Bovicin HJ50-like lantibiotics, a novel subgroup of lantibiotics featured by an indispensable disulfide bridge

Abstract

Lantibiotics are ribosomally-synthesized and posttranslationally modified peptides with potent antimicrobial activities. Discovery of novel lantibiotics has been greatly accelerated with the soaring release of genomic information of microorganisms. As a unique class II lantibiotic, bovicin HJ50 is produced by Streptococcus bovis HJ50 and contains one rare disulfide bridge. By using its precursor BovA as a drive sequence, 16 BovA-like peptides were revealed in a wide variety of species. From them, three representative novel lan loci from Clostridium perfringens D str. JGS1721, Bacillus cereus As 1.348 and B. thuringiensis As 1.013 were identified by PCR screening. The corresponding mature lantibiotics designated perecin, cerecin and thuricin were obtained and structurally elucidated to be bovicin HJ50-like lantibiotics especially by containing a conserved disulfide bridge. The disulfide bridge was substantiated to be essential for the function of bovicin HJ50-like lantibiotics as its disruption eliminated their antimicrobial activities. Further analysis indicated that the disulfide bridge played a crucial role in maintaining the hydrophobicity of bovicin HJ50, which might facilitate it to exert antimicrobial function. This study unveiled a novel subgroup of disulfide-containing lantibiotics from bacteria of different niches and further demonstrated the indispensable role of disulfide bridge in these novel bovicin HJ50-like lantibiotics.

Figure 1. Revealing of bovicin HJ50-like lantibiotic gene clusters.

(A) Sequence analysis of precursor peptides of BovA-like peptides including BovA, SuiA, PerA, BceA and ThuA. The residues in red of yellow background indicated identical amino acids in all aligned precursors. The thioether bridges (black) or disulfide bridge (red) illustrated the ring topology of bovicin HJ50. The vertical arrow indicated the processing site (between Gly-1 and Ala1) for BovT to release mature bovicin HJ50. (B) Bovicin HJ50-like lantibiotic gene clusters. Percentages in horizontal arrows represented the identities of the lantibiotic biosynthesis-related proteins with that of bov locus.

Figure 2. Tricine-SDS-PAGE of mature bovicin HJ50-like lantibiotics.

Lane 1, protein marker; lane 2, perecin; lane 3, cerecin; lane 4, thuricin.

Figure 3. Structure and antimicrobial activity of bovicin HJ50-like lantibiotics.

(A) Comparison of structure of bovicin HJ50-like lantibiotics with lacticin 481. Gray residues indicated identical amino acids and the Cys residues in red of yellow background indicated disulfide-forming Cys in bovicin HJ50, suicin, perecin, cerecin and thuricin. (B) Comparison of antimicrobial activity of bovicin HJ50-like lantibiotics. In each hole, 25 µL of 10 µg/mL compounds were applied.

Figure 4. MS and HPLC analysis of bovicin HJ50 and its in vitro produced counterparts.

(A) a, bovicin HJ50 produced by BovM in vitro in the absence of DTT; b, main product produced by BovM in vitro in the presence of DTT. (B) a, HPLC analysis of reduced bovicin HJ50; b, HPLC analysis of reduced bovicin HJ50 after exposed to air for 2 days. The white star indicated reduced bovicin HJ50 and the black star indicated the wild type bovicin HJ50. (C) MS analysis of reduced bovicin HJ50 (a) and wild type bovicin HJ50 (b).

Figure 5. Mutation, substitution and alkylation of disulfide bridge in bovicin HJ50-like lantibiotics.

(A) Antimicrobial activity of wild type bovicin HJ50 (25 µL, 5 µg/mL) when treated respectively with 0, 2, 4, 8 mM TCEP. (B) MS analysis of purified NEM-alkylated bovicin HJ50. (C) Antimicrobial activity of wild type bovicin HJ50 (WT) and alkylated bovicin HJ50 (WT-NEM). (D) Antimicrobial activity of disulfide-related mutants. D/K represents substitution of disulfide-forming Cys residues to Asp and Lys, e.g., bovicin HJ50 D/K means bovicin HJ50 mutant C21D/C29K. L/L or F/F is referred as the same condition as D/K. 25 µL peptide samples of 20 µg/mL were applied to each hole.

Figure 6. Inhibition of bacterial growth and disruption of membrane by bovicin HJ50.

(A) Influence of bovicin HJ50 and its mutant C21A on bacterial growth. The growth curve of M. flavus NCIB8166 was recorded when bovicin HJ50 of 5×IC₅₀ (square dotted curve), its mutant C21A of 50×IC₅₀ (circle dotted curve) and H₂O (triangle dotted curve) was added at the time point of 5 h. (B) Influence of membrane potential when bovicin HJ50 and its mutant C21A were added to M. flavus NCIB8166. The fluorescence intensity was monitored in a time duration of 15 min. bovicin HJ50 (square dotted curve) and its mutant C21A (circle dotted curve) was added at the time point of 5 min. Nisin Z (triangle dotted curve) was used as positive control.

Figure 7. Circular dichroism (CD) analysis of bovicin HJ50 and its mutants.

Dash-dot-dash line represented the secondary structures of bovicin HJ50 (black), its mutant C21A (red) and reduced form in H₂O, while solid line represented the secondary their structures in 1% SDS.

Figure 8. Comparison of hydrophobicity of bovicin HJ50 and its mutants by ANS binding analysis.

Bovicin HJ50 (black curve), its reduced form (blue) and mutant C21A (red). H₂O was used as negative control (green).