Identification of Sinorhizobium meliloti Genes Influencing Synthesis of Surface Polysaccharides and Competitiveness

Abstract

A new approach to isolating mutants with altered composition of capsular polysaccharides (CPS) and lipopolysaccharides (LPS) in nodule bacteria of alfalfa Sinorhizobium meliloti based on analysis of their respiratory activity was proposed. Random Tn 5mob mutants of symbiotically effective strain SXM1-105 were tested for slime-production ability and coloration on diagnostic media containing the indicator of reducing equivalents, triphenyltetrasolium chloride (TTC), the inhibitor of respiratory activity, 2-methyl-4-chlorphenoxybutyric acid and Congo Red, the stain for LPS and exopolysaccharides (EPS). Electrophoretic analysis (SDS-PAGE) of polysaccharides in seven mutants, labeledly differing from the parental strain with respect to their growth on diagnostic media demonstrated that (1) the production of CPS was either decreased (in mutants T64 and T835) or blocked (in T71, T94, T124, T134, and T170); (2) the R form of LPS had changed mobility (in T134); (3) the S form of LPS contained only one component instead of two (T71). In symbiosis with alfalfa Medicago sativa , all mutants exhibited a decreased competitive ability for adsorption on roots of host alfalfa plant, compared to the parental strain. At early stages of symbiosis, mutants had a tendency toward a decrease in the number of nodules, in comparison with the parental strain (a statistically significant decrease was observed in mutants T71 and T64). In mutants T64, T71, and T134, nodulation competitiveness was lower than in the parental strain. Analysis of Tn 5mob tagged sequences of the mutants revealed homologies with the genes encoding methionine synthase, luciferase-like monooxygenase, UDP-glucuronic acid epimerase, sensor hystidine kinase, acetoacetyl-CoA-reductase, oligopeptide uptake ABC transporter, and a transcription activator.

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

“…on the Diagnostic Media Tn5 mutants were preselected by SE using diagnostic media: medium 79 (g/L: K 2 HPO 4 , 0.5; MgSO 4 ⋅ 7H 2 O, 0.5; NaCl, 0.1; yeast extract 1.0; CaCO 3 traces; manni tol 10.0; pH 7.0) containing 2,3,5 triphenyltetrazo lium chloride (TTC, 0.01%) for the selection of mutants based on respiratory activity (Yurgel et al, 1998); medium 79, containing herbicide 2 methyl 4 chloro phenoxy butyric acid (2M 4XM, 0.1%), which inhib its respiration of bacteria; as well as a minimal medium (g/L: K 2 HPO 4 , 0.5; MgSO 4 ⋅ 7H 2 O, 0.2; NaCl, 0.1; NH 4 NO 3 , 0.1; CaCO 3 traces; mannitol 10.0, pH 7.0) with a Congo red dye (0.02%) for the selection of mutants with altered production of polysaccharides (Onishchuk et al, 2005). The SE of the mutants which showed the altered phenotypes on these media (growth or coloration of the colonies compared to the parental strain CXM1 105) were studied in symbiosis with alfalfa under the conditions of a micro vegetation experiment.…”

Root Nodule Bacteria: Perspectives of Monitoring Symbiotic Properties by Applying Genetic Markers

“…Аналогичные группы fix и eff генов у S. meliloti расположены на мегаплазмидах, а также на хромосоме. Анализ Tn5 мутантов устойчивого к стрептомицину штамма СХМ1-105, который был получен на основе производного штамма 425а, широко использовавшегося для приготовления биопрепаратов в 1980-1990-х годах, позволил впервые выявить 12 новых генов, вовлеченных в контроль симбиотической эффективности (57)(58)(59). Прибавки сухой массы растений люцерны, инокулированных такими транспозантами, составляла от 15 до 34 %.…”

“…Гены, детерминирующие синтез полисахаридов (exo-exp-lps), сосредоточены преимущественно на второй мегаплазмиде, которая не содержит гены nod-niffix. Показано, что мутации по генам синтеза полисахаридов приводят к нарушению микробно-растительного взаимодействия уже на ранних этапах, в результате чего не происходит образования клубеньков (58,67,74,76). Вместе с тем мутации по некоторым из указанных выше групп генов (например, exoB, exoY или lpsL) могут, наоборот, усиливать клубенькообразование (77).…”