Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

Subclass 1: Lanthipeptides

Lanthipeptides are peptides containing unusual amino acids, such as dehydroalanine/dehydrobutyrine, lanthionine/methyl-lanthionine residues, introduced by different kinds of PTMs [15]. Lanthipeptides with antimicrobial activity form the so-called lantibiotics [17], which can be subdivided into four subclasses, following the classification scheme of lanthipeptides [18]. The main differences between class I, II, III and IV lanthipeptides are the PTM enzymes involved. Class I lanthipeptides are modified by two distinct enzymes that carry out the PTM process: dehydratase LanB and cyclase LanC, while class II peptides are modified by a bifunctional lanthionine-introducing enzyme, called LanM. There are also two-component lanthipeptides consisting of two peptides, which belong to class II lanthipeptide, because they are processed by a single modifying enzyme, called LanM [19–23]. For other lanthipeptides (class III and IV), the dehydration and cyclization reactions are catalyzed by multifunctional enzymes (RamC/LabKC or LanL) or they lack significant antibiotic activity, which are not further described here [24].

Subtilin is a well-investigated class I lanthipeptide produced by B. subtilis, the encoding gene cluster of which is also found in the genome of Bacillus sp. YP1. The gene encoding subtilin encodes a 56-residue peptide precursor that is processed to yield the 32-residue mature peptide, which is structurally related to the lantibiotic nisin of Lactococus lactis [25]. The subtilin gene cluster includes the structural gene spaS, encoding its prepeptide; PTM genes spaB and spaC, encoding a dehydratase and a cyclase for lanthionine formation, respectively; transporter gene spaT for modified precursor export and immunity genes spaIFEG (Additional file 2: Table S2) [26–28]. The presubtilin will be converted to mature subtilin by serine proteases secreted by B. subtilis [29]. Subtilin exhibits bactericidal activity against a broad spectrum of Gram-positive bacteria, based on pore formation in the cytoplasmic membrane, using cell wall precursors such as lipid II and undecaprenyl pyrophosphate, the hydrophobic carrier module for peptidoglycan monomers, as docking module and as a central constituent of the pore [30, 31]. The class II lanthipeptide mersacidin produced by several B. amyloliquefaciens strains [32–34], with a more globular structure comprising 20 amino acid residues, inhibits cell wall biosynthesis by binding to lipid II [35, 36]. The mersacidin gene cluster includes the structural gene mrsA, two modification genes (Additional file 2: Table S2), i.e. mrsM coding for both dehydration and cyclation and mrsD coding for a C-terminal S-[(Z)-2-aminovinyl]-3methyl-D-cysteine formation enzyme, and the gene mrsT coding for a transporter with an associated protease domain, as well as three genes, mrsEFG, coding for immunity and three genes, mrsR1, R2, K1, coding for regulation [37–39].

A total of 105 putative lanthipeptide gene clusters were discovered in Bacillales in this study (Table 1). Among them, gene clusters of class I lanthipeptides distribute over the genomes of B. subtilis, B. thuringinensis, B. cereus, B. megaterium, B. mycoides, B. clausii, Bacillus sp., Geobacillus thermodenitrificans, Geobacillus kaustophilus, Paenibacillus polymyxa, Paenibacillus larvae, Paenibacillus peoriae and Paenibacillus durus, while gene clusters of class II lanthipeptides distribute over the genomes of B. thuringinensis, B. cereus, B. amyloliquefaciens, B. licheniformis, B. mycoides, B. halodurans, B. methylotrophicus, B. paralicheniformis, B. endophyticus, B. pseudomycoides, Bacillus sp., G. thermodenitrificans, P. polymyxa, P. durus and Paenibacillus sp. (Additional file 2: Table S2 and Fig. 2). Class I lanthipeptides identified by BAGEL3 includes subtilin, clausin, subtilomycin and geobacillin I [22, 40–42]. Gene clusters of entianin, ericinA/S, paenibacillin, paenicidin A, B, thuricin 4A and its derivative thuricin 4D were not found by genome mining tools (because whole genome sequences of the producing organisms were not available in most cases) but were also added to the list (Additional file 2: Table S2) [43–47]. Class II lanthipeptides usually exhibit a globular structure, including mersacidin, amylolysin, pseudomycoicidin, cerecidin A1-A6 and geobacillin II; also two-component class II lanthipeptides including haloduracin and lichenicidin were identified [19, 22, 23, 48–52]. It is notable that gene clusters of two novel subtilin-like lantibiotics were found in several P. polymyxa strains. By further analysis, both of their sequence of core peptides showed high similarity with the N-terminal part of subtilin but were quite different in the C-terminal part. Moreover, we report a novel gallidermin/nisin-like lantibiotic from genomes of Bacillus mycoides ATCC 6462, B. mycoides 2048 and B. cereus AH1272. Looking into the sequence of its precursor peptide (see Additional file 2: Table S2), it has the conserved F(N/D)LD motif in its leader and theoretically could form the same rings as gallidermin/nisin according the position of serine and cysteine residues. All of the three putative lantibiotics have lanBC genes in their gene clusters, which suggest they are involved in their production. A gene cluster of a two-peptide bacteriocin was found in the genome of B. cereus Q1. Due to the existence of a lanM gene, it was predicted to be a class II lanthipeptide. Interestingly, the C-terminal parts of both its core peptides are similar to lichenicidin and haloduracin, and the N-terminal part of one of the core peptides shows high similarity with one of cytolysins produced by Enterococcus faecalis [53].

Subclass 2: Head to tail cyclized peptides

Head to tail cyclic peptides are named by their unifying feature, which is the head to tail circularization of their peptide backbones by direct linkage of their N- and C-terminal amino acids, resulting in a well-defined three-dimensional structure, by folding in α-helical manner [54–57]. To our knowledge, these peptides contain no lanthionine, β-methyl-lanthionine, and dehydrated residues, making them clearly distinguishable from lanthipeptides [58].

Amylocyclicin was recently reported to be produced by B. amyloliquefaciens FZB42 and identified as a novel circular bacteriocin [59], which is derived from the 112 amino acid precursor AcnA (Additional file 2: Table S2) encoded by acnA, with a 48 amino acid derived leader cleaved by a protease that is still unknown, and then circularization occurring between Leu-1 and Trp-64 [59]. There are gene clusters present, regulating their maturation (e.g. circularization and cleavage), transportation and self-protection. The first gene of the putative operon, acnB, encodes a membrane-anchored protein comprising five transmembrane helices with unkown function. acnD is likely to encode the transporter complex, whereas AcnC might act as circularization enzyme showing high similarity with the sequence of UclB, which brings uberlysin to maturation [60]. AcnEF are proposed to be the putative immunity genes. Amylocyclicin has the ability to inhibit Gram-positive bacteria like B. subtilis, but not against Gram-negative bacteria.

There are 52 gene clusters of putative head to tail cyclized peptides identified in this genome-mining study, which distribute over the genomes of B. thuringiensis, B. cereus, B. coagulans, B. pumilus, B. paralicheniformis, B. gobiensis, Bacillus sp., Kyrpidia tusciae, Geobacillus stearothermophilus, G. kaustophilus, Geobacillus sp., P. larvae and Paenibacillus mucilaginosus (Table 1 and Fig. 2). An amylocyclicin-like circular bacteriocin gene cluster was found in the genomes of B. coagulans. The core peptide sequence is identical to that of amylocyclicin of B. amyloliquefaciens FZB42, but the leader peptide sequence is quite different (Additional file 2: Table S2). It is noteworthy that a gene cluster of an uberolysin-like peptide was detected in the genome of Bacillus sp. 1NAL3E and gene clusters of circularin A/bacteriocin AS-48 like peptide were detected in several Geobacillus sp., while uberolysin was produced by Streptococcus uberis, circularin A was produced by Clostridium beijerinckii and bacteriocin AS-48 was produced by E. faecalis [54, 60–63]. From the core peptide sequences, their circularization is most likely being formed between leucine and tryptophan (Additional file 2: Table S2). There are also other putative gene clusters of head to tail cyclized peptides found in this study, but notably these show no similarity with reported peptides. Whether these are real circular bacteriocins or not, need to be further investigated experimentally.

Subclass 3: Sactipeptides

Sactipeptides form a class of cyclic antimicrobial peptides with unusual sulfur to α-carbon cross-links, which are catalyzed by radical S-adenosylmethionine (SAM) enzymes in a leader peptide-dependent manner [64, 65]. Posttranslational linkage of a thiol to the α-carbon of an amino acid residue responsible for their antimicrobial bioactivities is rare in ribosomal synthesized peptides and they are classified as an independent group [66–68]. These unusual linkages differ from lanthionine bridges containing sulfur to β-carbon linkages.

Subtilosin A is a 35-residue peptide, formed by cleavage of a seven amino acid leader peptide, cyclization of the N- and C-terminal parts, and further modification of cysteine, threonine and phenylalanine residues. The maturation of subtilosin A begins with the transcription and translation of the sbo-alb genes (Additional file 2: Table S2), resulting in the precursor peptide SboA [69, 70]. Subsequently, the radical SAM enzyme AlbA generates the thioether linkages between the sulfur atom of the cysteine residue and the α-carbon of the threonine residue [68]. Afterwards, either AlbE or AlbF (putative proteases) cleaves off the leader peptide. In the last step, the peptide backbone is circularized by one of the two proteases, resulting in subtilosin A, which is subsequently exported by the putative ABC transporter AlbC. The operon is induced under anaerobic conditions and is controlled by the transition state regulatory protein AbrB [4]. It shows antibacterial activity against Bacillus spp., E. faecalis, Gardnerella vaginalis and Listeria monocytogenes by targeting their membranes and forming pores [71–73].

In this study, we found 87 putative gene clusters of sactipeptides in the genomes of Bacillales (Table 1), most of which belong to three reported types of sactipeptides (Additional file 2: Table S2): subtilosin A from B. subtilis, B. atrophaeus, B. simthii and Bacillus sp. strains; sporulation killing factor (SKF) from B. atrophaeus, B. pumilus and B. subtilis strains; and thuricins, such as thuricin H (17) and thuricin CD from B. thuringiensis and B. cereus [67, 74–77]. We found several other putative gene clusters of sactipeptides in the genomes of B. clausii, G. stearothermophilus, Brevibacillus laterosporus, P. larvae, Paenibacillus odorifer, Paenibacillus graminis, Paenibacillus riograndensis and Paenibacillus sp. (Additional file 2: Table S2 and Fig. 2), which showed very limited similarity with reported sactipeptides, and that need to be further experimentally confirmed.

Subclass 4: Linear azole-containing peptides (LAPs)

The linear azole-containing peptides (LAPs), form an important subgroup of RiPPs with a distinguishing heterocyclic ring of oxazoles and thiazoles derived from serine/threonine and cysteine by enzymatic cyclodehydration and dehydrogenation [78–81]. Prominent natural products such as microcin B17 produced by Escherichia coli and streptolysin S produced by LAB, are model of representive LAPs peptides [82–86]. The LAP family has already been extended with plantazolicin A and B produced by B. amyloliquefaciens and B. methylotrophicus [80, 81].

Plantazolicin A (Additional file 2: Table S2) and its desmethyl analogue plantazolicin B represent an unusual type of thioazole/oxazole-containing peptide antibiotic with a hitherto unknown mechanism of action, which show inhibition against Bacillus [80, 87]. The mature product plantazolicin is a linear 41 amino acid precursor peptide with the 14 amino acid core-peptide encoded by the structural gene pznA. The trimeric protein complex PznBCD (cyclodehydratase, dehydrogenase, and docking/scaffolding protein) likely catalyzes PTMs of ten cyclodehydrations followed by nine dehydrogenations. After the protease PznE cleaves off the leader peptide to yield desmethylplantazolicin plantazolicin B, a final N, N-bismethylation by methyltransferase PznL gives plantazolicin A [80, 81].

A total of 117 putative gene clusters of LAPs occupy 20 % of the total putative gene clusters of bacteriocins in this study and are widely distributed in more than 20 species of Bacillales (Table 1 and Fig. 2). However, only plantazolicin A and B produced by B. amyloliquefaciens and B. methylotrophicus have been reported before (Additional file 2: Table S2). This means that many novel LAPs can be found and need further experimental investigation.

Subclass 5: Thiopeptides

Thiopeptides, or thiazolyl peptides are highly modified via either non-ribosomal or ribosomal assembly, with a six membered nitrogenous macrocycle being central of piperidine/pyridine/dehydropiperidine and including additional thiazoles and dehydrated amino acid residues [15, 88, 89]. Because of the trithiazolyl (tetrahydro) pyridine core, they display high affinity binding to either the 50S ribosomal subunit or elongation factor Tu.

In the thiocillins, found in the producer B. cereus ATCC 14579, at least 10 and up to 13 of the 14 C-terminal residues undergo PTM to generate a set of eight related antibiotics. The thiocillin gene cluster contains four identical copies of a gene encoding a 52-residue precursor peptide (tclE-H), which is thought to be posttranslationally modified to yield the mature antibiotic scaffold (Additional file 2: Table S2). Four of the eight thiocillins produced abundantly by B. cereus display similar efficacies against B. subtilis and two methicillin-resistant Staphylococcus aureus (MRSA) strains [90, 91].

Thiopeptide gene clusters involved in ribosomal synthesis are found in the genome sequences of several B. cereus, B. subtilis and Lysinibacillus sphaericus (Additional file 2: Table S2 and Fig. 2), which might go beyond the classification for LAB bacteriocins [14].

Subclass 6: Glycocins

Glycocins are bacteriocins with glycosylated residues. There are various unique and diverse putative glycopeptide containing bacteriocins named glycocins in Firmicutes [15, 92].

There is one model glycopeptide bacteriocin, sublancin 168 (Additional file 2: Table S2), produced by B. subtilis with a β-S-linked glucose moiety attached to cysteine22 and two disulfides [92–95]. The sublancin 168 biosynthetic gene cluster contains the precursor gene sunA coding a 56-residue polypeptide consisting of a 19-residue leader peptide and a 37-residue mature peptide and genes bdbA and bdbB encoding two thiol-disulfide oxidoreductases, i.e. BdbA and BdbB [95, 96]. In addition, it contains two open reading frames of unknown function, yolJ and yolF. YolF was recently suggested to be important for immunity of the producing strain and was renamed SunI; the function of YolJ has not yet been reported [97]. SunT is responsible for transport. The antimicrobial activity spectrum of sublancin 168 was like that of lantibiotics, inhibiting Gram-positive bacteria, but not Gram-negative bacteria; and acts also similar to the lantibiotics nisin and subtilin in its ability to inhibit both bacterial spore outgrowth and vegetative cell growth [17].

In addition to sublancin 168 found in B. subtilis, genome-mining study indicated that nine other putative gene clusters of glycocins were found in genomes of B. thuringiensis, B. cereus, B. weihenstephanensis, B. lehensis, Bacillus sp., Geobacillus sp. and Paenibacillus sp., which need further characterization (Table 1 and Fig. 2).

Subclass 7: Lasso peptides

Lasso peptides, which form an emerging class of RiPPs from bacteria, were first described in 1991 [98]. Their defining structural feature is an N-terminal macrolactam ring that is threaded by the C-terminal tail resulting in a unique lasso structure–the so-called lariat knot. The ring is formed by an isopeptide bond between the N-terminal α-amino group of a glycine, alanine, serine, or cysteine and the carboxylic acid side chain of an aspartate or glutamate, which can be located at positions 7, 8, or 9 of the amino acid sequence [16, 99].

In general, lasso peptide production requires at least three genes encoding a precursor peptide A, a cysteine protease B, and an ATP-dependent lactam synthetase C. Gene clusters might contain additional genes, but so far no system was proven to be in need of an additional enzyme to produce mature lasso peptides [100–104]. Microcin J25 produced by E. coli AY25 has served as a model for studies of lasso peptides [105]. Known lasso peptides display antimicrobial activity by enzyme inhibition [106, 107].

Genome mining of Bacillales indicated 48 gene clusters of hypothetical peptides, which are likely lasso peptides in the genomes of 20 Bacillales species (Table 1 and Fig. 2), but these still need to be experimentally confirmed.

Subclass 1: Pediocin-like peptides

The pediocin-like bacteriocins are antilisterial peptides that have a YGNGVXC consensus motif [110, 111]. Coagulin produced by B. coagulans I₄ is a peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1 [109, 112]. Coagulin and pediocin differ only by a single amino acid at their C-terminus (asparagine41threonine). Gene clusters of coagulin are located on a plasmid including the structural gene coaA, immunity gene coaB, and ABC transporter genes coaC and coaD [113].

Subclass 2: Other unmodified peptides

Subclass 2 includes other unmodified peptides, such as lichenin produced by B. licheniformis, or cereins produced by B. cereus, which have already been described in a previous review although not yet detected in the reported complete genome sequences [4]. We found a lactobin A family protein [114] and a lactococcin A1 family protein [115] belonging to class II bacteriocins from Anoxybacillus flavithermus WK1. Here, we mainly added some new members of Bacillus class II bacteriocins detected by BAGEL3, in particular holins and holin-like peptide BhlA, antimicrobial peptide LCI, and leaderless bacteriocin aureocin A53 (Additional file 2: Table S2).

Analysis of all Bacillales genome sequences revealed the presence of a structural gene encoding a holin in Geobacillus sp. WCH70 and BhlA encoding genes in most of B. subtilis, B. amyloliquefaciens, B. mycoides, B. pseudomycoides, B. licheniformis, B. pumilus and B. thuringiensis, and further structural analysis of their sequence revealed features similar to holin (Additional file 2: Table S2) [116, 117]. Holins are phage-encoded proteins involved in the disruption of bacterial membrane to facilitate the release of progeny phage particles [118–121]. However, the functions of these specific ORFs have not yet been identified. The bacteriocin-related holin-like peptide BhlA from Bacillus showed antibacterial activity against several Gram-positive bacteria, including MRSA and Micrococcus luteus by destroying cell membranes [122]. BhlA consists of 70 amino acid residues with a single transmembrane domain at the N-terminus, a number of highly charged amino acid residues at the C-terminus. The presence of hydrophilic residues and the membrane topology of BhlA make it different from holins [122].

The lci gene encoding LCI was found in the genomes of B. amyloliquefaciens and B. methylotrophicus strains (Additional file 2: Table S2), sharing 98–100 % identity with the LCI sequence of B. subtilis. The antimicrobial peptide LCI was first identified and isolated by Liu et al. [123] from a B. subtilis strain named A014 that possesses very strong antagonistic activity against the Gram-negative pathogen Xanthomonas campestris pv oryzea causing rice leaf-blight disease, which is a serious threat to rice production and causes great losses in yields in most rice fields annually. LCI is a β-structure antimicrobial peptide containing 47 residues of 5460 Da with no disulfide bridge or circular structure. It also contains a hydrophobic core formed by valine5, tyrosine41 and tryptophan44 as well as 23 H-bonds which contribute to its considerable thermal stability [124, 125]. According to our BAGEL3 gene cluster mining results, there are two genes: a structural gene lci and an immunity/transporter-like gene which was still unknown. LCI’s positively charged residues lead to a short-lived channel in the bacterial membrane of sensitive strains [126].

Another new member of Bacillus class II bacteriocins is leaderless aureocin A53, whose gene cluster was identified in the genome sequence of B. pumilus strains (Additional file 2: Table S2). It is active against L. monocytogenes by dissipating the membrane potential and simultaneously stopped biosynthesis of DNA, polysaccharides, and protein [127]. Aureocin A53 is a highly cationic 49-residue peptide containing six lysine and four tryptophan residues. Unlike most class II bacteriocins, aureocin A53 is synthesized without a leader peptide and retains a formylated N terminus. Notably, genes for biosynthetic enzymes, immunity functions, or regulation of biosynthesis are not found in the vicinity of the aureocin A53 structural gene [128].