Study On The Promoter Of Zur Gene In Xanthomonas Campestris Pv. Campestris

In bacteria,the zinc uptake regulator Zur is encoded by zur gene and typically functions as a repressor to repress the expression of zinc uptake systems when the zinc concentration inside the cell reached a threshold. However,our previouse work showed that,in Xanthomonas campestris pv.campestris(Xcc),in addition to involved in zinc homeiostasis,zur (ORF number XC1430)also play an important role in pathogenesis and EPS production of this pathogen.Furthermore,our unpublished data suggested that Xcc zur may contain two independent promoters,P1, which is located upstream of the annotated start coden ATG,and P2, which is upstream of GTG located inside the coding region ofzur(63 bp downstream of ATG).To confirm this hypothesis,in this study,we constructed the promoter(P1 and P2)-gusA transcriptional fusion reporter plasmids pL6PlgusA and pL6P2gusA,and tested their GUS activity.The result showed that both P1 and P2 displayed promoter activity,and expression of P1 and P2 is not affected by zinc concentration.In order to identify the transcriptional start site of P1 and P2,we carried out the 5'-RACE analysis and the result show that the A base located 23 bp upstream of the annotated start coden ATG is the transcriptional start site for P1,but we can't detect the transcriptional start site of P2 under the experimental conditions.Based on the fact that P1 and P2 are functional promoters,we speculated that the two promoters can lead to the sythesis of two different functional Zur proteins,one(designated as ZurL)was translated from the ATG and the other(designated as ZurS)was translated from GTG.To confirm this,the 2 start codens,ATG and GTG,were respectively mutated(ATG→ATA,GTG→GTA)by site-directed mutagenesis,and the function of the mutated zur were assay by complementation of the zur deletion mutant.Result showed that,the plasmid pL6zurATG which expresses ZurL can restore all of the phenotypes(sensitive to high zinc concentration,reduced virulence and EPS production)of the zur mutant, while the plasmid pL6zurGTG which expresses the ZurS can restore EPS production and virulence but not high zinc sensitivity,indicating that ZurL and ZurS have overlap and different functions:ZurL has zinc homeostasis regulation,EPS production and virulence function ZurS has EPS production and virulence function.To test if the ZurL and ZurS do exist in Xcc cell,we performed Western blotting analysis and the result show that one hybrid band was observed in the cell extractes of each one site-directed complement strain,but only one hybrid band was observed in the cell extractes of the wild type strain 8004.Based on the above results,we conclude that:(1)it is very likely that Xcc zur has two different promoters:except for the promoter upstream of ATG(P1),the region upstream of GTG is also a functional promoter(P2).(2)ZurL(translated from ATG)and ZurS(translated from GTG)have overlap and different function.