Pseudomonas mosselii E1铁载体合成相关基因cysI的克隆与功能初步分析

Cloning and functional analysis of cysI gene involved in siderophores biosynthesis in Pseudomonas mosselii E1

摘要:

从棉花根际分离的铁载体产生菌E1,其16SrDNA与Pseudomonas mosselii ATCCBAA-99的同源性为100%。采用三亲本杂交方法将携带转座子Tn5-1063的质粒pRL1063a导入E1中进行转座子插入诱变。利用CAS法,从1000个突变株中,筛选到一株铁载体合成缺失突变株E1-185。利用TAIL-PCR方法,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到E1的cysI基因内。该基因与Pseudomonas entomophila L48的cysI同源性为96%,其CysI氨基酸序列相似性为97%。该基因与半胱氨酸的合成密切相关,而在加有半胱氨酸的CAS平板上,突变株恢复了铁载体产生能力,证明cysI在E1铁载体合成过程中具有重要作用。据推测,cysI可能与铁载体合成途径中关键蛋白acyl-S-PCPs的形成有关。

Abstract:

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.