Cerecidins, novel lantibiotics from Bacillus cereus with potent antimicrobial activity

Abstract

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are widely produced by Gram-positive bacteria, including many species of the Bacillus group. In the present study, one novel gene cluster coding lantibiotic cerecidins was unveiled in Bacillus cereus strain As 1.1846 through genomic mining and PCR screening. The designated cer locus is different from that of conventional class II lantibiotics in that it included seven tandem precursor cerA genes, one modification gene (cerM), two processing genes (cerT and cerP), one orphan regulator gene (cerR), and two immunity genes (cerF and cerE). In addition, one unprecedented quorum sensing component, comQXPA, was inserted between cerM and cerR. The expression of cerecidins was not detected in this strain of B. cereus, which might be due to repressed transcription of cerM. We constitutively coexpressed cerA genes and cerM in Escherichia coli, and purified precerecidins were proteolytically processed with the endoproteinase GluC and a truncated version of putative serine protease CerP. Thus, two natural variants of cerecidins A1 and A7 were obtained which contained two terminal nonoverlapping thioether rings rarely found in lantibiotics. Both cerecidins A1 and A7 were active against a broad spectrum of Gram-positive bacteria. Cerecidin A7, especially its mutant Dhb13A, showed remarkable efficacy against multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant Enterococcus faecalis (VRE), and even Streptomyces.

FIG 1

Organization of the cerecidin gene cluster and comparison of putative cerecidins with other known lantibiotics. (A) Putative cerecidin gene cluster. Gray arrows indicate genes for lantibiotic biosynthesis: precursor gene (cerA), modification gene (cerM), regulation gene (cerR), transporter gene (cerT), processing gene (cerP), immunity genes (cerFE). White arrows indicate quorum sensing component genes: pre-ComX modification and processing gene (comQ), ComX precusor gene (comX), histidine kinase gene (comP), and response regulator gene (comA). (B) Alignment of leader peptides of CerA1 and CerA7 with similar known lantibiotics. The vertical arrows indicate the putative two processing sites, i.e., a double glycine motif (bold residues) and a hexapeptide. (C) Alignment of core peptides of cerecidins with similar known lantibiotics. The bold Ser or Thr residues are dehydrated residues, and the underlined ones are involved in ring formation with the bold Cys residues.

FIG 2

mRNA expression of genes related to cerecidin synthesis. (A) Transcriptional analysis of cerA in B. cereus As 1.1846 at different incubation times. Lane 1, 2 h; lane 2, 4 h; lane 3, 6 h; lane 4, 8 h; lane 5, 12 h; lane 6, genomic DNA; lane 7, H₂O. (B) Transcriptional analysis of cerM, cerR, cerT, cerP, cerF, and cerE. The “c” means cDNA of B. cereus As 1.1846 of 12 h, and the “g” means genomic DNA. (C) Transcriptional analysis of 16S rRNA. The DNase I-treated mRNA was used as a negative control (−).

FIG 3

MS analysis and antimicrobial activity of cerecidin isolated from a B. cereus As 1.1846 transformant. (A) Antimicrobial activity assay of culture of B. cereus As 1.1846 (a) and its pHY-P_aprN-cerM transformants (b). (c) Cerecidin A7 obtained via a semi-in vitro way was used as a positive control. (B) MS analysis of isolated cerecidins A1 and/or A2 to A6 from an agar plate with an inhibition zone.

FIG 4

Semi-in vitro synthesis of cerecidin A7. (A) Schematic presentation of cerecidin A7 biosynthesis. (B) SDS-PAGE of His₆-mCerA7 and His₆-CerP_80–490. Lane 1, His₆-mCerA7; lane 2, His₆-CerP_80–490; lane 3, protein marker. (C) C₁₈ RP-HPLC analysis of His₆-mCerA7 treated with GluC (a) and cerecidin A7′ treated with His₆-CerP_80–490 (b). Peak 1, cerecidin A7′; peak 2, cerecidin A7. (D) Antimicrobial assay against M. flavus NCIB8166 of cerecidin A7′ (1, peak 1 in panel C) and cerecidin A7 (2, peak 2 in panel C).

FIG 5

MALDI-TOF MS and antimicrobial activity analyses of cerecidins. (A) MS analysis of cerecidin A7′ (2,982.32 Da). MS analysis and antimicrobial assay of cerecidin A7 (B) and cerecidin A1 (C). (D) MS analysis of cerecidin A7′ derivatives with 1 or 2 ethanethiol adducts (panel a, 3,044.43 Da or 3,106.46 Da, respectively) and 3 or 4 ethanethiol adducts (panel b, 3,168.42 Da or 3,230.44 Da, respectively).

FIG 6

Structure dissection of cerecidins. (A) MS/MS analysis of cerecidin A7. (B) Fragmentation pattern of cerecidin A7. (C) Comparison of structures of cerecidins A1 and A7. The vertical arrows indicate the different residues between cerecidins A1 and A7.

FIG 7

Antimicrobial activity of mutants of cerecidin A7. (A) The relative antimicrobial activity of Ala scanning mutants of cerecidin A7 against M. flavus NCIB8166. The pentacle indicates wild-type cerecidin A7 (WT). (B) Antimicrobial activity of WT and its mutant Dhb13A against S. scabiei CGMCC 4.1765 and S. griseus NBRC 13350. Wild type of 60 μg/ml and Dhb13A of 20 μg/ml were applied to each strain. In each case, 25-μl compounds were used.