Cloning And Functional Analysis Of Genes Involved In Siderophores Biosynthesis In Pseudomonas Mosselii E1 And P.pseudoalcaligenes B50

B50 and E1 were isolated from the the rhizosphere of cotton. Both of them produce siderophores under iron-limiting conditions. By morphologic observation and physiological, phenotypic characterization, B50 and E1 were Gram negative, non spores and Pseudomonas. By 16S rDNA sequencing, 16S rDNA of B50 is identical to that of Pseudomonas pseudoalcaligenes at 100% level; 16S rDNA of E1 is identical to that of Pseudomonas mosselii at 100% level. Tested by the plate of nutrition agar with antibiotics, Pseudomonas pseudoalcaligenes B50 and Pseudomonas mosselii E1 were sensitive to kanamycin, but could tolerate 10μg/mL chloromycetin.The Tn5-1063a was used to mutagenize E1 and B50, respectively. Two siderophore-deletion mutants from E1 were obtained, E1-185 and E1-332, and five mutants from B50, B50-472, B50-982, B50-1358, B50-1657 and B50-1723. Designing primers according to luxA sequences from Tn5-1063a, the DNA of wild strains, pRL1063a and the mutants as the templates, the sequences of luxA was obtained by PCR. The luxA of the mutants is identical to that of Tn5-1063a at 100% level, it indicates that Tn5-1063a was inserted directly into the genome of E1 and B50, respectively.Three specific primers were designed according to Tn5-1063a, and seven arbitrary degenerate primers were designed at the same time. By TAIL-PCR (thermal asymmetric interlaced PCR) the gene cysI involved in siderophore synthesis was obtained from the mutant E1-185. The gene of cysI was required for the synthesis of cysteine and it plays an important role during the biosynthesis of acyl-S-PCPs. This was the key step for the synthesis of siderophores through the path of non-ribosomal peptide synthesis (NRPS) and verification was done.From the mutant B50-1657, the gene pyrD involved in siderophore synthesis was obtained. The protein pyrD was dihydroorotate dehydrogenases, which catalyzes the oxidation of (S)-dihydroorotate to orotate. This is the fourth step of UMP synthesis. Verification suggested that the gene of pyrD is involved in biosynthesis of siderophores.In addition, the gene relA involved in siderophore synthesis was obtained from the mutant B50-1358. The gene of relA encoded RelA/SpoT for stringent control, it regulates the synthesis and hydrolysis of (p)ppGpp. Stringent control was a kind of stress reaction of bacterium under the nutrit-limiting conditions. The iron limitation induces the stringent control. Bacterum produce siderophores to alleviating the limitation.