Regulation On PhzM Gene Expression By Temperature And Carbon Catabolite Repression In Pseudomonads

Pseudomonas sp. M18, an effective biological control agent isolatedfrom the melon rhizosphere shares a similar genetic background to that of theopportunistic human pathogen P. aeruginosa PAO1. However thepredominant phenazine produced by strain M18is phenazine-1-carboxylicacid (PCA) rather than pyocyanin (PYO); the quantitative ratio between PCAand PYO is105to1at28C in strain M18, while the ratio is1to2at37C instrain PAO1. We first provided evidence that the differential production ofthe two phenazines in strains M18and PAO1is related to thetemperature-dependent and strain-specific expression patterns of phzM, agene involved in the conversion of PCA to PYO. Transcriptional levels ofphzM were measured by quantitative real-time PCR (qRT-PCR) and theactivities of both phzM-‘lacZ transcriptional and translational fusion weredetermined in strains M18and PAO1, respectively. Using lasI and ptsPinactivated M18mutants, we further show that the expression of phzM geneis positively regulated by the quorum sensing protein LasI and negativelyregulated by the phosphoenolpyruvate phosphotransferase protein PtsP.Surprisingly, the lasI and ptsP regulatory genes were also expressed in a temperature-dependent and strain-specific manner. The differentialproduction of the phenazines PCA and PYO by strains M18and PAO1maybe a consequence of selective pressure imposed on P. aeruginosa strain andits relative, M18in the two different niches over a long evolutionary process.In Pseudomonas strains, carbon catabolite repression is important for thecompetition of the different bacterial species in their natural habitats. Theregulatory processes allowing for this selection of preferred carbon sourceshave been named carbon catabolite repression (CCR) or catabolite repressioncontrol (CRC). Currently, the regulatory factors or systems known to beinvolved in CCR in Pseudomonas are the Crc protein together withtwo-component system CbrA/CbrB and small RNA CrcZ, which modulatesCrc availability. In this work, it was found that Crc repressed while CrcZactivated phzM gene in Pseudomonas aeruginosa PAO1. Much moresecondary metabolite pyocyanin was produced in Crc-deleted mutant, whilepyocyanin was abolished in CrcZ-mutant. A CA-rich motif was found to beimportant for the regulation by Crc and CrcZ on phzM gene, which located inthe phzM translational initiation region. When the CA-rich motif was mutatedby bases-substitution, the regulation of Crc and CrcZ on phzM was lost.Further Protein-RNA binding shift assay revealed, Crc protein can bind themRNA of phzM gene, and the binding site is the CA-rich motif.