Phloroglucinol mediates cross‐talk between the pyoluteorin and 2,4‐diacetylphloroglucinol biosynthetic pathways in Pseudomonas fluorescens Pf‐5

Abstract

The antibiotics pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) contribute to the biological control of soilborne plant diseases by some strains of Pseudomonas fluorescens, including Pf-5. These secondary metabolites also have signalling functions with each compound reported to induce its own production and repress the other's production. The first step in DAPG biosynthesis is production of phloroglucinol (PG) by PhlD. In this study, we show that PG is required at nanomolar concentrations for pyoluteorin production in Pf-5. At higher concentrations, PG is responsible for the inhibition of pyoluteorin production previously attributed to DAPG. DAPG had no effect on pyoluteorin production, and monoacetylphloroglucinol showed both stimulatory and inhibitory activities but at concentrations 100-fold greater than the levels of PG required for similar effects. We also demonstrate that PG regulates pyoluteorin production in P. aeruginosa and that a phlD gene adjacent to the pyoluteorin biosynthetic gene cluster in P. aeruginosa strain LESB58 can restore pyoluteorin biosynthesis to a ΔphlD mutant of Pf-5. Bioinformatic analyses show that the dual role of PhlD in the biosynthesis of DAPG and the regulation of pyoluteorin production could have arisen within the pseudomonads during the assembly of these biosynthetic gene clusters from genes and gene subclusters of diverse origins.

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

“…We also evaluated cultures of P. protegens Pf-5 growing in NBG (nutrient broth supplemented with 2% glucose). This medium supports bacterial growth but not pyoluteorin biosynthesis by strain Pf-5 (36). No spontaneous Gac Ϫ mutant was detected when strain Pf-5 was cultured in NBG (193,155, and 148 colonies were screened in three independent cultures), which further supports the conclusion that pyoluteorin biosynthesis contributes to accumulation of spontaneous Gac Ϫ mutants of Pf-5.…”

“…Rhizoxin and 2,4-diacetyl- phloroglucinol were not produced to detectable levels in NYB medium, so it is possible that the production of these secondary metabolites could influence the proportion of Gac Ϫ mutants of Pf-5 in a different medium. However, no Gac Ϫ mutants were detected when strain Pf-5 was cultured in NBG, which is conductive to 2,4-diacetylphloroglucinol production (36), indicating that production of 2,4-diacetylphloroglucinol may not influence the accumulation of Gac Ϫ mutants. Pyrrolnitrin was produced by strain Pf-5 on NYB (see Fig.…”

“…Therefore, we cannot exclude the possibility that an intermediate(s) in pyoluteorin biosynthesis, which is not present in the ΔpltA mutant, contributes to the differential fitness of Gac Ϫ mutants and wild-type Pf-5 in NYB cultures. Our recent work also showed that the intermediates of secondary metabolic pathways can function as signals regulating gene expression of P. protegens (10,36,46). Future experiments are needed to investigate the possible contribution of intermediates in pyoluteorin biosynthesis to the accumulation of spontaneous Gac Ϫ mutants.…”

“…For example, in addition to the positive regulation imposed by GacS-GacA (54) and the pathway-specific regulator PltR (30), synthesis of pyoluteorin is also kept in check by numerous negative regulators that operate at the transcriptional and posttranscriptional levels. Specifically, pyoluteorin production by P. protegens Pf-5 and other strains of Pseudomonas is moderated by the presence of nonpreferred codons in PltR (11), transcriptional repressor PltZ (55), RNAbinding protein RsmE (45), stationary-phase sigma factor RpoS (29), Lon protease (56), and phloroglucinol, an intermediate in the biosynthesis of 2,4-diacetylphloroglucinol (36,46). The presence of multiple layers of negative regulation suggests that controlling pyoluteorin production to moderate levels is important for the fitness of the bacterial cell.…”

RNA pentaloop structures as effective targets of regulators belonging to the RsmA/CsrA protein family

“…31,32 The phlA gene is the first gene of an operon specifying DAPG biosynthesis. 34 An RsmA/ RsmE-binding site with a typical hexaloop (AUGGAA) located on a potential 2-bp stem lies immediately upstream of the phlA SD sequence (Fig. 1A).…”