The Regulation Of Gaca On Two Phz Gene Clusters And Quorum Sensing In Pseudomonas Sp. M18

The Pseudomonas sp. M18, isolated from the watermelon rhizosphere, is antagonistic against a number of soil-borne pathogens. This capability is primarily due to its ability to produce several antibiotics such as pyoluteorin (Plt), phenazine-1-carboxylic acid (PCA) and so on. In this study, we try to identify the construction of phz gene clusters in P. sp. M18 and the regulation of GacA on two phz gene clusters and quorum sensing in Pseudomonas sp. M18. There are three main sections involved in this study:Firstly, given that P. sp. M18 has the same genetic background with P. aeruginosa and P. fluorescens on the one hand, and P. sp. M18 can produce PCA just as P. aeruginosa on the other hand, it is assumed that P. sp. M18 has the similar phz gene clusters with P. aeruginosa. Based on this, conservative primers were designed and two phz gene clusters in P. sp. M18 were amplified. Sequence analysis showed the structure genes of these two clusters shared a 99% homology with that in P. aeruginosa PAO1. However, in the uncoding region downstream the phzA2-G2, P. sp. M18 has a three-144bp-repeat sequence which does not exist in P. aeruginosa PAO1, which proves that genetic differences exist between P. sp. M18 and P. aeruginosa PAO1.Secondly, RT-PCR and phzA-lacZ transcriptional fusions were adopted to explore the expression features of two phenazine biosynthetic gene clusters and to study the function of global regulator GacA in P. sp. M18. The results indicated that phzA1-G1 expressed at a higher level than phzA2-G2 in the wide type M18 strain. It was found that GacA had a differential regulation over these two phz gene clusters, positively regulating the expression of phzA1-G1 while negatively regulating that of phzA2-G2, and regulating the expression of the two clusters negatively on the whole level. The regulatory activity of GacA on phz expression in P. sp. M18 was opposite to that in P. aeruginosa PAO1.Finally, further research into P. sp. M18's quorum sensing system showed that the inactivation of gacA gene can reduce the expression of lux-like gene rhlI while has no effect on lux-like gene lasI expression. This result, together with the study on the regulation of rhl and las system over PCA, indicated that GacA mainly regulates the phz expression through unknown pathway instead of quorum-sensing systems in P. sp. M18. The differential regulation of GacA activity on secondary metabolites and quorum-sensing systems between P. sp. M18 and P. aeruginosa PAO1 maybe caused by their different environmental and evolutionary pressure.