Polyyne production is regulated by the transcriptional regulators PgnC and GacA in Pseudomonas protegens Pf-5

Abstract

Polyynes produced by bacteria have promising applications in agriculture and medicine due to their potent antimicrobial activities. Polyyne biosynthetic genes have been identified in Pseudomonas and Burkholderia. However, the molecular mechanisms underlying the regulation of polyyne biosynthesis remain largely unknown. In this study, we used a soil bacterium Pseudomonas protegens Pf-5, which was recently reported to produce polyyne called protegenin, as a model to investigate the regulation of bacterial polyyne production. Our results show that Pf-5 controls polyyne production at both the pathway-specific level and a higher global level. Mutation of pgnC, a transcriptional regulatory gene located in the polyyne biosynthetic gene cluster, abolished polyyne production. Gene expression analysis revealed that PgnC directly activates the promoter of polyyne biosynthetic genes. The production of polyyne also requires a global regulator GacA. Mutation of gacA decreased the translation of PgnC, which is consistent with the result that pgnC leader mRNA bound directly to RsmE, an RNA-binding protein negatively regulated by GacA. These results suggest that GacA induces the expression of the PgnC regulator, which in turn activates polyyne biosynthesis. Additionally, the polyyne-producing strain of Pf-5, but not the polyyne-nonproducing strain, could inhibit a broad spectrum of bacteria including both Gram-negative and Gram-positive bacteria.IMPORTANCEAntimicrobial metabolites produced by bacteria are widely used in agriculture and medicine to control plant, animal, and human pathogens. Although bacteria-derived polyynes have been identified as potent antimicrobials for decades, the molecular mechanisms by which bacteria regulate polyyne biosynthesis remain understudied. In this study, we found that polyyne biosynthesis is directly activated by a pathway-specific regulator PgnC, which is induced by a global regulator GacA through the RNA-binding protein RsmE in Pseudomonas protegens. To our knowledge, this work is the first comprehensive study of the regulatory mechanisms of bacterial polyyne biosynthesis at both pathway-specific level and global level. The discovered molecular mechanisms can help us optimize polyyne production for agricultural or medical applications.

Keywords: Pseudomonas protegens; antimicrobial activity; gene regulation; polyyne biosynthesis.

Fig 1

P. protegens Pf-5 produces polyyne that inhibits the growth of Pseudomonas syringae DC3000 on NAGly plates. (A) Polyyne biosynthetic gene cluster of Pf-5. The regulatory gene pgnC is shown in gray color. Chemical structures of polyyne derivatives Protegenin A–D were shown. Protegenin A, C, and D were detected from Pf-5 cultures in this study and a previous report (19). (B) Inhibition of wild-type Pf-5, the sixfold, sevenfold-A, and sevenfold-B mutants against P. syringae DC3000 on NAGly. (C) Inhibition of the sixfold mutant, the sevenfold-B mutant, and its complementation strain against P. syringae DC3000 on NAGly. “+” and “−” Indicate inhibition and no inhibition, respectively. The experiments were repeated at least three times, which generated similar results.

Fig 2

Chemical analysis of Pf-5’s metabolites on NAGly medium by HPLC (A and B) and LCMS (C and D). (A) HPLC chromatograms (254 nm) of metabolite extracts of (I) sixfold/pME6010, (II) sevenfold-B/pME6010, and (III) sevenfold-B/pME6010-pgnE. (B) UV spectra of the peak eluting at 25.2 minutes in the extracts of (I) and (III). (C) Extracted ion chromatogram (267.135–267.145) for protegenin C in the extracts of (I), (II), and (III). (D) Mass spectrum of the major protegenin C peak (calc. [M-H]⁻ = 267.1391) as seen in the chromatogram of (I) (obs. [M-H]⁻ = 267.1383, 2.99 ppm error) and (II) (obs. [M-H]⁻ = 267.1382, 3.37 ppm error).

Fig 3

Antibacterial activity of Pf-5-produced polyyne. The assays were conducted by mixing the tested bacterial strains in NAGly and then challenged by Pf-5 derivatives, including the polyyne-producing sixfold mutant and the polyyne nonproducing sevenfold-B mutant. G+: Gram-positive, G−: Gram-negative. Red and yellow frames of the photo indicate inhibition and no inhibition, respectively, of the tested bacteria. The experiment was repeated at least two times independently.

Fig 4

Expression of polyyne biosynthetic genes is controlled by PgnC. (A) Mutation of pgnC abolished the inhibition of Pf-5 to the growth of P. syringae DC3000 on NAGly. (B) Expression of the pgnD_promoter:gfp transcriptional reporter fusion in the sixfold mutant and the sevenfold-C mutant in NBGly. (C) Growth curves of the sixfold mutant and the sevenfold-C mutant in NBGly. (D) Expression of the pgnD_promoter:gfp reporter fusion without pgnC (carried by the ppgnD_promoter:gfp construct) or with pgnC (carried by the ppgnC-pgnD_promoter:gfp construct) in P. fluorescens SBW25 (D) and other tested bacterial strains (E) in NBGly. * Indicates significant difference as determined by the Student t-test. The P value is either less than 0.01 or included. “+” and “−” Indicate activation and no activation of pgnD_promoter:gfp, respectively, by the regulator PgnC. NT: not tested. Data are means ± SD of three replications of each treatment. All the experiments were repeated at least three times, which generated similar results.

Fig 5

Expression of polyyne biosynthesis genes is controlled by the GacA global regulator. (A) The ΔgacA mutant did not inhibit the growth of P. syringae DC3000 on NAGly. (B) Expression of the pgnD_promoter:gfp transcriptional reporter fusion in the wild-type Pf-5 and ΔgacA mutant in NBGly. (C) Growth curves of the wild-type Pf-5 and ΔgacA mutant in NBGly. (D) Expression of the pgnC_promoter:gfp transcriptional reporter fusion in the wild-type Pf-5 and ΔgacA mutant in NBGly. (E) Expression of the pgnC_translation:rfp translational reporter fusion in the wild-type Pf-5 and ΔgacA mutant in NBGly. Data are means ± SD of three replications of each treatment. * Indicates significant difference as determined by the Student t-test (P < 0.01). All the experiments were repeated at least three times, generating similar results.

Fig 6

RsmE interacts with the pgnC leader mRNA. (A) Schematic representation of the upstream region of pgnC. The two black arrows indicate PCR primers to generate a 156 bp mRNA fragment that was labeled with biotin and used in the AlphaScreen assay. (B) Predicted secondary structure of the pgnC leader mRNA (sequences were shown in [A] with an underline) generated by Mfold. The putative RsmE binding site is indicated by gray circles. The start codon of pgnC is also indicated. (C) Evaluation of the synthesized pgnC leader mRNA via agarose gel electrophoresis analysis. (D) Evaluation of the purified RsmE::His protein via SDS-PAGE gel electrophoresis analysis. Lane 1: protein ladder; 2: protein profile of E. coli cells without IPTG induction; 3: protein profile of E. coli cells after IPTG induction; 4: soluble proteins from the lane 3 sample; 5: purified recombinant His-tag RsmE. (E) AlphaScreen assay shows the interaction between RsmE::His-tag and biotinylated pgnC leader mRNA. RsmE::His-tag (1 nM) was amended with different concentrations (1, 5, 10, 50, and 100 nM) of the pgnC leader mRNA. Data are means ± SD of three replications of each treatment. * Indicates significant difference as determined by the Student t-test (P < 0.01). The experiment was repeated at least three times, generating similar results.

Fig 7

Model for regulation of polyyne biosynthesis by PgnC and GacA in P. protegens Pf-5 (1). GacS sensor activates GacA through phosphorylation (2); phosphorylated GacA induces the expression of small regulatory RNAs RsmX, RsmY, and RsmZ (3); the small RNAs sequester the RNA-binding proteins RsmA and RsmE (4); RsmE binds pgnC leader mRNA and blocks its translation (5); pgnC mRNA is translated into PgnC due to the sequestration of RsmE by RsmX, RsmY, and RsmZ (6). PgnC directly activates the transcription of pgn genes, which leads to (7) the biosynthesis of polyyne compounds. The proposed regulation pathway is supported by the results of this study and previous reports (24, 25).