Poly-Gamma-Glutamic Acid Secretion Protects Bacillus subtilis from Zinc and Copper Intoxication

Abstract

Zinc and copper are essential micronutrients that serve as a cofactors for numerous enzymes. However, when present at elevated concentrations, zinc and copper are highly toxic to bacteria. To combat the effects of zinc and copper excess, bacteria have evolved a wide array of defense mechanisms. Here, we show that the Gram-positive soil bacterium, Bacillus subtilis, produces the extracellular polymeric substance, poly-gamma-glutamate (γ-PGA) as a protective mechanism in response to zinc and copper excess. Furthermore, we provide evidence that zinc and copper dependent γ-PGA production is independent of the DegS-DegQ two-component regulatory system and likely occurs at a posttranscriptional level through the small protein, PgsE. These data provide new insight into bacterial metal resistance mechanisms and contribute to our understanding of the regulation of bacterial γ-PGA biosynthesis. IMPORTANCE Zinc and copper are potent antimicrobial compounds. As such, bacteria have evolved a diverse range of tools to prevent metal intoxication. Here, we show that the Gram-positive model organism, Bacillus subtilis, produces poly-gamma-glutamic acid (γ-PGA) as a protective mechanism against zinc and copper intoxication and that zinc and copper dependent γ-PGA production occurs by a yet undefined mechanism independent of known γ-PGA regulation pathways.

Keywords: Bacillus subtilis; biofilm; copper; poly-gamma-glutamic acid; zinc.

FIG 1

Zinc and copper induce PGA production. (A) Colony morphology of WT B. subtilis 3610 comI in the presence of 250 μM ZnSO₄, 1 mM CuSO₄, 2 mM FeSO₄, 500 μM MnCl_2. (B) Colony morphology of WT, ΔpgsB, and ΔpgsB P_hyspank-pgsB grown on LB agar in the presence of 250 μM ZnSO₄ or 100 μM CuSO₄. IPTG (1 mM) was also added to ΔpgsB P_hyspank-pgsB to induce pgsB expression. (C) Concentration of γ-PGA produced by cells grown in LB and modified minimal medium in the presence of 250 μM ZnSO₄ or 100 μM CuSO₄. The γ-PGA concentration is expressed as mg of γ-PGA per gram of cell wet weight. Data shown are the mean and standard deviation from three independent experiments. P values were calculated by one-way ANOVA. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 2

PGA protects B. subtilis from zinc and copper intoxication. (A) Disk diffusion susceptibility test performed on WT, ΔpgsB, and ΔpgsB P_hyspank-pgsB grown on LB agar. Five μL of 500 μM ZnSO₄ or 5 μL of 100 μM CuSO₄ was added to a filter disk. The zone of inhibition was determined by measuring the diameter of the zone of clearing. Data shown are the mean and standard deviation from three different experiments. P values were calculated by one-way ANOVA. ns, nonsignificant; **, P ≤ 0.01. (B and C) Growth curves of WT, ΔpgsB, and ΔpgsB P_hyspank-pgsB in the presence and absence of either (B) 250 μM ZnSO₄ or (C) 100 μM CuSO₄. IPTG (1 mM) was also added to ΔpgsB P_hyspank-pgsB to induce pgsB expression. Data shown are the mean and standard deviation from three independent experiments.

FIG 3

Zinc and copper dependent PGA production is not controlled at the level of transcription. (A) Colony morphology of WT and ΔdegU grown in LB agar in the presence of 250 μM ZnSO₄ or 1 mM CuSO₄. (B) pgs operon (pgsEΩlacZ) and P_pgdS promoter activity in cells grown in LB media amended with various concentrations of ZnSO₄ or CuSO₄. Data shown are the mean and standard deviation from three independent experiments. No significant difference was detected between samples by one-way ANOVA.

FIG 4

Overexpression of PgsE enhances Zn and Cu-dependent γ-PGA production. Concentration of γ-PGA produced by WT and PgsE overexpressing cells grown in the presence of 250 μM ZnSO₄ or 100 μM CuSO₄. IPTG (1 mM) was added to induce PgsE overexpression from the hyspank promoter. The γ-PGA concentration is expressed as mg of γ-PGA per gram of cell wet weight. Data shown are the mean and standard deviation from three independent experiments. P values were calculated by two-way ANOVA (****, P ≤ 0.0001).