The Rhizobium leguminosarum bv. trifolii pssB gene product is an inositol monophosphatase that influences exopolysaccharide synthesis.

Abstract

The pssB gene of Rhizobium leguminosarum bv. trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophosphatases. The gene was cloned and overexpressed in Escherichia coli. The purified gene product of pssB showed inositol monophosphatase activity with a Km of 0.23 mM, and a Vmax of 3.27 mumol Pi min-1 (mg protein)-1. Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit association (dimerization) were studied and compared to those of other inositol monophosphatases. Western immunoblotting with anti-PssB antibodies showed the presence of PssB in R. leguminosarum bv. trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A. The presence of PssB protein in R. leguminosarum bv. trifolii TA1 was correlated with phosphatase activity with myo-inositol 1-phosphate as a substrate. Evidence for a regulatory function of PssB protein in exopolysaccharide (EPS) synthesis is presented. The mutation in pssB caused EPS overproduction, and introduction of pssB into the wild-type TA1 strain reduced EPS synthesis. The changes in the level of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.