Study On Sulfur Metabolic Relevant Gene PdxP In Sinorhizobium Fredii WGF03

Sulfur is one of the essential macroelements in vivo of life. Sulfur not only plays an important role in amino acids, proteins and lipids synthesis and functional formation, is also involved in the synthesis of many coenzymes. While the rhizobia, its host specificity is determined by the sulfurized modificaton to the reducted end of Nod factor.In the sulfate assimilation pathway, O-acetyl serine-(sulfhydryl)-lyase (OASTL) is required to catalyze sulfide (S2-) and O-acetyl serine (OAS) to synthesize cysteine(Cys).OASTL is belonged to pyridoxal 5'-phosphate (PLP)-dependent enzyme family,and pdxP encodes pyridoxine 5'-phosphate phosphatase,which specificaly catalyzes pyridoxine 5'-phosphate (PNP) and pyridoxal 5'-phosphate (PLP) off phosphate into pyridoxine and pyridoxal.Through study Sinorhizobium meliloti, we found,PdxP is likely to be the restricted enzyme to regulate the PNP and PLP content in vivo.In this study, I first used plasmid transposon (pTnMod-RKm') to randomized insert into Sinorhizobium fredii WGF03 genome and get 5000 mutants, then through the use of different sulfur sources selective screening,20 mutants relevant sulfur metabolism are selected.Followed by gene sequencing and bioinformatics analysis, a gene sequence shared 87% identities at nucleotide level and 88% positives at amino acid level with the reported pdxP gene of Sinorhizobium meliloti.The polar mutant (WTA) and non-polar mutant (WTB) of pdxP gene were constructed by using homologous single-crossover method with the suicide plasmid pK18mob. The mutants were measured by adopting MM medium with different sulfate as the sole sulfur source. The result showed that the both mutants were able to grow on MM medium with sulfate,sulfite and thiosulfate as the sole sulfur source,but all of the growth rate were very slow and all of the growth state were also poor.The plant tests showed that the nodulation time of the mutants was later than the original strain WGF03 and the nodulate competitiveness of the mutants were declined.However,there were no significant deviation in nodulating effciency and the ability of nitrogen-fixation between mutants and original strain.