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. 2022 Jul 29:13:943232.
doi: 10.3389/fmicb.2022.943232. eCollection 2022.

A novel of new class II bacteriocin from Bacillus velezensis HN-Q-8 and its antibacterial activity on Streptomyces scabies

Jing Zhao  1   2 , Zhijun Zhou  3 , Xuefei Bai  1   2 , Dai Zhang  1   2 , Likui Zhang  4 , Jinhui Wang  1   2 , Beibei Wu  1   2 , Jiehua Zhu  1   2 , Zhihui Yang  1   2
Affiliations

A novel of new class II bacteriocin from Bacillus velezensis HN-Q-8 and its antibacterial activity on Streptomyces scabies

Jing Zhao et al. Front Microbiol. .

Abstract

Potato common scab is a main soil-borne disease of potato that can significantly reduce its quality. At present, it is still a challenge to control potato common scab in the field. To address this problem, the 972 family lactococcin (Lcn972) was screened from Bacillus velezensis HN-Q-8 in this study, and an Escherichia coli overexpression system was used to obtain Lcn972, which showed a significant inhibitory effect on Streptomyces scabies, with a minimum inhibitory concentration of 10.58 μg/mL. The stability test showed that Lcn972 is stable against UV radiation and high temperature. In addition, long-term storage at room temperature and 4°C had limited effects on its activity level. The antibacterial activity of Lcn972 was enhanced by Cu2+ and Ca2+, but decreased by protease K. The protein was completely inactivated by Fe2+. Cell membrane staining showed that Lcn972 damaged the cell membrane integrity of S. scabies. Scanning electron microscope (SEM) and transmission electron microscope (TEM) observations revealed that the hyphae of S. scabies treated with Lcn972 were deformed and adhered, the cell membrane was incomplete, the cytoplasm distribution was uneven, and the cell appeared hollow inside, which led to the death of S. scabies. In conclusion, we used bacteriocin for controlling potato common scab for the first time in this study, and it provides theoretical support for the further application of bacteriocin in the control of plant diseases.

Keywords: Bacillus; Lcn972; Streptomyces scabies; potato; potato common scab.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Lcn972 secreted by Bacillus velezensis HN-Q-8. (A) Phylogenetic analysis of Lcn972; (B) comparison of the amino acid sequence of Lcn972 from B. velezensis HN-Q-8 with these of Lcn972 from other Bacillus spp.
FIGURE 2
FIGURE 2
Obtain of Lcn972 and its antibacterial activity. (A) Obtaining of Lcn972. Lane 1, Lcn972; Lane 2, control; (B) double-digestion verification. Lane 1, Lcn972; Lane2, double digestion of pET-30a-Lcn972; (C) expression of Lcn972. Lane 1, before expression of Lcn972; Lane 2, after expression of Lcn972; Lane 3, total protein containing Lcn972; (D) total protein containing Lcn972 purified by heating; (E) Lcn972 purified by nickel column; (F) antibacterial activity of total protein containing Lcn972 on Streptomyces scabies HP4 after heating; (G) minimum inhibitory concentration (MIC) of Lcn972 against S. scabies HP4, (H) optimal inhibitory concentration of Lcn972 against S. scabies HP4. Letters on the graph denote statistically significant differences (ANOVA, P < 0.05).
FIGURE 3
FIGURE 3
Sensitivity of Lcn972. (A) Effect of NaCl concentration on the antibacterial activity of Lcn972; (B) effect of pH on the antibacterial activity of Lcn972; (C) sensitivity of Lcn972 to UV; (D) sensitivity of Lcn972 to proteases; (E) sensitivity of Lcn972 to metal ions. Letters on the graph denote statistically significant differences (ANOVA, P < 0.05).
FIGURE 4
FIGURE 4
Effect of storage at different temperatures on the activity of Lcn972. (A) 4°C; (B) room temperature.
FIGURE 5
FIGURE 5
Fluorescent staining of cell membranes of Streptomyces scabies HP4 treated with Lcn972. The mycelia were treated with (A) Buffer A (negative control); (B) 60 ng/L kanamycin (positive control); (C) 0.5% TritonX-100 (positive control); (D) 2 × MIC Lcn972. Each column represents one sample.
FIGURE 6
FIGURE 6
Scanning electron micrographs of S. scabies treated with Lcn972. (a,b): non-inhibition zone; (c,d): edge of the inhibition zone; (e,f): inhibition zone.
FIGURE 7
FIGURE 7
Transmission electron micrographs of S. scabies treated with Lcn972. (A) without Lcn972; (B) with Lcn972.
FIGURE 8
FIGURE 8
Effect of Streptomyces scabies on the expression of Lcn972. (A) Growth curve of Bacillus velezensis HN-Q-8; (B) relative expression of Lcn972. CK: B. velezensis HN-Q-8 added to Gauze’s synthetic broth medium; Treatment: B. velezensis HN-Q-8 added to cell-free culture medium of S. scabies HP4. **P < 0.01, one-way ANOVA, followed by Dunnet post-test, compared to control.
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