Identification And Characterization Of Rhl Quorum-Sensing System And Regulatory Gene PltR 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 four main sections involved in this study:Firstly, we got several mutants with the transposon mini-Tn5 lacZ-ter/1 inserted into the quorum-sensing-related genes expressing during late growth cycle. Three mutants of 46-11, 49-2 and 58-17 showed different phenotypes, which were disruption of Plt production, declined swarming ability and decreased red pigment production, respectively. Subsequently, the mutated genes were identified and gotten preliminary study. The mutant 58-17 was the potential quorum-sensing mutant. In addition, the N-acyl homoserine lactones (AHLs) were found in M18 culture and their degradation would affect antibiotics production. These data indicated that the putative quorum-sensing system (QS) might exist in this strain and be involved in the regulation of antibiotics production.Secondly, a putative lux box, the binding site for LuxR family regulators, was identified in the promoter region of Plt biosynthetic gene cluster. It further suggested certain QS system might participate in the regulation of Plt biosynthesis directly. Based on the genetic background of M18 and the sequence analysis of the mutant gene in mutant 58-17, it was concluded that strain M18 would be unusual in sharing some distinct features of both P. aeruginosa and P. fluorescens. We designed conserved primers according to the sequence message of P. aeruginosa and got the rhlR and rhlI genes in M18. Sequence analysis of the PCR product indicated that their sequence and deduced protein shared high identity to those reported in P. aeruginosa PAO1. Considering such a remarkable identity, we named our QS system rhl.A null mutant M18IG (rhlI::Gm) with an inactivated chromosomal rhlI gene was constructed by inserting a single Gm resistance cassette by homologous recombination, which Plt production was increased 5-fold while PCA production was suppressed as compared with the wild-type M18. Another mutant M18RK (rhlR::Km) showed the similar phenotypes. According to the analysis of the pltLA'–'lacZ and phzA'–'lacZ translational fusions in M18 and rhl mutants, it is easy to conclude that RhlI and RhlR made up of the integrated rhl QS system then regulated plt genes expression. Another experiment also confirmed that it is BHL, instead of HHL, played the key role in these regulations.At the same time, disruption of rhl QS system promote expression of the Plt-specific ABC transporter, PltHIJKN, which was in charge of Plt transport to accelerate Plt production. However, this enhancement disappeared when another pltB mutation was introduced.β-galactosidase activity of the pltH′-′lacZ fusion was similar in the Plt-negative strains M18T, M18TI, and M18TR, approximate 50 % of that in wild-type strain M18. These results indicated that the rhl quorum-sensing system can not directly regulate this Plt-specific ABC transporter alone, but does so rather in a Plt-dependent manner.Thirdly, the pltR gene, coding a putative LysR-type regulator, was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp. M18 using bioinformatics technology. The analysis of Plt or PCA production and transcription of plt genes in pltR mutant M18TRG (pltR::Gm) or pltR overexpression M18 (pME6032PLTR) indicated that PltR could positively regulate expression of Plt biosynthetic genes at transcriptional level. In addition, the investigation on the pltR expression in gacA mutant M18G, rsmA mutant M18R and rhl mutants (M18IG or M18RK) disclosed that PltR was a more downstream regulator and involved in the positive regulation of gacA on Plt production while in the negative control caused by rhl QS system.Finally, Plt production increased in PCA-negative strain M18P (phzB::Km), whereas PCA production in Plt-negative strain M18T (pltB::Gm) remained unchanged. It is possible the enhanced Plt biosynthesis at least partially results from the suppressed PCA biosynthetic ability. Further experiment proved that PCA itself was not involved in this regulation. The mRNA level of Plt genes in M18P, however, was unchanged relative to genes in strain M18T. Unlike the regulation of rhl QS on Plt biosynthesis, this regulation happens primarily at the posttranscriptional level in stead of transcriptional level.