Phenazine-1-carboxylic Acid Produced by Pseudomonas chlororaphis YL-1 Is Effective against Acidovorax citrulli

Abstract

The bacterial pathogen Acidovorax citrulli causes the destructive fruit blotch (BFB) on cucurbit plants. Pseudomonas chlororaphis YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a sacB-based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants ΔphzE and ΔphzF almost lost the inhibitory effects against A. citrulli in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in A. citrulli. The inhibition effect of PCA against A. citrulli was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of katB from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.

Keywords: Acidovorax citrulli; Pseudomonas chlororaphis; phenazine-1-carboxylic acid.

Figure 1

Antibacterial activity of P. chlororaphis YL-1 and its seven mutants on LB plates. All strains precultured in liquid LB medium at 28 °C and 200 rpm for 24 h were resuspended in distilled liquid LB medium to the desired cell density of 10⁸ CFU/mL. After spraying the suspension of A. citrulli (10⁸ CFU/mL) onto the surface of LB plates for 1 s, 5 μL cells of strain YL-1 or mutants were injected into the central hole (5 mm) of LB plates and incubated at 28 °C for 48 h. Distilled LB medium was used as a control. The inhibitory zone diameters of strain YL-1 and mutants were measured when A. citrulli covered the control plates entirely. Each treatment was replicated three times, and each experiment was repeated thrice independently.

Figure 2

Reversed phase (RP)-HPLC chromatograms. An overlay of the chromatograms at 254 nm of STD sample (blue) and the final extraction step of the wild-type YL-1 (pink) (a), mutants ΔphzE (green), and ΔphzF (black) (b), using an XDB-C18 reversed-phase column (4.6 mm × 150 mm, 5 μm, Agilent), is shown. Here, STD sample (blue) was used as a positive control. The samples were eluted with acetonitrile (ACN)-5 mM ammonium acetate (60:40, v/v) at a flow rate of 0.7 mL/min. The experiment was repeated three times.

Figure 3

MS spectrum at 2.0 min. The flow rate was maintained at 0.4 mL/min, and the mobile phase comprised H₂O with 0.5% acetic acid (A) and ACN with 0.5% acetic acid (B) as follows: 0 min, 0% B; 0–5 min, 50% B; 5–13 min, 100% B; 13–23 min, 100% B, 23–25 min, 0% B, and 25–28 min, 0% B. The detecting wavelength was 254 nm and the sample volume injected was 10 µL. The ESI source parameters were set as follows: positive ion mode, 3 kV spray voltage, 350 °C capillary temperature, nitrogen served as both the sheath gas (35 units) and auxiliary gas (10 units). The proposed three characteristic fragment ions of PCA produced by P. chlororaphis YL-1 (blue) were consistent with STD sample (blue). The experiment was repeated three times.

Figure 4

Inhibition of PCA against A. citrulli on LB plates. The one-, ten-, and fifty-fold dilutions (1×, 10×, and 50×) of the bacterial suspension were spotted onto LB plates, which contained different concentrations of PCA at 0, 2, 4, 8, 16, and 32 μg/mL. Inoculated plates were incubated at 28 °C for 72 h. Each treatment was replicated three times, and each experiment was repeated thrice independently.

Figure 5

Inhibition effect of PCA at different concentrations on A. citrulli when cultured in liquid LB medium (200 rpm; 28 °C for 12 h). The experiment was repeated three times.

Figure 6

Expression of oxyR, soxR, and the genes involved in antioxidant systems in A. citrulli as affected by PCA. Strain XJX12 was grown in liquid LB medium containing acetone solution without or with PCA to OD₆₀₀ values of 0.1 (with culture time 6 h) (a), 0.2 (with culture time 8 h) (b), and 0.35 (with culture time 10 h) (c). The amount of RNA in acetone was used as the control and was set at 1.0. The XJ-RT gene (GenBank number: CP000512.1) was used as an internal control in the qRT-PCR assay. The experiment was repeated three times.

Figure 7

ROS accumulation (a) and CAT (b) activity of A. citrulli strain XJX12 induced by PCA. The treatments included PCA in acetone and acetone alone. ROS accumulation was measured based on fluorescence in microplates, the wells of which contained a bacterial suspension and no additional agent (the negative control) or one of the following: PCA at 0, 16, or 32 μg/mL, with 0.4% (v/v) acetone (as the solvent) in each case; acetone alone (0.4%, v/v); or Rosup reagent (a positive control provided with the assay kit). The experiment was repeated three times.

Figure 8

Inhibition effect of PCA against A. citrulli by adding exogenous CAT, 0, 25, 50, 75, or 100 U. The experiment was repeated three times.