The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening.
Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA). Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C) with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asp-Tyr, which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA.
A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.
Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a β-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway.
A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis.
In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.
Non-ribosomal peptide; Biosynthesis; Gene cluster; Antimicrobial agent
Here, we report the draft genome sequence of Paenibacillus elgiiB69, which was isolated from soil and has broad-spectrum antimicrobial activity. As far as we know, the P. elgiigenome is the largest of the Paenibacillusgenus for which genome sequences are available. Multiple sets of genes related to antibiotic biosynthetic pathways have been found in the genome.
Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential lantibiotic-encoding gene clusters in genome sequenced bacteria.
In total 49 novel type 1 lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with lantibiotic production.
Multiple type 1 lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.
Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C18 reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 ± 1.5 and 3,136 ± 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332–4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.
lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an
antimicrobial peptide of 20 amino acids which is produced by
Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits
bacterial cell wall biosynthesis by binding to the precursor molecule
lipid II. The structural gene of mersacidin (mrsA) and the
genes for the enzymes of the biosynthesis pathway, dedicated
transporters, producer self-protection proteins, and regulatory factors
are organized in a biosynthetic gene cluster. For
site-directed mutagenesis of lantibiotics, the engineered genes must be
expressed in an expression system that contains all of the factors
necessary for biosynthesis, export, and producer self-protection. In
order to express engineered mersacidin peptides, a system in which the
engineered gene replaces the wild-type gene on the chromosome was
constructed. To test the expression system, three mutants were
constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at
position 16 was replaced with the Ile residue found in the closely
related lantibiotic actagardine. S16I mersacidin was produced only in
small amounts. The purified peptide had markedly reduced antimicrobial
activity, indicating an essential role for Dha16 in biosynthesis and
biological activity of mersacidin. Similarly, Glu17, which is thought
to be an essential structure in mersacidin, was exchanged for alanine.
E17A mersacidin was obtained in good yields but also showed markedly
reduced activity, thus confirming the importance of the carboxylic acid
function at position 17 in the biological activity of mersacidin.
Finally, the exchange of an aromatic for an aliphatic hydrophobic
residue at position 3 resulted in the mutant peptide F3L mersacidin;
this peptide showed only moderately reduced
Salivaricin G32, a 2667 Da novel member of the SA-FF22 cluster of lantibiotics, has been purified and characterized from Streptococcus salivarius strain G32. The inhibitory peptide differs from the Streptococcus pyogenes—produced SA-FF22 in the absence of lysine in position 2. The salivaricin G32 locus was widely distributed in BLIS-producing S. salivarius, with 6 (23%) of 26 strains PCR-positive for the structural gene, slnA. As for most other lantibiotics produced by S. salivarius, the salivaricin G32 locus can be megaplasmid encoded. Another member of the SA-FF22 family was detected in two Streptococcus dysgalactiae of bovine origin, an observation supportive of widespread distribution of this lantibiotic within the genus Streptococcus. Since the inhibitory spectrum of salivaricin G32 includes Streptococcus pyogenes, its production by S. salivarius, either as a member of the normal oral microflora or as a commercial probiotic, could serve to enhance protection of the human host against S. pyogenes infection.
A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.
Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent.
The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens.
The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.
Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.
Lantibiotics are ribosomally synthesized, posttranslationally modified peptide antibiotics. Microbisporicin is a potent lantibiotic produced by the actinomycete Microbispora corallina and contains unique chlorinated tryptophan and dihydroxyproline residues. The biosynthetic gene cluster for microbisporicin encodes several putative regulatory proteins, including, uniquely, an extracytoplasmic function (ECF) σ factor, σMibX, a likely cognate anti-σ factor, MibW, and a potential helix-turn-helix DNA binding protein, MibR. Here we examine the roles of these proteins in regulating microbisporicin biosynthesis. S1 nuclease protection assays were used to determine transcriptional start sites in the microbisporicin gene cluster and confirmed the presence of the likely ECF sigma factor −10 and −35 sequences in five out of six promoters. In contrast, the promoter of mibA, encoding the microbisporicin prepropeptide, has a typical Streptomyces vegetative sigma factor consensus sequence. The ECF sigma factor σMibX was shown to interact with the putative anti-sigma factor MibW in Escherichia coli using bacterial two-hybrid analysis. σMibX autoregulates its own expression but does not directly regulate expression of mibA. On the basis of quantitative reverse transcriptase PCR (qRT-PCR) data, we propose a model for the biosynthesis of microbisporicin in which MibR functions as an essential master regulator and the ECF sigma factor/anti-sigma factor pair, σMibX/MibW, induces feed-forward biosynthesis of microbisporicin and producer immunity.
Lantibiotics are ribosomally synthesized peptide antimicrobials which contain considerable posttranslational modifications. Given their usually broad host range and their highly stable structures, there have been renewed attempts to identify and characterize novel members of the lantibiotic family in recent years. The increasing availability of bacterial genome sequences means that in addition to traditional microbiological approaches, in silico screening strategies may now be employed to the same end. Taking advantage of the highly conserved nature of lantibiotic biosynthetic enzymes, we screened publicly available microbial genome sequences for genes encoding LanM proteins, which are required for the posttranslational modification of type 2 lantibiotics. By using this approach, 89 LanM homologs, including 61 in strains not known to be lantibiotic producers, were identified. Of these strains, five (Streptococcus pneumoniae SP23-BS72, Bacillus licheniformis ATCC 14580, Anabaena variabilis ATCC 29413, Geobacillus thermodenitrificans NG80-2, and Herpetosiphon aurantiacus ATCC 23779) were subjected to a more detailed bioinformatic analysis. Four of the strains possessed genes potentially encoding a structural peptide in close proximity to the lanM determinants, while two, S. pneumoniae SP23-BS72 and B. licheniformis ATCC 14580, possess two potential structural genes. The B. licheniformis strain was selected for a proof-of-concept exercise, which established that a two-peptide lantibiotic, lichenicidin, which exhibits antimicrobial activity against all Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococcus strains tested, was indeed produced, thereby confirming the benefits of such a bioinformatic approach when screening for novel lantibiotic producers.
The diversity of dinitrogenase reductase gene (nifH) fragments in Paenibacillus azotofixans strains was investigated by using molecular methods. The partial nifH gene sequences of eight P. azotofixans strains, as well as one strain each of the close relatives Paenibacillus durum, Paenibacillus polymyxa, and Paenibacillus macerans, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two nifH sequence clusters, designated clusters I and II, in P. azotofixans. The data further indicated that there was sequence divergence among the nifH genes of P. azotofixans strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH cluster II sequences were similar to the alternative (anf) nitrogenase sequence. A nested PCR assay for the detection of nifH (cluster I) of P. azotofixans was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the P. azotofixans nifH gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of nifH in P. azotofixans strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different nifH genes. Moreover, nifH is probably a multicopy gene in P. azotofixans. Both similarities and differences were detected in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.
Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides. The recently discovered lantibiotic epilancin 15X produced by Staphylococcus epidermidis 15X154 contains an unusual N-terminal lactate group. To understand its biosynthesis, the epilancin 15X biosynthetic gene cluster was identified. The N-terminal lactate is produced by dehydration of a Ser residue in the first position of the core peptide by ElxB, followed by proteolytic removal of the leader peptide by ElxP, and hydrolysis of the resulting new N-terminal dehydroalanine. The pyruvate group thus formed is reduced to lactate by an NADPH dependent oxidoreductase designated ElxO. The enzymatic activity of ElxB, ElxP, and ElxO were investigated in vitro or in vivo and the importance of the N-terminal modification for peptide stability against bacterial aminopeptidases was assessed.
Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from a human intestinal microbiota. A 12.8-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumA, has been cloned and sequenced. It consisted of 13 open reading frames, organized in three operons with predicted functions in lantibiotic biosynthesis, signal transduction regulation, and immunity. One unusual feature of the locus is the presence of three almost identical structural genes, all of them encoding the RumA precursor. In order to determine the role of trypsin in RumA production, the transcription of the rum genes has been investigated under inducing and noninducing conditions. Trypsin activity is needed for the growth phase-dependent transcriptional activation of RumA operons. Our results suggest that bacteriocin production by R. gnavus E1 is controlled through a complex signaling mechanism involving the proteolytic processing of a putative extracellular inducer-peptide by trypsin, a specific environmental cue of the digestive ecosystem.
The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.
Bifidobacterium longum DJO10A was previously demonstrated to produce a lantibiotic, but only during growth on agar media. To evaluate the feasibility of production of this lantibiotic in broth media, a transcription analysis of the lanA gene was undertaken. Comparative microarray analysis of broth and agar cultures of B. longum DJO10A revealed that the lantibiotic production, modification, transport/peptidase, and immunity genes were significantly upregulated in agar cultures, while the two-component regulatory genes were expressed equally under both conditions. This suggested that the signal transduction regulatory system should function in broth cultures. Real-time PCR and Northern hybridization confirmed that lanA gene expression was significantly repressed in broth cultures. A crude lantibiotic preparation from an agar-grown culture was obtained, and its antimicrobial spectrum analysis revealed a broad inhibition range. Addition of this extract to broth cultures of B. longum DJO10A induced lanA gene expression in a dose-dependent fashion. Subinoculation using >10% of an induced broth culture maintained lanA expression. The expression of lanA was log-phase specific, being significantly downregulated in stationary phase. Transcription start analysis of lanA revealed a 284-bp 5′ untranslated region, which was proposed to be involved in repression of transcription, while an inverted repeat structure located at bp −75 relative to the transcription start was strategically located to likely function as a binding site for the two-component response regulator. Understanding the transcription regulation of this lanA gene is the first step toward enabling production of this novel and potentially interesting lantibiotic in broth cultures.
Nisin produced by Lactococcus lactis 6F3 is used as a food preservative and is the most important member of a group of peptide-antibiotics containing lanthionine bridges (lantibiotics) (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). Nisin is ribosomally synthesized, and its structural gene, nisA, encodes a prepeptide that is posttranslationally modified, revealing the active lantibiotic (C. Kaletta and K.-D. Entian, J. Bacteriol. 171:1597-1601, 1989). Adjacent to nisA, the additional genes nisB, nisT, and nisC were identified. Over their entire sequences, these genes were homologous to genes recently identified as important for the biosynthesis of lantibiotics, that is, subtilin from Bacillus subtilis ATCC 6633 and epidermin from Staphylococcus epidermidis Tü 3298. Genes nisB, nisT, and nisC corresponded to open reading frames of 993, 600, and 418 amino acid residues, respectively. The nisT open reading frame is homologous to proteins of the HlyB (hemolysin B protein of Escherichia coli) subfamily. Proteins of this subfamily are responsible for the secretion of a variety of compounds, including large polypeptides, polysaccharides, and anti-drug tumors, indicating that NisT may be involved in nisin transport. Northern (RNA) blot analysis revealed a 0.3-kb transcript for the nisA structural gene, and the transcriptional start point of the nisA gene was determined by primer extension. Additionally, a mRNA of at least 3 kb was identified by using a hybridization probe specific to nisB. Antibodies were raised against the NisB protein, and Western blot (immunoblot) analysis revealed a molecular weight of about 115 kDa, which is in accordance with the theoretical protein size of 117.5 kDa as calculated from the nisB open reading frame. Several amphipathic transmembrane alpha-helices indicated that NisB is associated with the membrane. This was confirmed by preparing L. lactis vesicles. The NisB protein was tightly associated with the vesicle fraction and was released by sodium dodecyl sulfate treatment only. These results suggest that NisB is membrane associated and that nisin biosynthesis occurs at the cell membrane.
The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.
Streptococcus bovis HJ50 produces a lacticin 481-like 33-amino-acid-residue lantibiotic, designated bovicin HJ50. bovK-bovR in the bovicin HJ50 biosynthetic gene cluster is predicted to be a two-component signal transduction system involved in sensing signals and regulating gene expression. Disruption of bovK or bovR resulted in the abrogation of bovicin HJ50 production, suggesting both genes play important roles in bovicin HJ50 biosynthesis. Addition of exogenous bovicin HJ50 peptide to cultures of a bovM mutant that lost the capability for bovicin HJ50 production and structural gene bovA transcription in S. bovis HJ50 induced dose-dependent transcription of the bovA gene, demonstrating that bovicin HJ50 production was normally autoregulated. The transcription of bovA was no longer induced by bovicin HJ50 in bovK and bovR disruption mutants, suggesting that BovK-BovR plays an essential role in the signal transduction regulating bovicin HJ50 biosynthesis. A phosphorylation assay indicated that BovK has the ability to autophosphorylate and subsequently transfer the phosphoryl group to the downstream BovR protein to carry on signal transduction. Electromobility shift assays (EMSA) and green fluorescent protein (GFP) reporter gene expression assays showed the specific binding of BovR to the bovA promoter, indicating that BovR regulates bovA expression by direct binding between them. Taken together, bovicin HJ50 biosynthesis is induced by bovicin HJ50 itself and regulated via the two-component signal transduction system BovK-BovR.
Paenibacillus sp. strain ICGEB2008 (MTCC 5639) is a Gram-positive cellulolytic bacterium, isolated from the gut of Helicoverpa armigera. Here, we report the draft genome sequence of Paenibacillus sp. ICGEB2008. The annotation of the ~5.7-Mb sequence indicated a cluster of genes related to the glycosyl hydrolase family and the butanediol biosynthesis pathway.
The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids which inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II and which is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene of mersacidin as well as accessory genes is organized in a biosynthetic gene cluster which is located on the chromosome and contains three open reading frames with similarities to regulatory proteins: mrsR2 and mrsK2 encode two proteins with homology to bacterial two-component systems, and mrsR1 shows similarity to a response regulator. Both mrsR2/K2 and mrsR1 were inactivated by insertion of an antibiotic resistance marker. Disruption of mrsR1 resulted in loss of mersacidin production; however, producer self-protection was not impaired. In contrast, inactivation of mrsR2/K2 led to an increased susceptibility to mersacidin whereas biosynthesis of the lantibiotic remained unaffected. Binding of mersacidin to intact cells was significantly enhanced in the mrsR2/K2 knockout mutant. Reverse transcription-PCR analysis from total RNA preparations showed that in contrast to the wild-type strain, the structural genes of the ABC transporter MrsFGE were not transcribed in the knockout mutant. It was therefore concluded that producer self-protection against mersacidin is conferred by the ABC transporter MrsFGE and that the transcription of mrsFGE is regulated by MrsR2/K2, whereas production of the antibacterial peptide is solely activated by MrsR1.
Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.
Identification of a new class of lanthionine synthetases provides insight into the mechanism and evolution of cyclic peptide biosynthesis.
Lantibiotic synthetases are remarkable biocatalysts generating conformationally constrained peptides with a variety of biological activities by repeatedly utilizing two simple posttranslational modification reactions: dehydration of Ser/Thr residues and intramolecular addition of Cys thiols to the resulting dehydro amino acids. Since previously reported lantibiotic synthetases show no apparent homology with any other known protein families, the molecular mechanisms and evolutionary origin of these enzymes are unknown. In this study, we present a novel class of lanthionine synthetases, termed LanL, that consist of three distinct catalytic domains and demonstrate in vitro enzyme activity of a family member from Streptomyces venezuelae. Analysis of individually expressed and purified domains shows that LanL enzymes install dehydroamino acids via phosphorylation of Ser/Thr residues by a protein kinase domain and subsequent elimination of the phosphate by a phosphoSer/Thr lyase domain. The latter has sequence homology with the phosphothreonine lyases found in various pathogenic bacteria that inactivate host mitogen activated protein kinases. A LanC-like cyclase domain then catalyzes the addition of Cys residues to the dehydro amino acids to form the characteristic thioether rings. We propose that LanL enzymes have evolved from stand-alone protein Ser/Thr kinases, phosphoSer/Thr lyases, and enzymes catalyzing thiol alkylation. We also demonstrate that the genes for all three pathways to lanthionine-containing peptides are widespread in Nature. Given the remarkable efficiency of formation of lanthionine-containing polycyclic peptides and the latter's high degree of specificity for their cognate cellular targets, it is perhaps not surprising that (at least) three distinct families of polypeptide sequences have evolved to access this structurally and functionally diverse class of compounds.
Many bacteria generate cyclic peptides, which have improved biological activities compared to linear peptides, including higher stability. Lanthionine-containing peptides are one such group, and different members of this group have antibiotic, anti-inflammatory, and anti-viral activities. For example, one lanthionine-containing peptide called nisin has been used to protect food items from harmful bacteria. Two different pathways for the biosynthesis of lanthionine-containing peptides have been described previously. By comparing the DNA sequences of bacterial genomes we reveal a third biosynthetic route that provides further insight into how the biosynthetic pathways for these cyclic peptides have evolved. We characterized the novel lanthionine synthetase utilized in this third pathway in the soil bacterium Streptomyces venezuelae and show that the purified enzyme catalyzes the chemical reactions necessary to turn a linear peptide into a peptide with multiple rings. The discovery of this third biosynthetic pathway widens the scope for the engineering of new lanthionine-containing peptides for potential use in human therapeutics.
Streptococcus mutans JH1000 and its derivatives were previously shown (J. D. Hillman, K. P. Johnson, and B. I. Yaphe, Infect. Immun. 44:141–144, 1984) to produce a low-molecular-weight, broad-spectrum bacteriocin-like inhibitory substance (BLIS). The thermosensitive vector pTV1-OK harboring Tn917 was used to isolate a BLIS-deficient mutant, DM25, and the mutated gene was recovered by shotgun cloning in Escherichia coli. Sequence analysis of insert DNA adjacent to Tn917 led to the identification of four open reading frames including two (lanA and lanB) which have substantial homology to the Staphylococcus epidermidis structural gene (epiA) and a modifying enzyme gene (epiB) for biosynthesis of the lantibiotic epidermin, respectively. Although the BLIS activity could not be recovered from broth cultures, high yields were obtained from a solid medium consisting of Todd-Hewitt broth containing 0.5% agarose that was stab inoculated with JH1140 (a spontaneous mutant of JH1000 that produces threefold-elevated amounts of activity). Agar could not substitute for agarose. Chloroform extraction of the spent medium produced a fraction which yielded two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster-migrating band was absent in chloroform extracts of the mutant, DM25. The amino acid sequence of this band was determined by Edman sequencing and mass spectroscopy. The results showed that it is a lantibiotic, which we have named mutacin 1140, and that the sequence corresponded to that deduced from the lanA sequence. We observed a number of similarities of mutacin 1140 to epidermin and an S. mutans lantibiotic, B-Ny266, but it appears to have significant differences in the positions of its thioether bridges. It also has other unique features with regard to its leader sequence and posttranslational modification. A proposed structure for mutacin 1140 is presented.