Characterization of Subtilin L-Q11, a Novel Class I Bacteriocin Synthesized by Bacillus subtilis L-Q11 Isolated From Orchard Soil

Abstract

Bacteriocins are peptides or proteins synthesized by bacterial ribosomes that show killing or inhibitory activities against different groups of bacteria. Bacteriocins are considered potential alternatives to traditional antibiotics, preservatives in pharmaceutical and food industries. A strain L-Q11 isolated from orchard soil was phylogenetically characterized as Bacillus subtilis based on 16S rRNA gene sequencing analysis. A novel class I bacteriocin (Subtilin L-Q11), was identified and purified from L-Q11 cell-free supernatant in a four-step procedure, including salt precipitation, cation exchange, gel filtration, and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass (3,552.9 Da) of this novel bacteriocin was determined by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The purified Subtilin L-Q11 exhibited optimal features in pH tolerance, thermostability, and sensitivity to proteases. Further, Subtilin L-Q11 showed inhibitory activities against a number of bacteria including some human pathogens and food spoilage bacteria, in particular Staphylococcus aureus. All these important features make this novel bacteriocin a potential candidate for the development of a new antibacterial drug or food preservative in the future.

Keywords: Bacillus subtilis; Subtilin L-Q11; antibacterial activity; antibacterial mechanism; bacteriocin.

FIGURE 1

Growth and dynamics of bacteriocin production by B. subtilis L-Q11. “” represents the growth curve of L-Q11; “” represents values of pH in the culture medium; “” represents inhibitory activity against the indicator strain S. aureus ATCC 29213.

FIGURE 2

Purification of Subtilin L-Q11 by chromatography. (A) cation exchange column; (B) gel filtration chromatography; (C) RP-HPLC. (a) process of purification; (b) the assay of antibacterial activity against the indicator strain from the absorbance peaks versus CK (control) by agar well diffusion assay; (c) Tricine-SDS-PAGE of the purified active fraction. The arrows indicate the active fraction after each step of purification on Tricine-SDS-PAGE gel.

FIGURE 3

Molecular mass determination and amino acid sequence alignment of Subtilin L-Q11. (A) The mass spectrum shown corresponded to the absorbance peak after purification using RP-HPLC in Figure 2C. (B) Alignment of published class I bacteriocins from Bacillus spp. and Subtilin L-Q11. Alignments were obtained by SnapGene V4.2.6 with default settings.

FIGURE 4

Subtilin L-Q11 demonstrated optimal thermostability, pH tolerance, and resistance to chemical reagents. The effects of pH (A), temperature (B) and surfactant (C) on the bacteriocin activity produced by L-Q11 were assayed. Relative ratios in percentages were applied to represent retained antibacterial activities of the bacteriocin samples after various treatments when compared to the untreated control group. “abcde” indicates the significant difference among different conditions, the same letter represents no significant differences among those groups and different letters represents significant differences (p < 0.05) among those groups.

FIGURE 5

Subtilin L-Q11 treatment triggered cell lysis in S. aureus. “” represents viable cell count of untreated S. aureus ATCC 29213; “Δ” represents viable cell count of S. aureus ATCC 29213 with the treatment of bacteriocin; “” represents cell optical density at the wavelength of 600 nm (O.D.₆₀₀) without bacteriocin treatment; “” represents optical density at the wavelength of 600 nm with bacteriocin treatment.

FIGURE 6

Scanning electron microscopy of S. aureus cells treated by Subtilin L-Q11. (A) S. aureus ATCC 29213 cells from the untreated control group; (B–D) S. aureus ATCC 29213 cells treated with 256 μg/mL (4 × MIC₅₀) of Subtilin L-Q11 for 1, 2, and 3 h, respectively. Scale bars: 500 nm. Black arrows: the cell hollowness and membrane disruption; red arrows: cell fragments. For the sample preparation of TEM and SME, cell cultures were concentrated by centrifuging. During the sample preparation, large amount of the lysed cell (cell fragments) would lost.

FIGURE 7

Transmission electron microscopy of S. aureus cells treated by Subtilin L-Q11. (A) S. aureus ATCC 29213 cells from the untreated control group; (B–D) S. aureus ATCC 29213 cells treated with 256 μg/mL (4 × MIC₅₀) of Subtilin L-Q11 for 1, 2, and 3 h, respectively. Scale bars: 200 nm. Arrows numbered 1: the damaged cell membrane. Arrows numbered 2: the leaked intracellular substance. The arrow numbered 3: the ruptured cells.