The pssB gene product of Rhizobium leguminosarum bv. trifolii is homologous to a family of inositol monophosphatases

The Rhizobium leguminosarum bv. trifolii region encoding pssA and pssB genes was cloned. The pssB gene located upstream of the pssA encoded a 28.36-kDa protein which displayed 97.5% identity with the PssB of R. leguminosarum bv. viciae. Inactivation of the pssB gene by insertion of the lacZ-Gmr cassette resulted in the significant increased production of exopolysaccharide in comparison to the wild-type level. A mutant strain was also defective in nitrogen fixation suggesting a regulatory role of pssB in symbiosis with clover.     

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

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.     

The products of Rhizobium genes, psi and pss, which affect exopolysaccharide production, are associated with the bacterial cell surface

Translational fusions between a mutant phoA (lacking its promoter, ribosomal binding site and signal peptide sequence) and Rhizobium 'symbiotic' genes were isolated. Since these fusions expressed alkaline phosphatase (AP), the product of phoA, the genes into which phoA was inserted apparently specify proteins located in the bacterial periplasm or cell membrane, the compartment in which AP has activity. These genes were psiA and genes upstream of psiA (psiA is required for normal nodule development and strains with multicopy psiA fail to make exopolysaccharide (EPS) and to nodulate). Fusions between phoA and pss (exo) genes, which are required for EPS production, also resulted in the expression of AP indicating that products of these pss genes were located at the cell surface. Using gus fusions to psiA and pssA, we found that the former was expressed in N2-fixing bean root nodules but the latter was not.     

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.     

Topological and transcriptional analysis of <Emphasis Type="Italic">pssL</Emphasis> gene product: a putative Wzx-like exopolysaccharide translocase in <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii</Emphasis> TA1

An identified pssL gene is yet another one, besides the pssT, pssN and pssP genes, encoding for a protein engaged in polysaccharide polymerization and export in Rhizobium leguminosarum bv. trifolii strain TA1 (RtTA1). Amino acid sequence similarity and hypothetical protein secondary structure placed the PssL protein within Wzx (RfbX) translocases with putative flippase function that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the PssL membrane protein was examined with a series of PssL-PhoA and PssL-LacZ translational fusions. The results support the hypothesis of PssL being a member of PST protein family comprising transporters with 12 membrane spanning segments and amino and carboxyl termini located in the cytoplasm. Results of semi-quantitative RT-PCR showed that the initial abundance of mRNA encoding PssL protein was relatively lower when compared to the quantity of the previously identified PssT membrane protein. PssL might be a good candidate for Wzx-like protein that together with PssT (Wzy protein) could be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation in RtTA1.