For the production of proteins and peptides three basic enzymes are required: tRNA synthetases, tRNAs and the ribosome [19]. First the aa-tRNA synthetase selects the cognate amino acid and loads it onto the 2’- or 3’- hydroxyl group of the corresponding t-RNA [20, 21]. Subsequently, with help of the elongation factor (EF-Tu), the ribosome selects the correct aa-tRNA during each cycle of polypeptide elongation, according to the mRNA sequence [22]. Therefore a complex comprising aa-tRNA, EF-Tu and GTP enters the acceptor site of the ribosome. The large ribosomal subunit stimulates GTP hydrolysis when there is complementary base-pairing between the mRNA and the cognate aa-tRNA. Peptide bond is formed when the aa-tRNA has been accommodated to the acceptor site, whereupon translocation can occur regenerating the ribosome. Eventually, post-translational modification events lead to the completion of synthesis of these peptide antibiotics [23].

Lantibiotics are the major group of ribosomally synthesized antibiotics in Bacilli. They contain lanthionine, which is formed post-translationally through dehydration of serine or threonine residues followed by addition of neighbouring cysteine thiol groups, leading to inter-residual thioether bonds [24, 25]. Based on structural properties, two types of lantibiotics are distinguished: type A, with a more linear secondary structure, and type B, with a more globular one [26].

Subtilin and ericin are members of the type A group and are lethal against Gram-positive bacteria by forming voltage-dependent pores into the cytoplasmic membrane [27]. Mersacidin belongs to the type B group and inhibits cell wall biosynthesis by complexing lipid II [28]. Other unusual lantibiotics produced by Bacilli are sublacin and subtilosin, which also act against Gram-positive bacteria through yet unknown mechanisms. The organization of these gene clusters is shown in figure 2.

Subtilin biosynthesis is mediated by the prepeptide SpaS [29], which is post-translationally modified by SpaBC [30]. Furthermore, the translocator SpaT exports the lantibiotic. Immunity to the producer strain is conferred by the lipoprotein SpaI and the ABC transporter SpaFEG [31]. In a positive feedback loop, subtilin activates the two component regulatory system SpaRK (response regulator and sensor histidine kinase) and directly stimulates expression of genes involved in biosynthesis and immunity [32, 33]. SpaRK expression is also controlled by the sporulation transcription factor SigH [33].

Ericin has the same gene cluster organization as subtilin, but surprisingly two structural genes, eriA and eriS. However, the production of ericin A and ericin S, that differ in amino acid composition and ring structure, is under the control of the same synthetase (EriBC) [34].

Figure 2: Bacillus subtilis lantibiotics, lantibiotic-like peptides and specifying gene clusters.

The organisation of gene clusters (boxed) specifying lantibiotic and lantibiotic-like peptides are presented along with schematic structure representations of the mature peptides. The size of gene clusters is given in kilobases (kb). Black boxes indicate structural genes and genes involved in post-translational modification and transport; grey boxes indicate regulatory genes; filled boxes stand for immunity genes. The figure is reproduced from [35].

mrsA is the structural gene in the mersacidin gene cluster, whereas the genes mrsM and mrsD are involved in its post-translational modification [36]. Furthermore, mrsT, coding for a transporter with an associated protease domain, mediates the transport while the operon mrsFGE, an ABC transporter, confers self-protection against the lantibiotic. mrsR1 is a response regulator that controls biosynthesis of mersacidin whereas the putative two component regulatory system mrsR2K2 controls immunity [36, 37].

The structural gene for sublancin biosynthesis is sunA and it belongs to the B. subtilis temperate bacteriophage SPβ. An ABC transporter (SunT) and two thiol-disulphide oxidoreductases (BdbAB) belong to the same locus [38]. Until now, only BdbB is proven to be involved in the sublancin production, most probably for the formation of the disulphide bonds [39]. The genes conferring immunity are unidentified.

Finally, the gene cluster of subtilosin (sbo-alb) encodes AlbA protein, probably involved in post-translational modification of presubtilosin, and AlbBCD proteins, a putative ATP-binding transporter, involved in immunity [40]. The expression of alb genes is under the negative control of AbrB [41].

In spite of their structural heterogeneity, the peptide antibiotics of this group share a common mode of synthesis, the multicarrier thiotemplate mechanism [43]. According to this model, peptide bond formation takes place on multienzymes designated nonribosomal peptide synthetases (NRPS), which are arranged in modules. Modules are the units responsible for the incorporation and/or modification of a specific amino acid into the peptide product, and their arrangement and number are usually colinear to the amino acid sequence and the length of the peptide respectively (colinearity rule) [44], [45, 46]. Modules are further divided into domains; the enzymatic units involved in a specific step of synthesis, such as substrate activation, covalent binding, elongation etc [47].

According to the multicarrier thiotemplate mechanism, the carboxy group of amino acid is activated to the corresponding adenylate by ATP hydrolysis and then it is transferred onto the free thiol-group of an enzyme bound 4'-phosphopantetheinyl cofactor (4'-PP), forming a thioester. At this stage, the substrates can undergo modifications such as epimerization or N- methylation. Peptide assembly is achieved via peptide bond formation steps, by binding of the thioester-activated carboxyl group of the upstream module to the free amino group of the adjacent downstream module. During this N to C stepwise elongation, the intermediates are covalently attached to the multienzyme complex. The termination of the synthesis is induced by the release of the thioester-bound peptide product by hydrolysis, cyclization or transfer to a functional group [19, 44, 48]. As an example figure 3 shows a prototype NRPS assembly line for the cyclic lipoheptapeptide surfactin [49].

Figure 3: Surfactin assembly line.

The multienzyme complex consists of seven modules which are responsible for the incorporation of seven amino acids. 24 domains catalyse the same number of chemical reactions. The peptide chain is elongated stepwise from N to C end. The last domain is responsible for release and cyclization surfactin. The figure is reproduced from [48].

Domains are not just imaginary sections in the module. They are enzymatically active, as well as structurally and catalytically independent. They can be excised from the peptide chain and still retain their activity [45].

Nonribosomal peptide synthetases require posttranslational modification to be functionally active. As it has been already mentioned, thiolation domains are unable to serve as transport proteins immediately after translation, resulting in blocking of peptide synthesis. A modification by transfer of the 4'-PP moiety of coenzyme A onto a conserved serine residue of each T-domain, converts the latter from apo- to holo-form and unblocks the synthesis. The mobile 4'-PP prosthetic group is about 20Å in length and since it is covalently bound as a phosphothioester to the multienzyme [91], it serves as a “flexible arm”, which initially accepts the activated substrates and later on delivers them to the next building-block [43, 55, 57]. The conversion of T-domain is catalyzed by a dedicated 4'-phosphopantetheinyl transferase (4'-PPTase) in a Mg⁺²-dependent way, thereby releasing 3', 5'-ADP (Fig. 6) [92, 93]. Sfp and Gsp proteins control this reaction in B. subtilis and B. brevis, respectively [56, 92, 93].

Recent studies have shown that Sfp accepts as substrates CoA derivatives, such as acetyl-CoA and aminoacyl-CoA [60, 94]. It is therefore likely that PPTases also modify the T-domains of NRPSs with acyl-4'-PP, rendering the enzyme inactive, as misprimed transport units are unable to accept activated amino acids. The activity can be restored by thioesterases II (TE-II) which hydrolyze the acyl-4'-PP, leaving only the 4'-PP bound, and are found in association with the peptide synthetases [95]. TE-IIs contribute as proofreading enzymes, since they preferentially hydrolyze acetyl-Ts versus aminoacyl or peptidyl-Ts [96]. Consequently, the capable of nonribosomal peptide synthesis holo-Ts are made either by direct priming of the apo-derivatives, catalyzed by PPTases ,or by deblocking misprimed derivatives, catalyzed by TE-IIs.

Figure 6: Conversion of thiolation domain from apo- to holo-form.

The 4'-phosphopantetheine moiety of coenzyme A is covalently attached onto an invariant serine residue of the thiolation domain (PCP) by dedicated phosphopantetheinyl transferases; thus PCP-domains are activated. The figure is reproduced from [48].

In recent years increasingly more peptide synthetases have been identified that contain domains normally present in fatty acid (FASs) or polyketide (PKSs) synthases. The first determined mixed NRPS-PKS biosynthetic gene cluster was that of rapamycin in Streptomyces hydroscopius, that contains a NRPS module for the incorporation of pipecolic acid into the polyketide[97, 98]. In addition, synthesis of melithiazole and myxothiazole requires six multifunctional enzymes that switch back and forth between NRPS and PKS [99, 100]. Furthermore, hybrid systems of peptide synthetase and fatty acid synthase, such as mycosubtilin and iturin were characterized in various Bacillus strains [63, 101]. Most recently, a genomic island (54kb) that consists of three nonribosomal peptide synthetases, three polyketide synthases and two hybrid NRPS/PKS synthases was identified among pathogenic E. coli strains of the B2 group. Interestingly, it was shown that E. coli strains expressing this gene cluster induce double-strand breaks in eukaryotic cells leading to cell death [102].

Nonribosomally synthesized peptide antibiotics are widespread among Bacilli. Some of them are characteristically produced by only one member of the genus whereas others are more conserved. Nowadays more information concerning their diversity and distribution has accumulated, partly as a result of the increased number of sequenced genomes. Due to their conserved genetic structure and huge size, these synthetases can be easily recognized. Together with the polyketide synthases, they are the largest operons in the genome. In this section, an attempt will be made to summarize the current knowledge in respect with how the most well studied antibiotics of this group are organized and operate.

Different Bacillus strains produce small cyclic peptides with long fatty moiety, the so-called lipopeptides. Based on their structure, they can be generally classified into three different groups: i) the surfactin [112], ii) the fengycin [76, 113, 114] and iii) the iturin group [115].

Surfactin is a heptapetide linked via lactone bond to a β-hydroxy fatty acid composed of 13 to 15 carbon atoms (Fig. 8A) [116, 117]. Its operon comprises four open reading frames (ORFs) codifying the proteins SrfAA, SrfAB, SrfAC, SrfAD (Fig. 9A) [49, 118, 119, 120, 121]. SrfAC protein ends with a TE-domain, responsible for peptide release and cyclization, whereas the following protein SrfAD shows high homology to TE-IIs. Remarkably, disruption of this gene leads to severe reduction but not abolishment of the antibiotic’s production [95, 96]. Furthermore, SrfAD acts in a double manner by hydrolyzing 4'-PP bound acetyl groups of misprimed NRPSs, according to the TEII ability [95] as well as by mediating the transfer of the fatty acid substrate to the Glu-module and stimulating β-hydroxyacyl-glutamate formation [122]. In general, the number of amino acids and their configuration agrees totally with the organization of modules and domains on the surfactin synthetase, confirming the colinearity rule mentioned earlier. An example is the presence of two D-configurated amino acids that correspond exactly to the position of two epimerization domains.

Surfactin is one of the best characterized lipopeptides, since it possesses various beneficial abilities. Firstly, surfactin is able to lower surface and interfacial tension, thanks to its amphiphilic structure. In particular, surfactin produced by B. subtilis ATCC 21332 is considered one of the most powerful biosurfactans, since it can lower the surface tension of water from 72 to 28 mN/m at concentrations as low as 24μM [123, 124]. Furthermore, surfactin is responsible for inhibition of fibrin clotformation [124] and for erythrocytes lysis [125]. Other beneficial properties, with potential biotechnological and pharmaceutical applications are. i) antitumor activity [126], ii) activity against enveloped viruses [127], iii) antibiotic function against the protoplast of B. megaterium [128] and Mycoplasma [129, 130]. Furthermore, the srf operon encodes the regulatory gene, comS [131], which is involved in the development of genetic competence, an active process aimed at acquiring new genetic material that enables the cell to survive under changing environmental conditions [1].Surfactin is also essential for swarming motility [132, 133, 134, 135], a flagellum-driven social form of surface locomotion, as well as for formation of biofilms, i.e. surface-associated multicellular communities [136, 137].

Fengycin, synonymous to plipastatin, is a cyclic decapeptide linked to a β-hydroxy fatty acid moiety, with lengths that vary from 14 to 18 carbon atoms (Fig. 8E, 9B) [138, 139, 140]. Fengycin demonstrates strong surface activity, although lower compared to surfactin [141]. Fengycin is active against filamentous fungi [76, 139, 140], and inhibits the enzymes phospholipase A2 [142] and aromatase [143].

Iturin, mycosubtilin and bacillomycin belong to the same group of lipopeptides. These compounds consist of seven α-amino acids and one β-amino fatty acid, that distinguishes them from the already mentioned groups. The peptide moiety contains a tyrosine in the D-configuration at the second amino acid position as well as two additional D-amino acids at positions three and six (Fig. 8B, 8C, 8D). Gene sequences encoding enzymes for biosynthesis of iturin A and mycosubtilin, but not bacillomycin D, have been reported (Fig. 9C) [63, 101]. Thereby it has been revealed that these lipopeptides are synthesized on hybrid synthases, since domains homologous to fatty acid and polyketide synthases are situated at their N- terminus [63]. These domains are absent from the peptide synthetases of surfactin and fengycin groups, so it appears very likely that these domains are involved in the incorporation of the β-amino fatty acid moiety into the peptides of the iturin group lipopeptides [63]. Moreover, these antibiotics exhibit strong antifungal and hemolytic activities, whereas their antibacterial function is more limited [76, 115].

Figure 8: Schematic structure of various lipopeptides produced by Bacilli.

A Surfactin, n = 10-12, B Iturin A, n = 10-13, C Mycosubtilin, n = 10-13, D Bacillomycin D, n = 10-13, E Fengycin, n = 13-17, F Lichenysin, n = 9 -14

The above mentioned lipopeptides are produced by different Bacilli, such as B. subtilis and B. cereus. However, one lipopeptide with similar structure to surfactin is exclusively composed by B. licheniformis [144, 145, 146]. It is designated as lichenysin and is a cyclic heptapeptide with a β-hydroxy fatty acid moiety, composed of 12-17 carbon atoms (Fig. 8F, 9D) [146]. It demonstrates antimicrobial properties and reduces the surface tension of water [144, 146]. In particular, lichenysin A can cause a similar reduction in water surface tension as surfactin from B. subtilis ATCC 21332, albeit in lower concentration (12μM versus 24μM) [145].

Another nonribosomally synthesized antibiotic compound is bacitracin found in B. licheniformis [147, 148]. This thiazoline ring-containing dodecapeptide is synthesized by the large multienzyme complex BacABC (Fig. 9E) [149]. Bacitracin is a prominent inhibitor of cell wall biosynthesis and most active against Gram-positive bacteria [147]. However, B. licheniformis and several other Gram-positive bacteria are not susceptible to this antibiotic suggesting the existence of specific resistance mechanisms [150]. Its primary mode of action is the formation of a tight ternary complex with the peptidoglycan carrier C₅₅-isoprenyl pyrophosphate (IPP) and a divalent metal cation. This carrier is responsible for the translocation of cell envelope building blocks from the cytosol to the external side of the cytoplasmic membrane, where they are incorporated to the macromolecular network of the cell envelope (i.e. peptidoglycan, teichoic acids and polysaccharide capsule). Binding of bacitracin to IPP prevents its recycling by dephosphorylation to the monophosphate form that is normally reloaded on the inner face of the membrane [150, 151].

Another member of the Bacillus genus, B. brevis, produces two cyclic decapeptides, tyrocidine and gramicidin S (Fig. 5D, 9F) [52, 84, 152]. The first one characteristically contains a nonproteinogenetic residue, the L-ornithine and acts as antibiotic by membrane perturbation [17, 52]. Gramicidin S is synthesized on the enzymes GrsTAB, where only five amino acids are activated and incorporated. However, the peptide is dimerized to the decapeptide prior to its release. Furthermore, gramicidin S exhibits strong antibacterial activities against Gram positive and negative bacteria [153, 154], probably due to an interaction with membrane phospholipids. Thereby, gramicidin S causes a phase separation of negatively charged phospholipids from other lipids leading to a disturbance of the membrane’s osmotic barrier [155, 156].

Figure 9: Schematic representation of peptide synthetase operons in Bacilli.

The genes comprising each peptide synthetase operon and their sizes are indicated. Organisation within the modules is presented, while the respective activated amino acid are depicted within the adenylation domains. A. Surfactin operon in B. subtilis [49]. B. Fengycin operon in B. subtilis [140]. C. Iturin A and mycosubtilin operons in B. subtilis [63, 101]. D. Lichenysin A operon in B. licheniformis [146]. E. bacitracin operon in B. licheniformis [149]. F. Tyrocidine and gramicidin S operons in B. brevis [52, 152]. The figure is adapted from [157].

In the last few decades, the pathways that govern the synthesis of antibiotics on large multienzymes have been thoroughly studied. Significant progress has been made on the functional analysis of various domains as well as on the role of their assembly in the peptide synthetases. Moreover, high resolution structures obtained for several enzymatic subunits from different antibiotics led to a better understanding of their architectural organization, substrate specificity and catalytic action [70, 158, 159, 160]. In contrast, our knowledge concerning how the organism regulates expression of these systems or the mechanisms which govern export of the peptides and/or resistance to them is rather limited. An exception is the case of surfactin, for which studying the regulation of gene expression received increased attention due its connection with the development of genetic competence.

The expression of surfactin is growth-phase dependent and is induced during transition to stationary phase [161]. Its transcription is driven by a σ^A-dependent promoter [161] and its expression is regulated via a complex network, including the two component regulatory system, ComAP [161, 162]. ComP is the sensor histidine kinase that is autophosphorylated after sensing increase in the concentration of the pheromone ComX [163]. The phosphoryl group is then transferred to the response regulator, ComA and activates it. Phosphorylated ComA can bind upstream of the srf operon and induce its expression. Therefore, systems involved in the phosphorylation / DNA-binding ability of ComA (ComXQ, RapC-CSF, RapF) modify indirectly the antibiotic’s expression [163, 164, 165, 166, 167]. PerR, a general repressor of the peroxide stress regulon, is shown to positively regulate surfactin in a direct manner, independently of ComA [168]. In contrast CodY, a GTP-activated global regulator, acts as a direct repressor under casamino acids rich conditions [169]. Furthermore, YerP, a protein homologous to the RND (resistance, nodulation and cell division) family of efflux pumps in Gram-negative bacteria, seems to contribute in secretion of surfactin and self-resistance of the producer strain against it [170].

Knowledge on transcriptional regulation of the remaining lipopeptides is rather limited. The promoters of fengycin and iturin operons have been successfully identified and show similarity to a housekeeping σ^A promoter [101, 113]. Furthermore, deletion of degQ, a pleiotropic regulator gene that controls the production of several secreted and degradative enzymes [171], reduces severely the production of these antibiotics, via an unidentified mechanism [172, 173].

All these lipopeptides are post-translationally regulated by sfp, a 4'-phosphopantetheinyl transferase which converts T-domains to their active form (see corresponding chapter; [92, 137]. The importance of this gene is demonstrated in strains that contain intact synthetases but dysfunctional sfp. B. subtilis strain 168 contains intact srf and fen operons but is unable to produce the antibiotics, due to a frameshift mutation on the sfp gene [174]. However, when complemented with a functional 4'-PPTase, the antibiotic production of the strain is restored [172, 175].

Mechanisms that govern regulation of lichenysin and bacitracin are studied only in a preliminary basis. Lichenysin expression is dependent on the two component regulatory system ComAP [176]. In the case of bacitracin, an ABC transporter (BcrABC) conferring resistance to the producer strain against the antibiotic was determined [177, 178]. It is located about 3 kb downstream of the bacitracin biosynthetic operon bacABC and its expression is induced by the dodecylpeptide [150, 179]. Moreover, a two component regulatory system BacRS, situated between the bac operon and the bcrABC genes, negatively regulates expression of the transporter genes [150].

Transcription of the tyrocidine operon is driven by a typical σ^A promoter and its expression is induced at the end of exponential phase of growth. Spo0A, Spo0B and Spo0E, involved in the sporulation process, are required for full activation of the operon, whereas AbrB, a transition-phase regulator, acts as its repressor [180]. Further studies revealed that AbrB inhibits tyrocidine expression directly by binding to the upstream region of tycA [181]. Moreover, tycD and tycE, which are located downstream of the operon, show high similarity to members of the ABC transporter family and thus may confer immunity to the producer strain [52]. However, their role remains to be verified.

Years of research revealed that NRPS and NRPS-PKS hybrids can produce biologically active compounds exhibiting high antimicrobial activity. Their modular architecture allows the possibility to manipulate the enzymatic machinery in order to increase or alter their biological action. In the last decade, successful steps have been made in creating novel improved antibiotics by genetically redesigning natural synthesized compounds.

Genetic engineering has been achieved using different approaches. The first approach was based on exchanging the A-T units of the terminal module of surfactin synthetase that is originally responsible for the incorporation of leucine. Different A-T units have replaced the already existing one and novel surfactins with aliphatic (Val), charged (Orn) and aromatic (Phe) residues at position 7 were created. However, their hemolytic activity did not differ significantly from that of the wild type product [182]. Nevertheless, swapping of numerous domains indicated for the first time that a rational design of antibiotics is accomplishable [183].

A further strategy for constructing synthetic antibiotics involves entire module swapping as well asinsertions or deletions of modules. Conistent to this concept, deletion of the second module of the srf operon produced a new hexapeptide surfactin [184]. Alternatively, the manipulation of the A-domain’s specificity via point mutagenesis can also result in novel antibiotics. The altered A-domain recognizes and activates a different amino acid, which is then incorporated in the polypeptide chain to yield a new product [185]. Another pathway to novel antibiotic production involves the replacement of TE-domains on the synthetase to force earlier release and cyclization [186]. It has been already shown that the bioactivity of many peptide antibiotics is attributed to small heterocyclic compounds, such as thiazoline and oxazoline, which are composed by heterocyclization domains present on the synthetases. Therefore incorporation of such domains on peptide synthetases could lead to new pharmaceutical substances [187].

Nowadays, there are an increasing number of examples for functional engineered peptide synthetases. Genetic redesign requires well-defined sequence information about the biosynthetic system that will be altered. Although this is often provided, manipulation has been unsuccessful in some cases, due to possible disruptions on some catalytic site(s) [182]. Therefore, information on domain structures as well as on possible protein-protein interaction sites between domains would improve manufacturing of novel antibiotic compounds.