UNLABELLED: In many Pseudomonas, RsmA mediates the production of a set of secondary metabolites or virulence factors. OBJECTIVE: Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA. With the insertion of gentamycin resistance cassette (aacC1), the deletion mutant PA-RG was created and verified with PCR. To complement and overexpress the rsmA gene, pME10R and pME32R were also constructed. By constructing the translational fusion plasmids phz1'-'lacZ pMEZ1 and phz2'-'lacZ pMEZ2, we introduced them into the wild type strain PAO1 and the mutant PA-RG, respectively. Activities of beta-galactosidase were determined with Miller method. RESULTS: In glycerol-alanine medium, overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain. In addition, beta-galactosidase activity of phz1'-'lacZ in the mutant PA-RG was much higher than that in the wild type strain. However, beta-galactosidase activity of phz2'-'lacZ in the wild type strain was 2fold more than that in the mutant PA-RG. CONCLUSION: The regulation mediated by RsmA on two phenazine loci is specific and differential.
UNLABELLED: In many Pseudomonas, RsmA mediates the production of a set of secondary metabolites or virulence factors. OBJECTIVE: Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA. With the insertion of gentamycin resistance cassette (aacC1), the deletion mutant PA-RG was created and verified with PCR. To complement and overexpress the rsmA gene, pME10R and pME32R were also constructed. By constructing the translational fusion plasmids phz1'-'lacZ pMEZ1 and phz2'-'lacZ pMEZ2, we introduced them into the wild type strain PAO1 and the mutant PA-RG, respectively. Activities of beta-galactosidase were determined with Miller method. RESULTS: In glycerol-alanine medium, overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain. In addition, beta-galactosidase activity of phz1'-'lacZ in the mutant PA-RG was much higher than that in the wild type strain. However, beta-galactosidase activity of phz2'-'lacZ in the wild type strain was 2fold more than that in the mutant PA-RG. CONCLUSION: The regulation mediated by RsmA on two phenazine loci is specific and differential.