Extracellular polysaccharide protects Rhizobium leguminosarum cells against zinc stress in vitro and during symbiosis with clover: EPS protects rhizobial cells against zinc stress

Rhizobium leguminosarum bv. trifolii is a soil bacterium that establishes symbiosis with clover (Trifolium spp.) under nitrogen-limited conditions. This microorganism produces exopolysaccharide (EPS), which plays an important role in symbiotic interactions with the host plant. The aim of the current study was to establish the role of EPS in the response of R. leguminosarum bv. trifolii cells, free-living and during symbiosis, to zinc stress. We show that EPS-deficient mutants were more sensitive to Zn2+ exposure than EPS-producing strains, and that EPS overexpression conferred some protection onto the strains beyond that observed in the wild type. Exposure of the bacteria to Zn2+ ions stimulated EPS and biofilm production, and increased cell hydrophobicity. However, zinc stress negatively affected the motility and attachment of bacteria to clover roots, as well as the symbiosis with the host plant. In the presence of Zn2+ ions, cell viability, root attachment, biofilm formation and symbiotic efficiency of EPS-overproducing strains were significantly higher than those of the EPS-deficient mutants. We conclude that EPS plays an important role in the adaptation of rhizobia to zinc stress, in both the free-living stage and during symbiosis.