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. 2021 Nov 24:12:789362.
doi: 10.3389/fmicb.2021.789362. eCollection 2021.

Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [ Camellia sinensis var. assamica (J.W.Mast.) Kitam.]

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Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [ Camellia sinensis var. assamica (J.W.Mast.) Kitam.]

Patthanasak Rungsirivanich et al. Front Microbiol. .

Abstract

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75-100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

Keywords: Miang; RT-qPCR; amylocyclicin; bacteriocin; biocontrol; gene cluster; plipastatin; surfactin.

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Conflict of interest statement

The authors declare this study received funding from Tea Gallery Group (Thailand) Co., Ltd. and Amazing Tea Limited Partnership. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. All authors declare no other competing interests.

Figures

Figure 1
Figure 1
Schematic representation of antimicrobial purification by (A) RP-HPLC and (B) by solvent extraction assay (for the partial purification of surfactin).
Figure 2
Figure 2
Schematic image of the gene clusters associated with the ribosomally synthesized peptides, Mersacidin (A) and amylocyclicin (B) of strains B. velezensis ML122-2 (red), B. velezensis FZB42 and B. sp. HIL Y-85,54,728. Predicted functions and sequence similarity are color-coded as indicated.
Figure 3
Figure 3
The gene clusters associated with the non-ribosomally synthesized peptides, bacilysin (A), bacillibactin (B), surfactin (C), macrolactin H (D), and plipastatin (E), and polyketide, bacillaene (F), of B. velezensis ML122-2 (red) compared to equivalent clusters in reference Bacillus strains. The predicted functions and sequence similarity are color-coded according to the legend.
Figure 4
Figure 4
MALDI-TOF mass spectra of IPA supernatant from cell extract of B. velezensis ML122-2 (A). Surfactin, amylocyclicin, and plipastatin were detected at m/z 1,059.25, 1,464.33 and 6,381.58, respectively. (B,C) Purification of amylocyclicin and plipastatin by RP-HPLC. The active fractions eluted at time interval of 25–28 and 31–37min in a gradient of 40–85% propan-2-ol 0.1% trifluoroacetic acid represented the peaks of purified amylocyclicin (m/z 6,381.58 [M+H]+ and 3,192.25, the doubly charged form) and plipastatin (m/z 1,462.57, 1,484.46 1,492.32, 1520.43 and 1542.54), respectively. (D) Partial surfactin purification via solvent extraction of B. velezensis ML122-2 from supernatant revealing corresponding masses at m/z 1,032.29, 1,046.25, 1,060.30 and 1,104.03.
Figure 5
Figure 5
Transcriptional activity of mrsA, ancA, ppsA, and srfAA genes in 24 and 48h of B. velezensis cultivation. The housekeeping gene rpsE was used as the reference gene.

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