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DATABASES |
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| BIOSYNTHESIS |
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Organization of the gene clusters:
The genes involved in biosynthesis of the model lantibiotic nisin are located on a 70 kb conjugative transposon,42 which also contains the genetic information for sucrose metabolism. The first gene of the nisin gene cluster, nisA, encodes the 57 amino acid nisin precursor, consisting of a N-terminal leader sequence followed by the propeptide, from which nisin A is matured. The structural gene is followed by ten other genes i.e.
nisB, nisT, nisC, nisI, nisP, nisR, nisK, nisF, nisE, nisG,43 encoding regulatory proteins, proteases, transport proteins and immunity proteins (Fig. 1).
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Fig. 1 Model for the biosynthesis of nisin. The nisin precursor is modified by the putative enzymes NisB and NisC and translocated across the membrane by the exporter NisT. The precursor is extracellularly processed by NisP, resulting in the release of mature nisin. NisK senses the presence of nisin in the medium and autophosphorylates. The phosphate-group is transferred to NisR, which activates transcription of the genes nisABTCIP and nisFEG. NisI, F, E, and G protect the cell from the bacteriocidal activity of nisin. P: promoter region, P*: nisin-regulated promoters.
The proteins that are encoded by these genes have been found to be homologous to gene products of the gene clusters of other lantibiotics, such as those of subtilin, epidermin and Pep5. Not all of the mentioned genes have been detected in all gene clusters, so far. Besides, there is no uniform order of the genes in the individual gene clusters. For an overview of the organization of the various gene clusters the reader is referred to Siezen et al.
Modification and transport:
The gene clusters for nisin, 45 subtilin, 46 epidermin, 47 Pep548 and epicidin 28038 all contain the genes lanB and lanC. These genes do not display significant homology to known genes in sequence databases and their function is not completely understood yet. It has been shown that both genes are essential for the production of lantibiotics, since their disruption results in cessation of lantibiotic biosynthesis.45,46,49 Most likely, LanB and LanC are involved in post-translational modification of the lantibiotics, involving the formation of dehydrated residues and interaction of these residues with the sulfhydryl group of a nearby Cys-residue to form lanthionine residues. The LanB proteins, generally around 1000 amino acid residues in size, are the putative enzymes that catalyze dehydration of the Ser and Thr residues in the propeptide domain of the prelantibiotic. LanC proteins are all about 400 amino acid residues long.
Deletion of the pepC gene in Staphylococcus epidermidis resulted in production of incorrectly modified Pep5 fragments, that contained only one of three expected lanthionine residues.47 This suggested that PepC is the thioether-forming protein. Investigations on the proteins SpaB48 and EpiB50 revealed that these proteins are membrane-associated, suggesting that lantibiotic biosynthesis occurs at the cytoplasmic membrane. Moreover, by use of the yeast two-hybrid system and coimmunoprecipitation techniques, it was shown that nisin51 and subtilin52 precursor peptides are processed by a multimeric protein complex located at the cytoplasmic membrane, consisting of LanB, LanC and LanT proteins. In the cyl, las, lct and mut gene clusters, for biosynthesis of cytolysin, lacticin 481, lacticin S and mutacin II, respectively, LanB is missing. Instead a LanM protein is found, from which the C-terminal part is homologous to LanC proteins.44 Possibly,
LanM is able to catalyze both the reactions assumed to be catalysed by LanB and LanC. The epidermin-gene cluster is unique in that it contains the gene epiD, encoding a amino acid protein that is also involved in post-translational modification of epidermin. To identify its function, EpiD was purified from Staphylococcus epidermidis and incubated in vitro with epidermin precursor peptide. The results showed that EpiD is a flavoenzyme that catalyzes the oxidative decarboxylation of the C-terminal Cys-residues of epidermin, a modification that is only found in epidermin and its natural variant gallidermin. Another experimental approach in which a variant epiA gene encoding His-tag-labelled epidermin was co-expressed with epiD established this role of EpiD. In the gene cluster of epicidin, which is produced by Staphylococcus epidermidis, an additional gene was found, designated epiO.
Since this gene has similarity to a family of oxidoreductases, it most likely codes for an enzyme that catalyzes the modification of the Nterminal residue of epicidin. All known lantibiotic gene clusters contain a lanT gene, which encodes a protein that is involved in transport of the lantibiotic precursor across the cellular membrane to outside the cell. It was first shown for subtilin that disruption of the gene spaT resulted in intracellular accumulation of subtilin. A number of transport protein-encoding genes have now been identified in several of the lantibiotic gene clusters, including those for nisin, lacticin lactocin S38 and recently for lacticin 3147. It should be noted that the transport gene in the cyl gene cluster is designated cylB. The LanT proteins share homology with a large family of transport proteins, characterized by the presence of a cytoplasmic ATP-binding domain and a membrane spanning domain.
For all lantibiotic transporters, with the exception of EpiT, both domains are present in one protein, of about 600 amino acid residues in size. The gene encoding EpiT is incomplete and it seems not to be required for transport of epidermin in Staphylococcus epidermidis. Possibly, transporter proteins of the host replace its function. Introduction of gdmT, the transporter gene of the related lantibiotic gallidermin into an epidermin producing strain strongly increased production yields. The epi and gdm gene clusters also contain a lanH gene, encoding a protein of about 330 amino acids containing several putative transmembrane sections. GdmH is assumed to be involved in secretion of gallidermin, in cooperation with GdmT.
Processing of precursor peptides:
The last step in the biosynthesis of lantibiotics is the removal of the leader peptide from the lantibiotic precursor. In the gene clusters for the lantibiotics nisin, epidermin, Pep5, epilancin K7, lacticin S, cytolysin and epicidin 28038 a lanP gene was found encoding a subtilisin-like protease. The location at which processing of the leader peptide occurs, varies with the lantibiotic. NisP, responsible for cleavage of the nisin precursor is anchored to the cellular membrane at the outside of the host cell. EpiP and CylP are also transported to the outside of the cell, but appear to lack a membrane anchor. In contrast, PepP, ElkP and LasP were reported to act in the cytoplasm of cells, possibly in association with LanB, C and T. Cleavage of lacticin 481 occurs by the protein LctT, which displays a dual function since it is responsible for proteolytic cleavage as well as secretion of the lantibiotic.
In contrast to the gene clusters discussed above, the spa gene cluster does not contain a gene encoding a peptidase, and it is assumed that processing of subtilin occurs by a general serine protease of the host Bacillus subtilis. The function of the leader peptide is unclear. The fully modified lantibiotic precursor is almost inactive, and it has been suggested that the leader sequence is of importance to keep the lantibiotic in an inactive state, to protect the producer cell from its activity. However, this does not hold for those lantibiotics that are cleaved intracellularly. NMR studies on the nisin precursor have demonstrated that the residues Ile1-Ala of the nisin precursor interact differently with membrane-mimicking micelles than the corresponding part of mature nisin. These results indicate that the low in vivo activity of the precursor is caused by a less efficient insertion of the peptide into a membrane.
The leader peptide might alternatively, or additionally, play a role in modification and excretion of the peptide, for instance by targeting the unmodified precursor to the modification and secretion machinery. This assumption was strengthened by the observation that specific mutations in the leader sequences of nisin63 and Pep564 strongly affected the production level of the lantibiotics. Obviously, a defined leader sequence is of importance for optimal biosynthesis of the lantibiotic.
Immunity of producer organisms:
One of the most intriguing questions in lantibiotic production concerns the mechanism of self-protection. Obviously, without efficient ways to protect themselves from the pore-forming activity of the peptides, the producing cells would be suicidal. In all gene clusters studied so far small (50–70 AA) or mediumsized (160–250 AA) hydrophobic proteins (LanI) are encoded that are attached to the outside of the membrane and are involved in the immunity process. So far, LanI proteins have been found in nisin A45,65 and Z,66 subtilin,67 Pep568 and epicidin 28038 producing cells. Possibly, these molecules play a role in the recognition of the active lantibiotic present at the outside of the cell, either directly from solution or upon adsorption to the membrane. Overexpression of LanI proteins in cells that do not possess the lantibiotic biosynthesis machinery only yields very low protection levels (1–4%) against the corresponding lantibiotic.
An in-frame disruption of nisI in Lactococcus lactis yielded a strain that could still produce nisin, albeit to levels five times lower than wild-type, which suggests that the immunity level could be the first limiting factor in reaching high production levels. These results already indicate that additional factors are involved in the acquirement of full self-protection. In several lantibiotic gene clusters three other proteins are encoded, named LanF, LanE and LanG. The LanFEG proteins belong to the group of ABC transporters, where LanF contains the intracellular ATP-binding domain and LanEG the mem- brane-spanning subunits. The genes lanFEG have been found in the gene clusters for nisin, subtilin, lacticin 481, epidermin and lacticin 3147. It is tempting to speculate that these proteins have a function in the removal of the corresponding lantibiotic at a certain stage of its membrane interaction.
Expression of the LanFEG proteins, without LanI, yields a significant level of immunity although it remains below the wild-type level. The synergistic function of all immunity proteins could thus reside in the first recognition and binding or ‘immobilization’ of the lantibiotic in the membrane by NisI, followed by active removal, in which the LanFEG proteins are involved. It cannot be excluded, however, that also other proteins encoded in the lantibiotic gene clusters display a synergistic effect in the immunity mechanism, for instance by being involved in a multi-protein complex that spans the membrane. Candidates to be involved in such a complex are LanBTC proteins, which have already been shown to form a membrane associated complex. Interestingly, in some cases, e.g. nisin and subtilin production, the expression of immunity genes is also regulated by the concentration of the lantibiotic in the medium,
which means that by sensing low (subinhibitory) amounts of the antimicrobial peptide in the medium, cells can rapidly increase their immunity level, concomitant with or even faster than the biosynthesis rate. The challenge still exists to unravel the complete mechanism of immunity at the molecular level, the understanding of which could in the end result in engineering increased immunity and possibly increased production levels of lantibiotics.
Regulation of biosynthesis:
In 1995 it was reported that apart from displaying a strong antimicrobial activity, the lantibiotic nisin also plays an important role in the regulation of its own biosynthesis. In fact nisin can be regarded as a peptide pheromone which is sensed by the histidine kinase NisK, which resides at the outer side of the membrane, probably by direct protein–peptide interaction. By analogy with other known two-component regulatory systems NisK will autophosphorylate at a specific histidine residue when it senses a certain nisin concentration in the medium and subsequently transfer the phosphate moiety to the response regulator NisR. The response regulator is assumed to get phosphorylated at a specific Asp residue, which is supposed to trigger its binding to two regulated promoters in the nisin gene cluster, i.e. the nisA and the nisF promoter,
thereby activating transcription of the structural gene nisA and the downstream genes nisBTCIP by limited readthrough from nisA, and the genes nisFEG located at the end of the gene cluster. The regulatory genes nisRK themselves are transcribed from their own promoter which is assumed to be not dependent on nisin induction. Although not all molecular details have been unraveled yet, it is clear that this autoregulatory process resembles the quorum-sensing phenomenon found in several Gram-negative bacteria, for which the bioluminescence phenotype of Photobacterium fischeri is the paradigm. The transcription from the nisA and nisF promoter in the nisin gene cluster is directly related to the concentration of nisin in the medium. This property is extremely useful for the development of controlled gene expression systems,
since a linear dose–response for the expression of target genes is highly desirable in industrially relevant production organisms. Especially when toxic gene products should be produced, the nisin-controlled expression (NICE) system is ideally suited, since the nisA promoter is tightly shut off in the uninduced state. Moreover, very high expression levels of up to 60% of total intracellular protein can be reached in Lactococcus lactis. The NICE system has also been succesfully implemented in some heterologous hosts like the lactic acid bacteria Leuconostoc lactis and Lactobacillus helveticus and the Gram-positive bacteria Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecalis. Since in many other lantibiotic and non-lantibiotic gene clusters the counterparts of the two-component regulatory proteins NisR and NisK are found,
it is reasonable to assume that also in these cases an autoregulatory process takes place. In fact for the production of several non-lantibiotic antimicrobial peptides, e.g. carnocin, plantaricin and sakacin the peptide pheromone concept has been shown to be valid. In the case of subtilin production the autoregulatory process has been shown to be very similar to the nisin case, because the spaB, spaI and SpaS promoters could be activated by subtilin in the medium.88 Knock-outs of spaRK and nisRK have been shown to be detrimental for lantibiotic production10,88,89 An exception to the general rule is found in the case of epidermin, since in that biosynthetic gene cluster only the response regulator encoding EpiQ was found to be present.90 Possibly, a high expression level of only the response regulator results in effective expression of the target genes,
probably because this protein will be phosphorylated in an aspecific way. Also by overexpression of nisR in a strain lacking nisK, efficient transcription from the nisA promoter was observed. Both inducer and sensor engineering have been used to study the molecular interaction between these molecules. First, it was shown that variants and fragments of nisin were able to act as inducer with variable efficiencies and that their induction capacity was unrelated to their antimicrobial activity, demonstrating that the mechanisms for induction and pore formation are different. In a recent study a plasmid containing the reporter gene gusA under control of the nisA promoter was introduced in a strain containing the nisR gene integrated on the chromosome. Introduction of a nisK expressing plasmid in this strain resulted in a fully functional nisin induction system.
Surprisingly, also introduction of spaK, combined with subtilin as inducer, led to a functional signal transduction, showing that cross-talk between SpaK and NisR occurs, albeit with decreased efficiency. In an extension of this study hybrids of NisK and SpaK were constructed. It was demonstrated that the subdomain needed for inducer interaction is located in the N-terminal domain of the sensor protein and includes at least both transmembrane domains and the external loop. A better understanding of these interactions will open the way to the rational design of more effective sensors and perhaps even to sensors with a loosened specificity for the inducer molecule. Undoubtedly, in the coming years the autoregulatory processes found to occur in many lantibiotic biosynthesis processes will yield valuable spin-off for a variety of industrial applications, e.g. increased production levels,
development of novel biosensors and improved controlled gene expression systems.
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| SLIDE SHOW |
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