The Bacillus cereus Group Is an Excellent Reservoir of Novel Lanthipeptides

Abstract

Lantibiotics are ribosomally synthesized peptides that contain multiple posttranslational modifications. Research on lantibiotics has increased recently, mainly due to their broad-spectrum antimicrobial activity, especially against some clinical Gram-positive pathogens. Many reports about various bacteriocins in the Bacillus cereus group have been published, but few were about lantibiotics. In this study, we identified 101 putative lanthipeptide gene clusters from 77 out of 223 strains of this group, and these gene clusters were further classified into 20 types according to their gene organization and the homologies of their functional genes. Among them, 18 types were novel and have not yet been experimentally verified. Two novel lantibiotics (thuricin 4A-4 and its derivative, thuricin 4A-4D) were identified in the type I-1 lanthipeptide gene cluster and showed activity against all tested Gram-positive bacteria. The mode of action of thuricin 4A-4 was studied, and we found that it acted as a bactericidal compound. The transcriptional analysis of four structural genes (thiA1, thiA2, thiA3, and thiA4) in the thuricin 4A gene cluster showed that only one structural gene, thiA4, showed efficient transcription in the exponential growth phase; the other three structural genes did not. In addition, the putative transmembrane protein ThiI was responsible for thuricin 4A-4 immunity. Genome analysis and functional verification illustrated that B. cereus group strains were a prolific source of novel lantibiotics. Bacteriocins are ribosomally synthesized peptides produced by bacteria and exhibit antimicrobial activity against other bacteria (either in the same species or across genera) (1); they include posttranslationally modified bacteriocins and unmodified bacteriocins (2). Lanthipeptides (lantibiotics) are peptides that undergo multiple posttranslational modifications, and these are the best-characterized modified bacteriocins (3). Modifications include the formation of meso-lanthionine and 3-methyllanthionine as well as dehydrated amino acids (Dha and Dhb). Lanthipeptides are classified into four different classes on the basis of their biosynthetic enzymes (4). In class I lanthipeptides, the dehydration is carried out by a dehydratase (LanB) and cyclization is catalyzed by a cyclase (LanC). Class II lanthipeptides are modified by LanM proteins, which perform both dehydration and cyclization. In class III and IV lanthipeptides, the dehydration and cyclization reactions are catalyzed by multifunctional enzymes (RamC/LabKC or LanL).The Bacillus cereus group currently contains eight species: B. anthracis, B. cereus, B. thuringiensis, B. cytotoxicus, B. weihenstephanensis, B. mycoides, B. pseudomycoides, and B. toyonensis (5, 6). To date, bacteriocins of the B. cereus group have been identified or characterized mainly in strains of B. cereus and B. thuringiensis. More than 20 bacteriocins have been reported in these two species (7,8). They are active against closely related bacilli, such as the foodborne pathogen B. cereus...

Three Novel Lantibiotics, Ticins A1, A3, and A4, Have Extremely Stable Properties and Are Promising Food Biopreservatives

“…1A). This gene cluster was also present in the genome of B. cereus F65185, and it was regarded as a type I-5 lanthipeptide gene cluster in B. cereus group strains (11). The second lanthipeptide gene cluster consists of 11 genes, including 4 highly homologous structural genes (tinA1, tinA2, tinA3, and tinA4), 1 gene (tinM) encoding a modification enzyme, 3 genes (tinEFG) encoding an ABC transporter that could be involved in immunity, 1 gene (tinT1) encoding a transporter with an associated protease domain, 1 gene (tinT2) encoding an HlyD family secretion protein, and 1 regulatory gene (tinR) (Fig.…”

“…The B. cereus group contains diverse novel lanthipeptides, and recently reported lantibiotics in this group have been shown to exhibit high efficacy against certain Gram-positive pathogens (11,13). Therefore, we screened more than 100 strains (mainly B. cereus and B. thuringiensis) for antimicrobial activity against two important foodborne pathogens, B. cereus ATCC 49064 and L. monocytogenes LM201 (14,15), and tested the stability of the cell-free supernatant of antimicrobial-producing strains under high-temperature, strong-acid, and strong-base conditions.…”

“…Antimicrobial activity was detected by the agar well diffusion method (11). The MICs of the bacteriocins for the various indicator strains were measured by serial 2-fold HPLC-purified bacteriocin solutions.…”

“…Our previous work proved that the B. cereus group is an excellent reservoir of novel lanthipeptides (11). All three lantibiotics reported to date within this group exhibit broad-spectrum antimicrobial activity against some clinically important Gram-positive pathogens, including multidrug-resistant strains (11)(12)(13). In this study, we applied a targeted screening method to identify and characterize three novel class II lantibiotics, ticins A1, A3, and A4, in B. thuringiensis BMB3201.…”

“…These can be isolated from diverse environments, such as soil, water, and animal hosts (9, 10). Our previous work proved that the B. cereus group is an excellent reservoir of novel lanthipeptides (11). All three lantibiotics reported to date within this group exhibit broad-spectrum antimicrobial activity against some clinically important Gram-positive pathogens, including multidrug-resistant strains (11)(12)(13).…”