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

Abstract

Aims 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 (H 2 O 2 ). Methods The levels of enzymatic (catalase, superoxide dismutase, pectinase and β-glucosidase) and nonenzymatic (superoxide anion generator, formaldehyde, phenolic compounds) biomarkers were monitored using biochemical methods in both the supernatants and rhizobial cells after treatment with 0.3mM MQ and 1.5mM H 2 O 2 . 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. Results The tested stress factors significantly changed enzymatic patterns of the rhizobial strains, and the wildtype 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 wildtype strain. H 2 O 2 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. Conclusions 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.

Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

“…However, rhizobia must survive often a long period of time in the soil as free-living organisms until they meet their symbiotic partner. During this period, they are exposed to the action of several environmental factors (e.g., drought, pH, temperature, salinity, nutrient limitation, heavy metals, oxidative stress) (Frey and Blum 1994;R s nen et al 2004;Soussou et al 2013;Talbi et al 2013;Suzuki et al 2014;Cazenave et al 2014;Pini et al 2014;Burchill et al 2014;Barthelemy-Delaux et al 2014;Jaszek et al 2014). Extracellular polysaccharide secreted in large amounts by rhizobial cells plays a significant protective role in their adaptation to stress conditions.…”

“…Several functions are ascribed to rhizobial EPS in symbiosis such as facilitation of bacterial adhesion to and invasion of host roots, a structural function as a component of matrix inside infection threads, and protection against plant defense reactions (Downie 2010;Cheng and Walker 1998;Jaszek et al 2014). D'Haeze et al (2004) indicated for Azorhizobium caulinodans that a mutant strain producing significantly less EPS that the wild-type bacterium was more sensitive to oxidative stress and induced only ineffective pseudonodules on its host plant, Sesbania rostrata.…”

“…Diana) were surface sterilized as described previously (Jaszek et al 2014). Next, the seeds were placed on plates with Fåhraeus agar (Vincent 1970), incubated for 2-3 days at 22°C, and seedlings were subsequently placed onto Fåhraeus slants.…”

“…ensures adaptation of bacterial cells to changing environmental conditions (Fujishige et al 2006;Flemming and Wingender 2010;Jaszek et al 2014;Janczarek et al 2015a). In the case of soil nitrogen-fixing bacteria, which are able to establish symbiotic interactions with legumes, EPS is also indispensable for effective infection and nodulation of many host plants (e.g., Medicago, Trifolium, Pisum, Vicia spp.)…”

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

“…clover, pea, vetch, and alfalfa) (Rolfe et al 1996;Becker and Pühler 1998;Janczarek and Urbanik-Sypniewska 2013). This polysaccharide is indispensable for protection against host plant defence reactions and initiation and elongation of infection threads, special tubular structures via which rhizobia colonize root nodules (Cheng and Walker 1998;Jaszek et al 2014). EPS-deficient mutant strains of R. leguminosarum bvs.…”