Cloning And Functional Analysis Of Genes Involved In Siderophores Biosynthesis In Pseudomonas Putida D15 And E19

Four hundred of bacterial isolates were obtained from rhizosphere of cotton using the plate culture method. A siderophores-producing bacterium E19 was screened from the isolates by the chrome azurol S (CAS) assay. In addition, a siderophores-producing bacterium D15 was screened from the peanut rhizosphere in the same way. According to the morphologic physiological and biochemistry characterization, E19 and D15 should belong to Pseudomonas. The16S rDNA sequences of them are identical to that of Pseudomonas putida at 100% level.Tested by the nutrition agar plate with antibiotics, Pseudomonas putida E19 and D15 were sensitive to kanamycin, but could tolerate 50μg/mL chloromycetin.The Tn5-1063 was used to mutagenize E19 and D15, respectively. We obtained 22 mutants deficient in siderophore biosynthesis of E19, and one mutants of D15. Designing primers according to luxA sequences from Tn5-1063, 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 Tn5-1063 at 100% level, it indicates that Tn5-1063 was inserted directly into the genome of E19 and D15, respectively.According to the flank sequence of Tn5-1063, three specific primers were designed. On the orther hand, seven arbitrary degenerate primers were designed. By TAIL-PCR (thermal asymmetric interlaced PCR), the flanking sequence was obtained from the mutant D15-4. By TAIL-PCR and specific PCR, the gene cluster cysPTWA involved in siderophore biosynthesis of P. putida D15 was obtained. The cysPTWA encode subunits of an ABC transporter responsible for the uptake of sulfate which offer–SH for cysteine, respectively. It is found that Tn5-1063 was inserted into the interval sequence between cysP and cysT. Because of the polarity effect, sulfate uptaking and cysteine biosynthesis are decreased and then the efficiency of siderophores biosynthesis was reduced . Sulfate uptake assays confirmed that mutant was defective in sulfate transport From the mutant E13-32, the gene psvA involved in siderophore synthesis was obtained. The protein psvA encod pyoverdine synthetase A,Deletion of this gene, encoding a predicted non-ribosomal peptide synthetases (NRPSs) involved in PVD chromophore biosynthesis confirmed the necessity of the gene for PVD production and for normal growth in iron-limited media.