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

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 (H₂O₂).

Methods

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.

Results

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.

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 Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships.     

Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation

Strain ANU1173 is an acid-tolerant Rhizobium leguminosarum biovar trifolii strain that is able to nodulate subterranean clover plants growing in agar culture at pH 4.4 At pH 6.5, its symbiotic effectiveness in association with Trifolium subterraneum cv. Mt. Barker was 80% relative to that of strain ANU794, a Sm^r derivative of the commercial inoculant R. leguminosarum bv. trifolii TA1. Strain ANU1173 contained four indigenous megaplasmids, the smallest of these being the symbiotic (Sym) plasmid. The critical pH requirement for growth of strain ANU1173 in laboratory media was shown not to be associated with this plasmid. When the Sym plasmid of strain ANU1173(pSym-1173) was mobilized into a Nod^- strain of R. leguminosarum bv. viciae, the plasmid conferred to the transconjugant a level of symbiotic effectiveness in association with T. subterraneum that was similar to that observed with ANU1173. The symbiotic effectiveness of strain ANU1173 was improved by first curing pSym-1173 (generating strain ANU1184) and replacing it with another R. leguminosarum bv. trifolii Sym plasmid, pBR1AN. Subterranean clover plants inoculated with strain ANU1184 (pBR1AN) exhibited a 35 or 53% increase in acetylene reduction activity and a 20 or 17% increase in dry weight when grown at pH 6.5 and pH 4.4, respectively, compared with plants inoculated with strain ANU1173 and grown under the same pH conditions. It was further shown that pBR1AN was stably maintained in strain ANU1184 under free-living and symbiotic conditions. These results indicate that it is possible to construct an acid-tolerant strain of R. leguminosarum bv. trifolii with an enhanced capacity for nitrogen fixation.     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

Characterization of Rhizobial Isolates of Phaseolus vulgaris by Staircase Electrophoresis of Low-Molecular-Weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.