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

The pssB gene of Rhizobium leguminosarum by. trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophospha tases. The gene was cloned and overexpressed in Escherichia coli. The purif ied gene product of pssB showed inositol monophosphatase activity with a K- m of 0.23 mM, and a V-max of 3.27 mu mol P-i min(-1) (mg protein)(-1). Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit associ ation (dimerization) were studied and compared to those of other inositol m onophosphatases. Western immunoblotting with anti-PssB antibodies showed th e presence of PssB in R. leguminosarum by. trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A. The presence of PssB protein in R. leguminosarum by. trifolii TA1 was correlated with phosphatase activi ty 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 i nto the wild-type TA1 strain reduced EPS synthesis. The changes in the leve l of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.