Elevated Levels of Synthesis of over 20 Proteins Results after Mutation of the Rhizobium leguminosarum Exopolysaccharide Synthesis Gene pssA

Abstract

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.Soil bacteria belonging to the genera Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium, and Azorhizobium (collectively termed rhizobia) are able to infect the roots of leguminous plants in a host-specific way and stimulate the formation of nodules. Inside these nodules, rhizobia differentiate into bacteroids that reduce atmospheric nitrogen to ammonia, which is used by the plant. One major characteristic of many rhizobia is the production of large amounts of acidic exopolysaccharide (EPS) molecules which serve a variety of roles in free-living rhizobia and in the establishment of symbiosis.EPS forms a biofilm layer on the cell surface which is thought to contribute to the following processes: cellular protection against environmental stresses, attachment to surfaces, nutrient gathering, and the preferential absorption of plant secreted flavonoids along th...

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum: From Genes to Functions

“…The differences identified in the pssE and pssD mutants of the Rlv VF39 strain were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant (9 out of 22 changes) [110]. Genetic complementation of pssA restored wildtype protein synthesis levels.…”

“…The N-terminal sequence analysis of 15 members of the pssA mutant stimulon was performed and the unique amino acid sequences were determined for 11 proteins [110]. However, our attempts to identify these proteins were unsuccessful, and it was clear why.…”

“…Data were taken from Table 2 [110]. ** Data on N-terminal amino acid sequences were taken from Table 3 [110]. Identical amino acids in the matched sequences in the ortholog proteins are shown in bold letters, and similar amino acids are underlined.…”

“…Observed Mr and pI values of the protein based on the migration of proteins in gels. Data were taken from Table 2 [110]. ** Data on N-terminal amino acid sequences were taken from Table 3 [110].…”

Proteome-wide analysis of the functional roles of bacilysin biosynthesis in Bacillus subtilis

“…These types of analyses have been used to explore the mechanisms behind several pathways in bacteria, including, exopolysaccharide biosynthesis (Guerreiro et al, 2000), response to nutrient limitation (Eberl et al, 2000), inactivation of sigma-factors (Elias et al, 2000) and quorum-sensing (Callahan et al, 2000).…”