Circular and Leaderless Bacteriocins: Biosynthesis, Mode of Action, Applications, and Prospects

Bacteriocins are a huge family of ribosomally synthesized peptides known to exhibit a range of bioactivities, most predominantly antibacterial activities. Bacteriocins from lactic acid bacteria are of particular interest due to the latter’s association to food fermentation and the general notion of them to be safe. Among the family of bacteriocins, the groups known as circular bacteriocins and leaderless bacteriocins are gaining more attention due to their enormous potential for industrial application. Circular bacteriocins and leaderless bacteriocins, arguably the least understood groups of bacteriocins, possess distinctively peculiar characteristics in their structures and biosynthetic mechanisms. Circular bacteriocins have N-to-C- terminal covalent linkage forming a structurally distinct circular peptide backbone. The circular nature of their structures provides them superior stability against various stresses compared to most linear bacteriocins. The molecular mechanism of their biosynthesis, albeit has remained poorly understood, is believed to possesses huge application prospect as it can serve as scaffold in bioengineering other biologically important peptides. On the other hand, while most bacteriocins are synthesized as inactive precursor peptides, which possess an N-terminal leader peptide attached to a C-terminal propeptide, leaderless bacteriocins are atypical as they do not have an N-terminal leader peptide, hence the name. Leaderless bacteriocins are active right after translation as they do not undergo any post-translational processing common to other groups of bacteriocins. This “simplicity” in the biosynthesis of leaderless bacteriocins offers a huge commercial potential as scale-up production systems are considerably easier to assemble. In this review, we summarize the current studies of both circular and leaderless bacteriocins, highlighting the progress in understanding their biosynthesis, mode of action, application and their prospects

Kunkecin A, a New Nisin Variant Bacteriocin Produced by the Fructophilic Lactic Acid Bacterium, Apilactobacillus kunkeei FF30-6 Isolated From Honey Bees

Apilactobacillus kunkeeiFF30-6 isolated from healthy honey bees synthesizes the bacteriocin, which exhibits antimicrobial activity againstMelissococcus plutonius. The bacteriocin, kunkecin A, was purified through three-step chromatography, and mass spectrometry revealed that its relative molecular mass was 4218.3. Edman degradation of purified kunkecin A showed only the N-terminal residue, isoleucine. Hence, alkaline alkylation made the subsequent amino acid residues accessible to Edman degradation, and 30 cycles were sequenced with 11 unidentified residues. Whole genome sequencing ofA. kunkeeiFF30-6, followed by Sanger sequencing, revealed that the genes encoding the proteins involved in lantibiotic biosynthesis were within the plasmid, pKUNFF30-6. Most of the identified proteins exhibited significant sequence similarities to the biosynthetic proteins of nisin A and its variants, such as subtilin. However, the kunkecin A gene cluster lacked the genes corresponding tonisI,nisR, andnisKof the nisin A biosynthetic gene cluster. A comparison of the gene products ofkukAandnisA(kunkecin A and nisin A structural genes, respectively) suggested that they had similar post-translational modifications. Furthermore, the structure of kunkecin A was proposed based on a comparison of the observed and calculated relative molecular masses of kunkecin A. The structural analysis revealed that kunkecin A and nisin A had a similar mono-sulfide linkage pattern. Purified kunkecin A exhibited a narrow antibacterial spectrum, but high antibacterial activity againstM. plutonius. Kunkecin A is the first bacteriocin to be characterized in fructophilic lactic acid bacteria and is the first nisin-type lantibiotic found in the familyLactobacillaceae

Generation and Characterization of Novel Bioactive Peptides from Fish and Beef Hydrolysates

Bioactive peptides were successfully produced from fish (Gadidae) and beef skeletal muscles after being hydrolyzed for 8 h with pepsin. Subsequently, they were purified using a Sep-Pak C18 cartridge and reversed-phase high-performance liquid chromatography (RP-HPLC). The molecular weights of pure fish and beef peptides were determined to be 2364.4 and 3771.8, respectively. According to Edman degradation, the fish peptide was composed of 21 amino acid residues (F21), while the beef peptide was composed of 34 amino acid residues (B34). F21 and B34 displayed angiotensin-converting enzyme inhibitory activity with a half maximal inhibitory concentration (IC50) values of 7.3 µg/mL and 5.8 µg/mL, respectively. F21 exhibited antioxidant activity with an IC50 value of 389.9 µg/mL, whereas B34 exhibited no antioxidant activity. Moreover, F21 and B34 displayed antimicrobial effects against a wide spectrum of food-borne pathogens and spoilage bacteria. Bioactive peptides derived from muscle proteins are a promising strategy for the production of functional food materials and safe food preservatives

Characterization of the Biosynthetic Gene Cluster of Enterocin F4-9, a Glycosylated Bacteriocin

Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in Enterococcus faecalis JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, E. faecalis F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from E. faecalis F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, enfC was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, enfI was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins

Lactococcin Q, a Novel Two-Peptide Bacteriocin Produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, α and β, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qα plus Qβ) and lactococcin G (Gα plus Gβ) clarified that hybrid combinations (Qα plus Gβ and Gα plus Qβ) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qα plus Gβ was 32 times higher than that of Qα plus Qβ, suggesting that the difference in β peptides was important for the intensity of antibacterial activity

Enterocin X, a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides▿

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known