PRODIGIOSIN INDUCES AUTOLYSINS IN ACTIVELY GROWN Bacillus subtilis CELLS

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 B. 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 two hours. 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

Prodigiosin - A Multifaceted Escherichia coli Antimicrobial Agent.

Despite a considerable interest in prodigiosin, the mechanism of its antibacterial activity is still poorly understood. In this work, Escherichia coli cells were treated with prodigiosin to determine its antimicrobial effect on bacterial physiology. The effect of prodigiosin was concentration dependent. In prodigiosin treated cells above MIC value no significant DNA damage or cytoplasmic membrane disintegration was observed. The outer membrane, however, becomes leaky. Cells had severely decreased respiration activity. In prodigiosin treated cells protein and RNA synthesis were inhibited, cells were elongated but could not divide. Pre-treatment with prodigiosin improved E. coli survival rate in media containing ampicillin, kanamycin and erythromycin but not phleomycin. The results suggest that prodigiosin acts as a bacteriostatic agent in E. coli cells. If prodigiosin was diluted, cells resumed growth. The results indicate that prodigiosin has distinct mode of antibacterial action in different bacteria

Survival of <i>E</i>. <i>coli</i> MG1655 cells pre-treated with prodigiosin followed by treatment with antibiotics.

<p>Cells were pre-treated with 120 mg L^-1 prodigiosin (PG) or in control with ethanol (EtOH). Tested antibiotic concentrations were 100 mg L^-1 ampicillin (A), 100 mg L^-1 erythromycin (B), 100 mg L^-1 kanamycin (C) and 5 mg L^-1 phleomycin (D). Data are presented as averages and standard deviations (n = 3). Asterisks represent statistically significant differences between ethanol and prodigiosin pre-treatment (p < 0.05). Note the difference in y-axis ranges.</p

The influence of different prodigiosin concentrations on <i>E</i>. <i>coli</i> MG1655 growth.

<p>Cells were grown in LB medium at 37°C and 200 rpm and treated in the middle of the exponential phase with 10 (A), 60 (B) and 120 mg L^-1 (C) of prodigiosin (filled symbols) or non-treated control (open symbols). The arrows represent the time of prodigiosin or ethanol addition. Data are presented as averages and standard deviations (n = 3).</p

Viability of <i>E</i>. <i>coli</i> MG1655 cells treated with prodigiosin.

<p>Cells were grown in LB medium at 37°C, 200 rpm, and inspected under inverted epifluorescence microscope before, 1, and 5 hours after addition of either 120 mg L^-1 of prodigiosin or 4.44% (V/V) of ethanol in control samples in the middle of the exponential phase. Images were obtained using fluorescence filters for green fluorescent dye SYTO 9 and red fluorescent dye propidium iodide. Data are presented as averages and standard deviations (n ≥ 3).</p

Protein and total RNA content in <i>E</i>. <i>coli</i> MG1655 cells treated with prodigiosin.

<p>Cells were treated with 120 mg L^-1 prodigiosin, 4.44% (V/V) ethanol or sterile deionized water as a control in the middle of the exponential phase. Data are presented as averages and standard deviations (n ≥ 3).</p

The modified comet assay for studying prodigiosin addition to <i>E</i>. <i>coli</i> MG1655 cells grown in LB medium at 37°C and 200 rpm.

<p>A—non-treated bacterial cells; B—control cells treated with 4.44% (V/V) of ethanol; C—cells treated with 120 mg L^-1 of prodigiosin; D—positive control cells treated with 100 mg L^-1 of ampicillin. Cells were treated in the middle of the exponential phase and were inspected 1 h after the treatment. Cells were stained with GelRed^™ and observed by epifluorescence microscopy. The scale bar represents 1 μm.</p

CO₂ production of <i>E</i>. <i>coli</i> MG1655 cells treated with prodigiosin.

<p>Cells were treated with 120 mg L^-1 of prodigiosin (filled symbols) during incubation in LB medium at 37°C and 200 rpm, control—ethanol treated (open symbols). CO₂ was measured by gas chromatography. The arrow represents the time of prodigiosin or ethanol addition. Data are presented as averages and standard deviations (n = 4).</p

β—lactamase activity of <i>E</i>. <i>coli</i> MG1655 cells determined as nitrocefin hydrolysis.

<p>The control (white columns), cells treated with 120 mg L^-1 prodigiosin (black columns). Cells were grown in LB medium at 37°C and 200 rpm. Data are presented as averages and standard errors (n = 3).</p