Temperature-responsive Expression Of Two Phz Gene Clusters Is Differently Regulated By Sensor RetS In Pseudomonas Aeruginosa Strain M18

Pseudomonas aeruginosa M18, an effective biological control agent isolated from the melon rhizosphere shares a similar genetic background to that of the opportunistic human pathogen P. aeruginosa PAO1 by the whole genome analysis, and both of them have two phenazine biosynthetic gene clusters, which can produce phenazine compounds. Since previous observations in the clinical originating isolates P. aeruginosa PAO1 has shown that a functional GacS/GacA two-component system positively controls phenazine synthesis, while the rhizosphere originating P. aeruginosa strain M18 has a distinctive feature that PCA production is negatively regulated by GacS/GacA system. By studying the relationship between the function of sensor RetS and phenazine production at different temperature, it is found that the reason for distinctive regulation of phenazine production between pseudomonas aeruginosa M18 and PAO1 is different temperature, and it is also necessary for the GacA/RsmA signal pathway to involve in.Firstly, retS gene was cloned in pseudomonas aeruginosa M18 by PCR and retS inactive mutant strain M18RS was constructed by the homelogens DNA technology. The production of PCA and PYO in wide type M18 and retS mutant M18RS were measured at two different temperature of 28℃and 37℃, it was found that the sensor RetS had different effect on PCA production under the two temperature, when cultivated in the lower temperature of 28℃, RetS played a positive regulatory role in PCA synthesis, and retS gene inactivation resulted in the decrease of PCA production, while RetS had negative regulation on the synthesis of PCA and PYO when the temperature was 37℃, and retS gene inactivation prompted the rise of the production PCA and PYO. At the same time, the above-mentioned phenomenon had been further confirmed through trans-complementation experiment.Secondly, the previous published work has confirmed that the two phenazine biosynthetic gene clusters are responsible for the synthesis of PCA. Through the determination ofβ-gal activities of the transcriptional and translational fusion of the two gene clusters in wild-type M18 and retS mutant M18RS at 28℃and 37℃, it was found that RetS put positive regulation into effect mainly through phz2 gene cluster at 28℃, and the role of negative regulation was implemented by phz1 gene cluster at 37℃, both of them were at the translational level. Namely, the different regulation caused by temperature is completed by different phenazine biosynthetic gene cluster. The retS and gacA gene double mutant strain M18RSG,the over-expression plasmid and the translational fusion plasmid of gacA gene were further constructed to analyze the relationship between the sensor RetS and GacA / RsmA signal transduction pathway, it has found that GacA is located at the downstream of RetS, which mediate the signaling pathway involved in temperature-responsive distinctive expression of two phz gene clusters regulated by mutation of retS in Pseudomonas aeruginosa strain M18, whereas the expression of gacA itself is hardly affected by temperature both at the transcriptional and post-transcriptional levels.The existing studies have shown that multiple senor RetS, GacS and LadS have some relationship with the GacA-mediated signaling pathway, in order to further understand the mechanism of thermoregulation, the single mutant stains of gacS and ladS, named M18GS and M18LS, were constructed to determine the temperature-dependent production of PCA and PYO, it is found the sensors GacS and LadS not only has their own regulation-specific, but also shares the similarity in thermoregulation to RetS.