Identification And Characterization Of Las Quorum-Sensing System And Regulatory Research Of Antibiotics Synthesis 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 Plt-related regulators by molecular technology and disclose the relationship between them and Plt biosynthesis, then to construct engineering strains with improved Plt production and biocontrol capability. There are two main sections involved in this study:Firstly, A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which was consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination enhanced the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) to 4 - 5 fold and 2 - 3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Expression of the translational fusions pltA'-'lacZ and phzA'-'lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.Secondly, two mutant strains M18R and M18IG have been constructed, which could product more Plt ,compared with the wide-type M18. In order to further investigate the regulation of Plt and PCA production by these two genes,the chromosomal inactivated strains of an rsmA mutant called M18R and an rsmA and rhlI double mutant, designated as M18RIG, were constructed in Pseudomonas sp. M18. It was found that Plt and PCA production in the double mutant strain M18RIG were both increased 10 folds and 4 folds than that in the wild- type strain M18. The negative control of both rsmA and rhlI gene together on Plt and PCA production in strain M18 was further confirmed by the analysis ofβ-galactosidase activities from the translational pltA'-'lacZ fusion and phzA'-'lacZ fusion. Furthermore, in PPM, the mutant strains M18R, M18IG, M18RIG grew better than the wide-type M18. Finally, by introducing a rhlI'-'lacZ translational fusion vector to strain M18, M18R, respectively, it was found thatβ-galactosidase activity was increased about 2-fold in M18R compared with the wide-type strain M18, suggesting that RsmA acted as a negative control of the rhlI gene.