Bacillus safensis APC 4099 has broad-spectrum antimicrobial activity against both bacteria and fungi and produces several antimicrobial peptides, including the novel circular bacteriocin safencin E

Abstract

Bacillus safensis APC 4099, isolated from bees' gut, has been identified as a promising candidate for food biopreservation. Antimicrobial activity screening revealed a broad-spectrum inhibition potential, ranging from gram-positive pathogenic bacteria to fungi responsible for food spoilage. Genomic analysis identified biosynthetic gene clusters coding for several antimicrobial peptides and secondary metabolites. Specifically, a novel, anionic, 6 kDa circular bacteriocin, named safencin E, was detected, showing 52.5% similarity to butyrivibriocin AR10. Additionally, gene clusters coding for the biosynthesis of bacteriocins such as pumilarin and plantazolicin and biosynthetic pathways for secondary metabolites, including pumilacidin A, bacilysin, and bacillibactin, were identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis detected molecular masses correlating to safencin E, plantazolicin, pumilarin, and pumilacidin A from the cell-free supernatant, cell extracts, or both. Overall, the broad-spectrum antimicrobial activity of B. safensis APC 4099 indicates that this strain is a promising candidate for the biological control of food ecosystems and thus has the potential to enhance food safety.

Importance: The present article highlights the importance of the strain Bacillus safensis APC 4099 as a potential biocontrol agent. The strain possesses biosynthetic gene clusters coding for various antimicrobial peptides and secondary metabolites, including a novel circular bacteriocin, safencin E, and the bacteriocins pumilarin and plantazolicin. This diversity in the production of antimicrobial peptides renders the producer with broad-spectrum antimicrobial activity, ranging from gram-positive pathogenic and spoilage bacteria to spoilage molds. Considering that 1.3 billion tons of food appropriate for human consumption is lost or wasted annually, identifying strains or novel antimicrobial peptides capable of biopreservation is highly relevant. This strain and its bioactive compounds offer a solution to this global problem as biocontrol agents for food ecosystems against spoilage and pathogenic microbes.

Keywords: Bacillus safensis; antimicrobial peptides; bacillibactin; bacilysin; circular bacteriocin; food biopreservation; plantazolicin; pumilacidin; pumilarin; safencin E.

Fig 1

Well diffusion assay of B. safensis APC 4099 CFS against a selection of bacterial and fungal indicator strains.

Fig 2

Effect of the active CFS (5%, 10%, 25%, and 50%) and no CFS (control) on the growth of bacterial and fungal indicators, according to the recorded OD_{590 nm} value over time (48 h).

Fig 3

Safencin E, pumilarin, and plantazolicin gene clusters. Genes are color-coded according to the putative role of each protein.

Fig 4

Schematic representation of the similarity between the gene clusters associated with lichenysin, bacilysin, and bacillibactin, and the identified gene clusters detected in B. safensis APC 4099 genome. Genes are color-coded according to the putative role of each protein.

Fig 5

Structural characteristics of safencin E, and pumilarin. (A) Sequence alignment among the Class IIc circular bacteriocins acidocin B, butyrivibriocin AR10, gassericin A, plantaricyclin A, plantacyclin_B21AG, and safencin E, highlighting in blue the conserved amino acids. (B) Large letters in the Web Logo represent the conserved amino acids among the Class IIc circular bacteriocins acidocin B, butyrivibriocin AR10, gassericin A, plantaricyclin A, plantacyclin_B21AG, paracircularin, and safencin E. The hydrophobic, hydrophilic, acidic, and basic amino acids are shown in black, green, blue, and red, respectively. (C) Phylogenetic tree showing the similarity of safencin E with other identified circular bacteriocins based on the amino acid sequences of the mature peptide. Bar 0.5 indicates nucleotide substitutions per site. Safencin E and pumilarin are colored red and blue, respectively. (D) Predicted tertiary structures of safencin E and pumilarin. The degree of lipophilicity ranges from −20 to 0 to 20, and is colored blue-white-brown, respectively. Root mean square distance (RMSD) indicates the average distance of pairs of residues between the model and template. (D1) Safencin E comprises four α-helices structures, among which coil structures interfere (C-score = 0.54, TM-Score = 0.79 ± 0.09, RMSD = 1.9 ± 1.6 Å). (D2) Pumilarin is composed of five α-helices structures, among which coil structures interfere (C-score = 1.19, TM-score = 0.88 ± 0.07, RMSD = 1.1 ± 1.1 Å). Safencin E and pumilarin create a circular structure, according to which the hydrophobic amino acids hide inside the structure. The C-score and the TM-scores evaluate the global accuracy of the 3D structure model and the range from −5 to 2 and >−1.5, respectively, implies a model with correct global topology.

Fig 6

Colony MALDI-TOF mass spectrum showing the molecular masses of putative lipopeptide (1,072 Da), plantazolicin (1,336 Da), safencin E (5,999 Da), and pumilarin (7,091 Da).

Fig 7

(A) HPLC chromatogram of the CFS of B. safensis APC 4099 showing elution of plantazolicin (1) at 31 min, pumilarin (2) at 64 min, lipopeptides (3) between 68 and 78 min and safencin E (4) at 81 min. (B) MALDI TOF MS of HPLC fractions showing the expected molecular mass for (1) plantazolicin (1,353 ± 3 Da modified 1,336 Da), (2) pumilarin (7,089.99 ± 3 Da, (3) lipopeptide 1,072 ± 3 Da, and (4) safencin E 5996.70 ± 3 Da. (C) Antimicrobial activity of HPLC fractions containing plantazolicin (1), pumilarin (2), lipopeptide (3), and safencin E (4) against L. bulgaricus LMG6901.