Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells

Abstract

Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

Keywords: Bacillus subtilis; antimicrobial; autolysin; autolysis; comet assay; lytic rate; mechanism; prodigiosin.

FIGURE 1

The effect of prodigiosin addition on Bacillus subtilis PS-216 wt growth in LB medium at 37°C and 200 rpm at different growth phases. The arrows represent the time of addition of 5.9 mg L^-1 prodigiosin (opened squares) or 0.19% (V/V) ethanol in the control samples (filled squares). (A) cells treated in the middle of the exponential phase; (B) cells treated in the late exponential phase; (C) cells treated in the stationary phase. Data are presented as averages and standard deviations (n = 3).

FIGURE 2

Bacillus subtilis PS-216 wt cells (A) and B. subtilis PS-216 amyE::P_hyperspank-mKate2(B) grown in LB medium at 37°C and 200 rpm observed by DIC and fluorescent microscopy at 0, 1, and 5 h after the addition of either 5.9 mg L^-1 of prodigiosin or 0.19% (V/V) of ethanol in control samples in the middle of the exponential phase. The scale bar represents 10 μm.

FIGURE 3

Relative β-galactosidase activity upon addition of 5.9 mg L^-1 prodigiosin and 0.01% (w/V) SDS in B. subtilis PS-216 srfA-lacZ (neo) cells compared with control [addition of 0.19% (V/V) ethanol]. Cells were grown in LB medium at 37°C and 200 rpm. Data are presented as averages and standard errors (n = 3–6).

FIGURE 4

Lytic rates of different lyt mutants measured as a rate of OD₆₅₀ decrease after treatment with prodigiosin (5.9 mg L^-1) in LB medium at 37°C and 200 rpm. Data are presented as averages and standard deviations (n = 3).

FIGURE 5

The modified comet assay for studying prodigiosin mediated cell lysis of B. subtilis PS-216 wt cells grown in LB medium at 37°C and 200 rpm. (A) non-treated bacterial cells; (B) control cells treated with 0.19% (V/V) of ethanol; (C) cells treated with 5.9 mg L^-1 of prodigiosin. Cells were treated in the middle exponential phase and were inspected 1 h after the treatment. The gels were stained with GelRed^TM. The images were observed with epifluorescent microscopy.

FIGURE 6

The modified comet assay on different autolysin deficient strains of B. subtilis PS-216 cells grown in LB medium at 37°C and 200 rpm treated with 0.19% (V/V) of ethanol (control) or treated with 5.9 mg L^-1 of prodigiosin. Cells were treated in the middle exponential phase and were inspected 1 h after the treatment. The gels were stained with GelRed^TM. The images were observed with epifluorescent microscopy.