Functional Analysis Of Exopolysaccharide Biosynthesis In Mesorhizobium Tianshanense-plant Host Interaction

Nitrogen-fixing soil bacteria that establish mutualistic associations with legumes are referred to as rhizobia. Rhizobia produce polysaccharides, such as exopolysaccharide (EPS), capsular polysaccharide (KPS), lipopolysaccharide (LPS), and cyclicβ-(1,2)-glucans which are necessary for establishing symbiotic associations. In addition to protecting bacteria against environmental stresses, polysaccharides of the rhizobia play vital roles in infection of legume roots. EPS may aid in the attachment of bacteria to roots, and play a structural role in infection thread formation and protecting bacteria against host defenses.Exopolysaccharide biosynthesis represents a multi-step process, in which many gene products are involved. In Sinorhizobium meliloti, many exo genes responsible for EPSI biosynthesis in S. meliloti, including all the glycosyl transferase genes involved in the synthesis of whole repeating carbohydrate units, as well as some genes involved in secretion (exsA) and polymerization(exoQ, exoT, and exoP). ExoS-ChvI quorum sensing system is involved in regulating EPSII synthesis.Mesorhizobium tianshanense, isolated from arid, saline, desert soil in northwestern China in 1995, acting as a nitrogen fixing symbiont for at least eight different plant species, including species of Glycyrrhiza, whose roots are one of the most important crude medicines in Asia. Little is known of the regulation of EPS biosynthesis in M. tianshanense.To study the regulation of EPS production, a transposon insertion mutant library of M. tianshanense was screened for mutants defective in exopolysaccharide production.we isolated seven nonmucoid mutants. The colony morphology of these mutants is distinct from that of wild type.We then used arbitrary PCR followed by subcloning and DNA sequencing to identify transposon insertion sites in these seven EPS mutants. Sequence analysis revealed that those transposons inserted in two clusters. The first gene cluster is similar to pssNOPT of R. leguminosarum bv. viciae. All these genes are highly conserved in many Rhizobium species and involved in the translocation of polysaccharides and polymerization of the repeating subunits of EPS. We thus propose to name these genes mtpABCD (Mesorhizobium tianshanense polysaccharide genes ABCD). Another gene isolated from the transposon screen was located upstream of the mtpABCD operon and divergently transcribed. This gene, which we named mtpE, was similar to the exo5 gene in R. leguminosarum bv. trifolii.To investigate how EPS genes are regulated in M. tianshanense, we mutagenized the M. tianshanense strain containing the mtpC-lacZ reporter using a transposon and screened for LacZ- transconjugants. The transposon insertion in one mutant (ES11) disrupted the sensor histidine kinase gene of a two-component regulatory system. Further sequencing indicated that a response regulator gene is located immediately downstream of the senor kinase. We named them mtpS and mtpR.To confirm the role of MtpRS in regulation of EPS biosynthesis in M. tianshanense, we constructed mtpR and mtpS insertional deletions in wild type and mtpC-lacZ strains. Both mtpR and mtpS mutants did not produce exopolysaccharides, similar to that of mtpC mutants. Mutations in either mtpR or mtpS gene also abolished mtpC-lacZ activity, indicating that this two-component regulatory system is crucial for mtpC activation.To examine whether EPS biosynthesis is involved in biofilm formation in M. tianshanense, we compared the biofilm formation of wild type and EPS mutants on glass surfaces. Wild type strains produced significant amount of biofilm, while mtpC, mtpR, and mtpE mutants all formed little biofilms. In nodulation assay, on average 11.3 nodules were formed per plant about 1 month after inoculation of wild type bacteria while o nodules formed on the plant roots inoculated with either mtpC or mtpR mutant cultures, indicating that M. tianshanense EPS mutants are profoundly defective in nodulation.We used E. coli mutator strain XL-1 Red to introduce mutations to the Plac-msiR plasmid. The mutated plasmids were then transformed into M. tianshanense TC3 strain containing msiA-lacZ, and the transformants were spread on X-gal plates with different amino acid, we got ten constitutive MsiR mutants that activated msiA independent.