RpoN (σ⁵⁴) Controls Production of Antifungal Compounds and Biocontrol Activity in Pseudomonas fluorescens CHA0

Abstract

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.

Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity

“…In the pseudomonads, RpoN regulates expression of genes involved in alginate, flagellin, and pilin synthesis (14,37,74). RpoN is a key regulator of biocontrol activity in P. fluorescens (58), and for phytopathogenic bacteria such as P. syringae, RpoN is an important virulence regulator and is required for the elicitation FIG. 6.…”

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

“…The bacterial strains and plasmids used in the present study are described in Table 1. P. fluorescens strains were routinely cultivated on nutrient agar (NA), in nutrient yeast broth (NYB), and in Luria-Bertani broth (LB), at 30°C (3,31,39). Production and degradation of antifungal compounds was monitored in King's medium B broth supplemented with 0.75% (wt/vol) malt extract (KMBmalt) (3).…”

“…Bacterial cells were transformed with plasmid DNA by electroporation (39). PCRs were carried out according to amplification protocols described in detail previously (3,31). Nucleotide sequences of PCR-derived constructs were determined on both strands by Microsynth (Balgach, Switzerland).…”

“…DAPG is synthesized and accumulated until the early stationary growth phase, as is typical for secondary metabolites (1,3,8,31,39). Thereafter, the metabolite appears to be degraded by the producing bacterium, with MAPG temporarily accumulating as an intermediary product of the degradation process (39).…”

“…Relative cellular levels of several factors may also profoundly affect DAPG synthesis (16). They include the housekeeping factor RpoD, the stationary-phase and stress response factor RpoS, and the alternative factor RpoN (31,36,38). In addition, many abiotic and biotic environmental factors may modulate levels of DAPG production, including different carbon and nitrogen sources, transition metal ions and other minerals, and metabolites released by bacteria, fungi, and plants (12,27,28).…”