Background: Our previously published reports have described an effective biocontrol agent namedPseudomonassp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those ofP. aeruginosa, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures.Results: The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conservedP. aeruginosacore genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains acapBgene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with otherP. aeruginosastrains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that ofP. aeruginosastrain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C.Conclusions: TheP. aeruginosastrain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche.

The rhizobacterium Pseudomonas aeruginosa M18 can produce a broad spectrum of secondary metabolites, including the antibiotics pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), hydrogen cyanide, and the siderophores pyoverdine and pyochelin. The antibiotic biosynthesis of M18 is coordinately controlled by multiple distinct regulatory pathways, of which the GacS/GacA system activates Plt biosynthesis but strongly downregulates PCA biosynthesis. Here, we investigated the global influence of a gacA mutation on the M18 transcriptome and related metabolic and physiological processes. Transcriptome profiling revealed that the transcript levels of 839 genes, which account for approximately 15% of the annotated genes in the M18 genome, were significantly influenced by the gacA mutation during the early stationary growth phase of M18. Most secondary metabolic gene clusters, such as pvd , pch , plt , amb , and hcn , were activated by GacA. The GacA regulon also included genes encoding extracellular enzymes and cytochrome oxidases. Interestingly, the primary metabolism involved in the assimilation and metabolism of phosphorus, sulfur, and nitrogen sources was also notably regulated by GacA. Another important category of the GacA regulon was secretion systems, including H1, H2, and H3 (type VI secretion systems [T6SSs]), Hxc (T2SS), and Has and Apr (T1SSs), and CupE and Tad pili. More remarkably, GacA inhibited swimming, swarming, and twitching motilities. Taken together, the Gac-initiated global regulation, which was mostly mediated through multiple regulatory systems or factors, was mainly involved in secondary and primary metabolism, secretion systems, motility, etc., contributing to ecological or nutritional competence, ion homeostasis, and biocontrol in M18.

Pseudomonas sp. strain M18, an effective biological control agent isolated from the melon rhizosphere, has a genetic background similar to that of the opportunistic human pathogen Pseudomonas aeruginosa PAO1. However, the predominant phenazine produced by strain M18 is phenazine-1-carboxylic acid (PCA) rather than pyocyanin (PYO); the quantitative ratio of PCA to PYO is 105 to 1 at 28°C in strain M18, while the ratio is 1 to 2 at 37°C in strain PAO1. We first provided evidence that the differential production of the two phenazines in strains M18 and PAO1 is related to the temperature-dependent and strain-specific expression patterns of phzM , a gene involved in the conversion of PCA to PYO. Transcriptional levels of phzM were measured by quantitative real-time PCR, and the activities of both transcriptional and translational phzM ′-′ lacZ fusions were determined in strains M18 and PAO1, respectively. Using lasI ::Gm and ptsP ::Gm inactivation M18 mutants, we further show that expression of the phzM gene is positively regulated by the quorum-sensing protein LasI and negatively regulated 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 differential production of the phenazines PCA and PYO by strains M18 and PAO1 may be a consequence of selective pressure imposed on P. aeruginosa PAO1 and its relative M18 in the two different niches over a long evolutionary process.

Pseudomonas aeruginosa M18, a rhizosphere-isolated bacterial strain showing strong antifungal activity, can produce secondary metabolites such as phenazine-1-carboxylic acid and pyoluteorin (Plt). The LysR-type transcriptional regulator PltR activates the Plt biosynthesis operon pltLABCDEFG, the expression of which is induced by Plt. Here, we identified and characterized the non-conserved pltL promoter (pltLp) specifically activated by PltR and its upstream neighboring lys box from the complicated pltR–pltL intergenic sequence. The 22 bp palindromic lys box, which consists of two 9 bp complementary inverted repeats interrupted by 4 bp, was found to contain the conserved, GC-rich LysR-binding motif (T-N₁₁-A). Evidence obtained in vivo from mutational and lacZ report analyses and in vitro from electrophoretic mobility shift assays reveals that the PltR protein directly bound to the pltLp region as the indispensable binding motif “lys box”, thereby transcriptionally activating the pltLp-driven plt operon expression. Plt, as a potential non-essential coinducer of PltR, specifically induced the pltLp expression and thus strengthened its biosynthetic plt operon expression.