Effect of the hfq gene on 2,4-diacetylphloroglucinol production and the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is an effective biological control agent of a number of soilborne plant diseases caused by pathogenic microorganisms. Among a range of secondary metabolites produced by strain 2P24, the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) is the major determinant of its disease-suppressive capacity. In this study, we performed random mutagenesis using mini-Tn5 in order to screen for the transcriptional regulators of the phlA gene, a biosynthase gene responsible for 2,4-DAPG production. The mutant PMphlA23 with significantly decreased phlA gene expression was identified from approximately 10,000 insertion colonies. The protein sequence of the interrupted gene has 84% identity to Hfq, a key regulator important for stress resistance and virulence in Pseudomonas aeruginosa. Genetic inactivation of hfq resulted in decreased expression of phlA and reduced production of 2,4-DAPG. Furthermore, the hfq gene was also required for the expression of pcoI, a synthase gene for the LuxI-type quorum-sensing signaling molecule N-acyl-homoserine lactone. Additionally, the hfq mutation drastically reduced biofilm formation and impaired the colonization ability of strain 2P24 on wheat rhizospheres. Based on these results, we propose that Hfq functions as an important regulatory element in the complex network controlling environmental adaption in P. fluorescens 2P24.

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24.

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of soil-borne plant diseases by Pseudomonas fluorescens 2P24. Two mutants (PM810 and PM820) with increased extracellular accumulation of 2,4-DAPG were isolated using transposon mutagenesis. The disrupted genes in these two mutants shared >80 % identity with the genes of the EmhR-EmhABC resistance-nodulation-division (RND) efflux system of P. fluorescens cLP6a. The deletion of emhA (PM802), emhB (PM803) or emhC (PM804) genes in strain 2P24 increased the extracellular accumulation of 2,4-DAPG, whereas the deletion of the emhR (PM801) gene decreased the biosynthesis of 2,4-DAPG. The promoter assay confirmed the elevated transcription of emhABC in the EmhR disrupted strain (PM801) and an indirect negative regulation of 2,4-DAPG biosynthetic locus transcription by the EmhABC efflux pump. Induction by exogenous 2,4-DAPG led to remarkable differences in transcription of chromosome-borne phlA : : lacZ fusion in PM901 and PM811 (emhA(-)) strains. Additionally, the EmhABC system in strain 2P24 was involved in the resistance to a group of toxic compounds, including ampicillin, chloramphenicol, tetracycline, ethidium bromide and crystal violet. In conclusion, our results suggest that the EmhABC system is an important element that influences the production of antibiotic 2,4-DAPG and enhances resistance to toxic compounds in P. fluorescens 2P24.