Primary Study Of The Tow-Component System ColR-ColS In Pseudomonas Putida CD2

The ColRS was identified to be a two-component system(TCS) in Pseudomonas.It reported that the function of the two-component system ColRS varied largely in four Pseudomonas strains.It was involved in the ability of bacteria to colonize plant roots in Pseudomonas fluorescens WCS365 and Pseudomonas putida kt2440 and was related to regulation of Tn4652 transposition in Pseudomonas putida PaW85 under starvation condition and was important for heavy metal resistance in Pseudomonas putida CD2. Though the ColRS proteins in different Pseudomonas stains showed highly homologous, the two-component system ColRS was involved in three different processes.Therefore,it is important to search for the checkpoint of response regulator ColR in these stains.To determine the role of the ColRS system in Pseudomonas putida CD2,we searched for target genes of response regulator ColR through our previous results and analyzing of genome.Using similar RT-PCR conditions,the amount of this cDNA fragment obtained was unchangeable about the gene znuCB1,znuA1 and znuACB2.When it came to the gene czcCBA1,the amount of this cDNA fragment obtained was highest when cells cultured in the presence of Cd²⁺.Then the expression in mutant M11 was not detected.These results suggested that the expression of czcCBA1 could be regulated by ColR.To investigate the relationship between ColR and the ColR-regulated genes,the upstream region of the oprQ and czcCBA1 were cloned into a low-copy-number GFP-based promoter probe plasmid.The czcCBA1 promoter was 4.8-fold induced in the presence of Cd²⁺,while the oprQ promoter was 17.6-fold repressed by the Cd²⁺.In contrast to promoters of czcCBA1 and oprQ,the czcCBA1 promoter was repressed in colS mutant stain,the oprQ promoter was slightly increased in colS mutant stain. Transcriptional analysis of czcCBA1 and oprQ promoters revealed that ColR regulated expression of the two genes in two different ways.ColR was a positive transcriptional regulator of the czcCBA1 gene cluster,but was a negative transcriptional regulator of the oprQ.In the present study,we reported a new ColR-regulted gene cluster czcCBA1 from P. putida reference stain CD2.Transcriptional analysis of czcCBA1 and oprQ promoters revealed that ColR regulated expression of the two genes in two different ways.To our knowledge this is a first report on a regulation mechanism between ColR-ColS and CzcCBA efflux pump.Hopefully,further experiments will give us more information of the regulation mechanism of ColR-ColS.