A siderophores-producing strain E1 was isolated from the rhizosphere of cotton. Its 16S rDNA is identical to that of Pseudomonas mosselii sp. nov. at 100% level. The suicide plasmid pRL1063a carrying Tn5-1063 was introduced into E1 by triparental mating and 1000 transposon insertion mutants were subsequently screened using CAS assay. One mutant deficiency in siderophores production was obtained, namely, E1-185. DNA sequences flanking Tn5-1063 of E1-185 was amplified by TAIL-PCR. According to the DNA sequencing results, it is found that Tn5-1063 was inserted into cysI gene. The cysI of E1 is identical to that of Pseudomonas entomophila. L48 at 96% level, and similarity of amino acid sequences of their CysI is 97% . The cysI gene is required for the synthesis of cysteine. However, The ability in siderophores production of E1-185 on the plate of CAS with cysteine was recovered. It is indicated that cysI play an important role during the synthesis of siderophores. It was supposed that cysI is involved in the synthesis of acyl-S-PCPs, which is the key protein in the synthesis pathway of siderophores.