Subtilomycin: a new lantibiotic from Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans

Abstract

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.

Figure 1

Antimicrobial activity of B. subtilis strain MMA7. (A) Growth of the wild type (WT) strain B. subtilis MMA7 (diamond) and the ∆sbo-albF::cat mutant strain (squares) in MB (top panel). Antimicrobial activity of the WT and the ∆sbo-albF::cat mutant strains, tested on a deferred antagonism assay against B. cereus (Bc), B. megaterium (Bm), A. hydrophila (Ah) and C. albicans (Ca) (bottom panel); (B) Kinetics of production of antimicrobial compounds by B. subtilis strain MMA7. Antimicrobial activity of concentrated cell-free supernatants from samples collected at different time points of the bacterial growth was tested on a well diffusion assay against the indicators B. cereus (Bc) and L. monocytogenes (Lm). All experiments were repeated twice and representative results are shown.

Figure 2

Purification of the B. subtilis strain MMA7 antimicrobial compound. (A) RP-HPLC purification of the antimicrobial compound present in ammonium sulphate crude extracts (ASCE) from 12 h MB cultures. A single peak was eluted with an acetonitrile gradient after the injection of 1 mL ASCE (retention time, 24.070 min/46% acetonitrile); (B) Mass spectrometry analysis of the RP-HPLC purified sample showing a single compound with a low molecular mass.

Figure 3

Thermal, pH, and proteolytic stability of the purified peptide. Activity of the bioactive peptide samples (~10 μM) treated in the different ways was assessed by a spot on lawn assay. Five μL of treated/control samples were spotted onto BHI agar plates seeded with L. monocytogenes to an OD₆₀₀ of 0.015. Control samples containing the different proteolytic enzymes and no peptide, had no detectable inhibitory effect on the indicator strain. All assays were repeated at least three times, and representative results are shown.

Figure 4

Structural organisation of the putative subtilomycin biosynthetic cluster and flanking regions: subA, subtilomycin structural gene; subP, serine protease; subB, lanthionine dehydratase; subC, lanthionine synthetase; subT, ABC transporter. The function of subI cannot be predicted from its sequence, although its genetic location and lack of homologues is consistent with a possible involvement in immunity. Predicted promoters are indicated by arrows. A comparison of the genomic location of the subtilomycin biosynthetic cluster in strain MMA7 with that of the skf operon in strain 168 is provided.

Figure 5

Comparison of the amino acid sequence between subtilomycin and nisin pro-petides and their respective closest homologue (A). Comparison of the amino acid sequence of the N-terminal leader peptide and the C-terminal core peptide of subtilomycin and different class I lantibiotics (B). Sequences obtained from Uniprot were aligned with ClustalX (Multiple Sequence Alignment version 2.0.11, [34]) and sequence analysis processed with GeneDoc (Multiple sequence Alignment Editor & Shading Utility, Version 2.7.000). Conserved amino acids are boxed in black and gaps are indicated by hyphen. A vertical arrow indicates the first amino acid of the pro-peptide *, indicates the conserved motif of class I leader peptides documented to be important for efficient production. Proposed conformational structure of the mature lantibiotic subtilomycin (C). Top, unmodified propeptide. Bottom, mature peptide, where Ser and Thr residues which are posttranslationally dehydrated to Dha and Dhb, or involved in the formation of Lan and MeLan, respectively, with cysteine residues, are shaded in grey. The location of the thioether bridges was estimated from the amino acid sequence and by comparison with that of paenibacillin [32]. The presence of the N-terminal 2-oxobutyrate residue is also indicated.

Figure 6

Neighbour joining phylogenetic tree of gyrA gene sequences from different B. subtilis strains. Coastal (MMA7, CC15, AF31) and deep water (230-19, 230-29, 230-27, 126-43, 243-3) marine sponge-associated isolates are highlighted in bold. + and -, after strain designation, indicates the presence and absence of the subtilomycin structural gene, subA.