these genes have pleiotropic effects, changing various cultural-biochemical properties of bacteria. Materials and methods. The object of research were the laboratory S. meliloti strain CXM1-105 and its Tn5-mutants with Eff++ phenotype, which are able to grow in diagnostic media containing indicator of cell redox potential ТТC, herbicide 2М-4ХМ or the Congo Red dye. New eff-genes were identified using the modified method of “inverted” PCR. Results. We obtained three Tn5 mutants with an increased SE in which the symbiotic phenotypes are dependent on the host. Two-factor analysis of variance demonstrated that the genotypic difference between mutants is most pronounced under the salt stress, while in its absence SE is determined mostly by the host genotype. Molecular-biological analysis revealed that the T4 mutant harbours the Tn5 insertion in the mtaD gene, T795 - in the thiC gene, and M3 - in the gene which encodes a protein belonging to the GntR family of transcription regulators. Conclusion. We demonstrated firstly that mutations in genes involved in transcription regulation, phosphonate metabolism and thiamine biosynthesis may result in a SE increase. The “inverted” PCR method enabled us to rapidly extract DNA fragments flanking the transposon, which suggests applicability of this method for identification of new rhizobia genes marked by Tn5.

Keywords

Sinorhizobium meliloti / Sinorhizobium meliloti / symbiotic efficiency / competitiveness / alfalfa / genotypic interactions between symbiotic partners / salt stress, method of “inverted” PCR

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RIGHTS & PERMISSIONS

Onishchuk O.P., Kurchak O.N., Chizhevskaya E.P., Andronov E.Y., Simarov B.V.