Abstract
We isolated a Bacillus strain, RX7, with inhibitory activity against Listeria monocytogenes from soil and identified it as Bacillus amyloliquefaciens based on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action of α-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungus Candida albicans. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH2-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1) from Bacillus halodurans, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin.
Highlights
Bacteriocins are ribosomally synthesized antimicrobial peptides which are secreted to act against closely related bacterial species without affecting the producing strain [1]
Lactic acid bacteria (LAB) bacteriocins are studied widely due to their potential use as biopreservatives in the food industry because many strains have been “generally recognized as safe” (GRAS) status [4]
It was expressed as the diameter of the inhibition zones around the wells using agar well diffusion assays with Listeria monocytogenes ATCC 19114 as the indicator strain
Summary
Bacteriocins are ribosomally synthesized antimicrobial peptides which are secreted to act against closely related bacterial species without affecting the producing strain [1]. To address increasing bacterial resistance to conventional antibiotics, bacteriocins are considered as alternative antimicrobials for the treatment of human (and possibly animal) infections [2]. Since minimally processed foods with no chemical preservatives are in demand by consumers, research into natural antimicrobial agents such as bacteriocins [3] has been increasing. Lactic acid bacteria (LAB) bacteriocins are studied widely due to their potential use as biopreservatives in the food industry because many strains have been “generally recognized as safe” (GRAS) status [4]. The search for novel bacteriocins with improved biochemical properties, including stability over a wide pH range, thermostability, and a broad antimicrobial spectrum, is of great interest for applications in foods
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