Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a nitrogen-fixing symbiosis with clover plants (Trifolium spp.). This bacterium secretes large amounts of acidic exopolysaccharide (EPS), which plays an essential role in the symbiotic interaction with the host plant. This polymer is biosynthesized by a multi-enzymatic complex located in the bacterial inner membrane, whose components are encoded by a large chromosomal gene cluster, called Pss-I. In this study, we characterize R. leguminosarum bv. trifolii strain Rt297 that harbors a Tn5 transposon insertion located in the pssZ gene from the Pss-I region. This gene codes for a protein that shares high identity with bacterial serine/threonine protein phosphatases. We demonstrated that the pssZ mutation causes pleiotropic effects in rhizobial cells. Strain Rt297 exhibited several physiological and symbiotic defects, such as lack of EPS production, reduced growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. These data indicate that the protein encoded by the pssZ gene is indispensable for EPS synthesis, but also required for proper functioning of R. leguminosarum bv. trifolii cells.