Co-production of surfactin and fengycin by Bacillus subtilis BBW1542 isolated from marine sediment: a promising biocontrol agent against foodborne pathogens.

Pathogenic bacteria contaminations and related diseases in food industries is an urgent issue to solve. The present study aimed to explore natural food biopreservatives from microorganisms. Using dilution-plate method, a strain BBW1542 with antimicrobial activities against various foodborne pathogenic bacteria was isolated from the seabed silt of Beibu Gulf, which was identified as Bacillus subtilis by the morphological observation and 16S rDNA sequences. The antimicrobial substances of B. subtilis BBW1542 exhibited an excellent stability under cool/heat treatment, UV irradiation, acid/alkali treatment, and protease hydrolysis. The genome sequencing analysis and antiSMASH prediction indicated that B. subtilis BBW1542 contained the gene cluster encoding lipopeptides and bacteriocin subtilosin A. MALDI-TOF-MS analysis showed that the lipopeptides from B. subtilis BBW1542 contained C14 and C15 surfactin homologues, together with fengycin homologues of C18 fengycin A/C16 fengycin B and C19 fengycin A/C17 fengycin B. In silico analysis showed that an eight-gene (sboA-albABCDEFG) operon was involved in the biosynthesis of subtilosin A in B. subtilis BBW1542, and the encoded subtilosin A presented an evident closed-loop structure containing 35 amino acids with a molecular weight of 3425.94 Da. Overall, the antagonistic B. subtilis BBW1542 displayed significant resource value and offered a promising alternative in development of food biopreservation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05864-3.

The presence of sboA and spaS genes and antimicrobial peptides subtilosin A and subtilin among Bacillus strains of the Amazon basin.

This report demonstrates the usefulness of PCR for the genes spaS and sboA as a means of identifying Bacillus strains with a potential to produce subtilin and subtilosin A. One collection strain and five Bacillus spp. isolated from aquatic environments in the Amazon basin were screened by PCR using primers for sboA and spaS designed specifically for this study. The sequences of the PCR products showed elevated homology with previously described spaS and sboA genes. Antimicrobial peptides were isolated from culture supernatants and analyzed by mass spectrometry. For all samples, the mass spectra revealed clusters with peaks at m/z 3300-3500 Da, corresponding to subtilosin A, subtilin and isoforms of these peptides. These results suggest that the antimicrobial activity of these strains may be associated with the production of subtilosin A and/or subtilin. The PCR used here was efficient in identifying novel Bacillus strains with the essential genes for producing subtilosin A and subtilin.

Crystal structure of the AlbEF complex involved in subtilosin A biosynthesis.

Subtilosin A is a sactipeptide bacteriocin produced by Bacillus subtilis strain 168, containing intramolecular thioether bonds and a head-to-tail macrocyclic peptide bond. Macrocyclization is presumably catalyzed by AlbE and AlbF proteins encoded by the subtilosin A biosynthesis gene cluster. However, the underlying mechanism of macrocyclization remains uncertain as the tertiary structures of the proteins are undetermined. Here, we report the crystal structures of AlbE and AlbF homologs in Quasibacillus thermotolerans, wherein the subtilosin biosynthesis gene cluster is highly conserved. Structural analysis and pull-down assays revealed that AlbE and AlbF form heterodimeric complexes. Although the AlbEF complex shows structural similarity to M16B family metalloproteases, the substrate-binding chamber is shallower and more open than the other M16B family proteins. The chamber surface showed electrostatic complementarity to the precursor of subtilosin. Our findings provide insights into the role of AlbEF in metalloprotease catalysis and macrocyclic peptide bond formation.

Oxygen-Limiting Growth Conditions and Deletion of the Transition State Regulator Protein Abrb in Bacillus subtilis 6633 Result in an Increase in Subtilosin Production and a Decrease in Subtilin Production.

It has been recently shown, that certain strains/isolates of Bacillus subtilis can be used as a probiotic for humans. The production of the macrocyclic sactibiotic subtilosin in B. subtilis ATCC 6633 is highly regulated. To improve the subtilosin productivity of B. subtilis, different growth conditions were compared for maximal expression of the sbo promoter that regulates the expression of the subtilosin biosynthetic gene cluster. Oxygen-limiting conditions led to a strong increase of sbo promoter activities compared to aerobic conditions, and accordingly, the subtilosin amount determined by reversed phase HPLC (7.8 mg/L) was 15-fold superior to the amount of aerobic grown cultures (0.5 mg/L). A further promising enhancement of the subtilosin yield was achieved using a deletion mutant that is avoiding the general transition state regulator protein AbrB. The subtilosin titer of 42 mg/L produced by ΔabrB cells grown under oxygen-limiting conditions corresponds to an 84-fold increase compared to the subtilosin titer obtained from B. subtilis wild type cells propagated in aerobic conditions. Furthermore, evidence is provided that oxygen-limiting conditions led to a strong decrease in the productivity of the lantipeptide subtilin suggesting contrary regulatory mechanisms for the B. subtilis antimicrobials subtilin and subtilosin.

Bacterial Labionin-Containing Peptides and Sactibiotics: Unusual Types of Antimicrobial Peptides with Potential Use in Clinical Settings (a Review).

One of the biggest challenges faced presently by clinicians is the emergence of multidrug -resistant pathogens that can infect humans and animals. To control the infections caused by such pathogens the development of new drugs is required. Bacteria are a rich source of ribosomally -synthesized antimicrobial peptides known as bacteriocins, which are characterized by the presence of a self-defense immunity system. Labionin-containing lantibiotics and sactibiotics are posttranslationally modified bacteriocins with peculiar features. Labionin-containing peptides belong to subclass Ic lantibiotics in which the carbacyclic triamino triacid labionin, a structural variant of lanthionine, and a methyl-substitute labionin derivative are found, giving the molecule a labyrinthine structure. Sactibiotics are circular or linear peptides belonging to a distinct bacteriocin class (class V) which is characterized by the presence of cross-linkages formed by the thiol group of cysteine residues and the α-carbon of acceptor amino acids. A few examples of these bacteriocins have been described in the literature to date, although putative gene clusters with the potential to encode such peptides can be found in the genome of many bacterial species. Some peptides already under study exhibit potential biotechnological applications because of their remarkable antibacterial or antiviral activities, as well as their analgesic activity. Therefore, in this review, the main findings concerning these peptides will be addressed and discussed, with an emphasis on their potential use in clinical settings.

Purification of Antilisterial Peptide (Subtilosin A) from Novel Bacillus tequilensis FR9 and Demonstrate Their Pathogen Invasion Protection Ability Using Human Carcinoma Cell Line.

This study focuses on isolation, screening, and characterization of novel probiotics from gastrointestinal tract of free-range chicken (Gallus gallus domesticus). Fifty seven colonies were isolated and three isolates (FR4, FR9, and FR12) were selected and identified as Lactobacillus gasseri FR4, Bacillus tequilensis FR9, and L. animalis FR12 by 16S rRNA sequencing. Three strains were able to survive in stimulated acidic and bile conditions and inhibit the growth of pathogens. Especially, FR9 exhibited maximum inhibition against Listeria monocytogenes and none of them exhibited hemolytic activity. Native-PAGE revealed the presence of low molecular weight (3.4-5.0 KDa) antimicrobial peptide. The peptide was further purified by Sephadex G-50 column and RP-HPLC using C18 column. N-terminal amino acid sequencing of antimicrobial peptide showed 100% consensus to antilisterial peptide Subtilosin A and SboA gene was amplified from FR9 genome. FR9 showed maximum aggregation activity, exopolysaccharide production (85.46 mg/L) and cholesterol assimilation (63.12 ± 0.05 µg/mL). Strong adhesion property (12.6%) and pathogen invasion protection ability was revealed by B. tequilensis FR9 towards HCT-116 human colon carcinoma cell line. This is the first study to demonstrate antilisterial Subtilosin A production of B. tequilensis. Our results indicate that B. tequilensis FR9 strain furnish the essential characteristics of a potential probiotics and might be incorporated into human and animal food supplements.

Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain.

The antimicrobial activity and probiotic properties of Bacillus subtilis strain KKU213, isolated from local soil, were investigated. The cell-free supernatant (CFS) of a KKU213 culture containing crude bacteriocins exhibited inhibitory effects on Gram-positive bacteria, including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. The antibacterial activity of the CFS precipitated with 40% ammonium sulfate (AS) remained even after treatment at 60 and 100 °C, at pH 4 and 10 and with proteolytic enzymes, detergents and heavy metals. When analyzed by SDS-PAGE and overlaid with the indicator strains B. cereus and S. aureus, the 40% AS precipitate exhibited inhibitory activity on proteins smaller than 10 kDa. However, proteins larger than 25 kDa and smaller than 10 kDa were still observed on a native protein gel. Purified subtilosin A was prepared by Amberlite XAD-16 bead extraction and HPLC and analyzed by Nano-LC-QTOF-MS. Its molecular mass was found to be 3.4 kDa, and it retained its antibacterial activity. These results are consistent with the detection of the anti-listerial subtilosin A gene of the sbo/alb cluster in the KKU213 strain, which is 100% identical to that of B. subtilis subsp. subtilis 168. In addition to stable and cyclic subtilosin A, a mixture of many extracellular antibacterial peptides was also detected in the KKU213 culture. The KKU213 strain produced extracellular amylase, cellulase, lipase and protease, is highly acid-resistant (pH 2) when cultured in inulin and promotes health and reduces infection of intestinally colonized broiler chickens. Therefore, we propose that bacteriocin-producing B. subtilis KKU213 could be used as a potential probiotic strain or protective culture.