The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

The aim of this study was investigation of the response of R. leguminosarum bv. trifolii wild-type and its two rosR and pssA mutant strains impaired in exopolysaccharide (EPS) synthesis to oxidative stress conditions caused by two prooxidants: a superoxide anion generator- menadione (MQ) and hydrogen peroxide (H2O2). The levels of enzymatic (catalase, superoxide dismutase, pectinase and β-glucosidase) and non-enzymatic (superoxide anion generator, formaldehyde, phenolic compounds) biomarkers were monitored using biochemical methods in both the supernatants and rhizobial cells after treatment with 0.3 mM MQ and 1.5 mM H2O2. The viability of bacterial cells was estimated using fluorescent dyes and confocal laser scanning microscopy. In addition, the effect of prooxidants on symbiosis of the R. leguminosarum bv. trifolii strains with clover was established. The tested stress factors significantly changed enzymatic patterns of the rhizobial strains, and the wild-type strain proved to be more resistant to these prooxidants than both pssA and rosR mutants. Significantly higher activities of both catalase and superoxide dismutase have been detected in these mutants in comparison to the wild-type strain. H2O2 and MQ also increased the levels of pectinase and β-glucosidase activities in the tested strains. Moreover, pre-incubation of R. leguminosarum bv. trifolii strains with the prooxidants negatively affected the viability of bacterial cells and the number of nodules elicited on clover plants. EPS produced in large amounts by R. leguminosarum bv. trifolii plays a significant protective role as a barrier against oxidative stress factors and during symbiotic interactions with clover plants.