Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions

Abstract

Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 DeltaplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K(2)HPO(4), 3.0 g KH(2)PO(4), 0.1 g MgSO(4).7H(2)O, 0.1 g (NH(4))(2)SO(4), 0.01 g CaCl(2), 0.001 g FeSO(4), 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the DeltaplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and DeltaplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the DeltaplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the DeltaplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.

FIG. 1.

Effect of nutrient conditions on colony morphology. Overnight cultures of B. cereus ATCC 14579 (WT [wild type]) and the ΔplcR mutant were spotted onto EPS, 5×YE EPS, and LB agar plates. (A) Colony morphology was observed after incubation at 32°C for 96 h. Bars, 0.5 cm. (B) Microscopic images of the colony margins of the wild type and the ΔplcR mutant grown on EPS, 5×YE EPS, and LB agars at 32°C for 96 h. Bars, 100 μm.

FIG. 2.

Effect of nutrient conditions on production of flagella. Overnight cultures of the wild type (WT) and the ΔplcR mutant were spotted onto EPS, 5×YE EPS, and LB agars. After incubation at 32°C for 24 and 96 h, cells were lifted from the colony margins, stained, and observed by microscopy for the production of flagella. Bar, 10 μm.

FIG. 3.

(A) Effect of precoating with surfactin on colony morphology. An overnight wild-type (WT) culture was spot inoculated onto EPS agar plates after the plates were coated with 10 μl of 0 or 0.1 mg of surfactin/ml. Colony morphology was observed after 40 h at 32°C (bar, 0.5 cm). Colony margins were observed by microscopy (bar, 100 μm). Cells were lifted from the colony margins, stained, and observed by microscopy for the production of flagella (bar, 10 μm). (B) Effect of precoating with purified biosurfactant from the ΔplcR mutant on colony morphology. Overnight cultures of the wild type and the complemented ΔplcR mutant strain (CM) were spot inoculated onto EPS plates after the plates were coated with 40 μl of Tris-HCl or the purified biosurfactant from the ΔplcR mutant. Colony morphology was observed after 40 h at 32°C (bar, 0.5 cm). Colony margins were observed by microscopy (bar, 100 μm). Cells were lifted from the colony margins, stained, and observed by microscopy for the production of flagella (bar, 10 μm). The results presented are representative of triplicate experiments.

FIG. 4.

TLC analysis of the biosurfactant(s). Partially purified biosurfactant from the wild type (WT), the ΔplcR mutant, or the complemented ΔplcR mutant strain (CM) was run on a silica gel TLC plate and stained with ninhydrin (A) or bromthymol blue (B). The biosurfactant(s) is indicated by the upper arrow. Surfactin (40 μg) was run as a positive control (indicated by the lower arrow). (C) Hemolytic assay. One-hundred-microliter volumes of Tris-HCl buffer, surfactin (5 μg/100 μl buffer), and the biosurfactant of the ΔplcR mutant purified from TLC plates (50 μl/100 μl of buffer) were added to wells of sheep blood agar plates and incubated at 37°C for 48 h. The hemolysis results presented are representative of duplicate experiments.

FIG. 5.

MALDI-TOF MS analysis of the biosurfactant from the ΔplcR mutant, showing an [M+H]⁺ peak at 1,278.1722. Inset, MS-MS fragmentation spectrum of the 1,278.1722 peak.

FIG. 6.

Effect of nutrient conditions on the colony morphology of B. subtilis NCIB 3610. An overnight culture was spotted onto EPS agar and incubated at 32°C. Colony morphology was observed at 12 and 24 h (bar, 0.5 cm). Colony margins were observed by microscopy at 24 h (bar, 100 μm). Cells were lifted from the colony margins, stained, and observed by microscopy for the production of flagella (bar, 10 μm).