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.

Matching population diversity of rhizobial nodA and legume NFR5 genes in plant–microbe symbiosis

“…The standard PCR protocol was used: initial denaturation at 95°C for 3 min, 30 cycles with denaturation at 94°C for 30 s, primer annealing at 48°C for 30 s, extension at 72°C for 1 min, and final extension for 4 min. PCR fragments were extracted from agarose gel (Onishchuk, Chizhevskaya, Kurchak, Andronov, & Simarov, ) and cloned into the plasmid pTZ57R/T (Thermo Scientific). For each plant species, 100 randomly selected cloned fragments of NFR5 genes were sequenced by Sanger method in an automated ABI 3500xL sequencer (Applied Biosystems) using standard M13 (−20) and (−26) primers.…”

Structural Insight Into the Role of Mutual Polymorphism and Conservatism in the Contact Zone of the NFR5–K1 Heterodimer With the Nod Factor

“…The standard PCR protocol used consisted of initial denaturation at 95°C for 3 min, 30 cycles with denaturation at 94°C for 30 s, primer annealing at 48°C for 30 s, extension at 72°C for 1 min and final extension for 4 min. PCR fragments were extracted from agarose gel (Onishchuk et al, 2015 ) and cloned into the plasmid pTZ57R/T (Thermo Scientific, Lithuania).…”

Supply of acetyl-CoA to N₂-fixing bacteroids: insights from the mutational and proteomic analyses of <em>Sinorhizobium meliloti</em>

“…Identification of Tn5-tagged genes was carried out using the inverted polymerase chain reaction technique (Onishchuk et al, 2014).…”

“…Moreover, activation of phosphoenolpyruvate carboxykinase in bacteroids leads to a 70 % reduction of N 2 -fixing activity compared to wild-type bacteroids (Mulley et al, 2010), suggesting that operation of this enzyme is disadvantageous for the symbiosis operation. Earlier, we revealed in alfalfa rhizobia (Sinorhizobi um meliloti) a number of eff genes whose inactivation by Tn5 insertions leads to an increased symbiotic efficiency (SE) -the impact of bacteria inoculation on host plant productivity (Sharypova et al, 1994;Onishchuk, Sharypova and Simarov, 1994). Among these genes, addressed as the negative regulators of symbiosis (Provorov et al, 2014), several groups were identified as being involved in: (i) accumulation of storage nutrients (glycogen, GABA), which reduces the catabolic activity of bacteroids; (ii) negative control of respiration, which restricts the energy flow to nitrogenase; (iii) synthesis of surface polysaccharides, which elicit host defense reactions, thereby reducing the persistence of the endo-symbiotic bacterial population.…”