Lantibiotic Gene Cluster Research Articles

Genome-Guided Mass Spectrometry Expedited the Discovery of Paraplantaricin TC318, a Lantibiotic Produced by Lactobacillus paraplantarum Strain Isolated From Cheese.

The quest for potent alternatives to the currently used antimicrobials is urged by health professionals, considering the rapid rise in resistance to preservatives and antibiotics among pathogens. The current study was initiated to search for novel and effective bacteriocins from food microbes, preferably lactic acid bacteria (LAB), for potential use as preservatives. Advances in genome-guided mass spectrometry (MS) were implemented to expedite identifying and elucidating the structure of the recovered antimicrobial agent. A LAB strain, OSY-TC318, was isolated from a Turkish cheese, and the crude extract of the cultured strain inhibited the growth of various pathogenic and spoilage bacteria such as Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, and Staphylococcus aureus. The antimicrobial producer was identified as Lactobacillus paraplantarum using MS biotyping and genomic analysis. Additionally, L. paraplantarum OSY-TC318 was distinguished from closely related strains using comparative genomic analysis. Based on in silico analysis, the genome of the new strain contained a complete lantibiotic biosynthetic gene cluster, encoding a novel lantibiotic that was designated as paraplantaricin TC318. The bioinformatic analysis of the gene cluster led to the prediction of the biosynthetic pathway, amino acid sequence, and theoretical molecular mass of paraplantaricin TC318. To verify the genomic analysis predictions, paraplantaricin TC318 was purified from the producer cellular crude extract using liquid chromatography, followed by structural elucidation using Fourier transform ion cyclotron resonance MS analysis. This genome-guided MS analysis revealed that the molecular mass of paraplantaricin TC318 is 2,263.900 Da, its chemical formula is C106H133N27O22S4, and its primary sequence is F-K-S-W-S-L-C-T-F-G-C-G-H-T-G-S-F-N-S-F-C-C. This lantibiotic, which differs from mutacin 1140 at positions 9, 12, 13, and 20, is considered a new member of the epidermin group in class I lantibiotics. In conclusion, the study revealed a new L. paraplantarum strain producing a novel lantibiotic that is potentially useful in food and medical applications.

Characterization of a Signaling System in Streptococcus mitis That Mediates Interspecies Communication with Streptococcus pneumoniae.

Streptococcus mitis is found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study, in silico analyses indicated the presence of an Rgg regulator and short hydrophobic peptide (Rgg/SHP) cell-to-cell communication system in S. mitis Although Rgg presented greater similarity to a repressor in Streptococcus pyogenes, autoinducing assays and genetic mutation analysis revealed that in S. mitis Rgg acts as an activator. Transcriptome analysis showed that in addition to shp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster in Streptococcus pneumoniae indicated that S. mitis lacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of autoinduction, growth was not significantly affected for the rgg mutant under normal or antibiotic stress conditions. The S. mitis SHP was, however, fully functional in promoting cross-species communication and increasing S. pneumoniae surface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in cocultures using a S. mitis reporter strain. In competitive assays, a slight advantage was observed for the rgg mutants. We conclude that the Rgg/SHP system in S. mitis regulates the expression of its own shp and activates an Rgg/SHP system in S. pneumoniae that regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multispecies interactions.IMPORTANCE Bacteria secrete signal molecules into the environment which are sensed by other cells when the density reaches a certain threshold. In this study, we describe a communication system in Streptococcus mitis, a commensal species from the oral cavity, which we also found in several species and strains of streptococci from the mitis group. Further, we show that this system can promote cross-communication with S. pneumoniae, a closely related major human pathogen. Importantly, we show that this cross-communication can take place during coculture. While the genes regulated in S. mitis are likely part of a futile cycle of activation, the target genes in S. pneumoniae are potentially involved in virulence. The understanding of such complex communication networks can provide important insights into the dynamics of bacterial communities.

Use of Lactococcus lactis as a production system for peptides and enzymes encoded by a Lantibiotic gene cluster from Bifidobacterium longum.

Bifidobacterium longum DJO10A was previously demonstrated to be able to produce a broad-spectrum lantibiotic, but production in media was very limited and only periodically on solid media. Given the difficulty of obtaining these lantibiotic peptides using B. longum DJO10A due to its tightly controlled production, genes predicted to be required for its production and immunity were designed and codon optimized according to the preferred codon used by Lactococcus lactis. Since the lanR1 gene within this lantibiotic gene cluster was the only one without a characterized analogue from other lantibiotic gene clusters, its annotation was re-examined as it was previously suggested to be a regulatory protein. Lack of DNA binding motifs did not support this, and one current analysis suggested a high likelihood of it interacting with LanD. Therefore, gene lanR1 together with lanADMIT were codon optimized and synthesized. Those genes were then cloned into an efficient dual-plasmid nisin-controlled expression system in L. lactis. The addition of the lanR1 gene exhibited toxicity in E. coli, specifically causing a shorter cell size as observed by SEM. No toxicity was observed in L. lactis. While this production system did not result in the production of a bioactive lantibiotic by L. lactis, it did successfully produce all the peptides and enzymes encoded by the original lantibiotic gene cluster from B. longum, as confirmed by LC-MS. This will now facilitate efforts into determining the proper conditions required for these enzymes to produce a bioactive lantibiotic.

CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus.

Cerecidins are small lantibiotics from Bacillus cereus that were obtained using a semi-in vitro biosynthesis strategy and showed prominent antimicrobial activities against certain Gram-positive bacteria. However, the parental strain B. cereus As 1.1846 is incapable of producing cerecidins, most probably due to the transcriptional repression of the cerecidin gene cluster. Located in the cerecidin gene cluster, cerR encodes a putative response regulator protein that belongs to the LuxR family transcriptional regulators. CerR (84 amino acids) contains only a conserved DNA binding domain and lacks a conventional phosphorylation domain, which is rarely found in lantibiotic gene clusters. To investigate its function in cerecidin biosynthesis, cerR was constitutively expressed in B. cereus As 1.1846. Surprisingly, Constitutive expression of cerR enabled the production of cerecidins and enhanced self-immunity of B. cereus toward cerecidins. Reverse transcription-PCR analysis and electrophoresis mobility shift assays indicated, respectively, that the cer cluster was transcribed in two transcripts (cerAM and cerRTPFE) and that CerR regulated the cerecidin gene cluster directly by binding to the two predicted promoter regions of cerA and cerR DNase I footprinting experiments further confirmed that CerR specifically bound to the two promoter regions at a conserved inverted repeat sequence that was designated a CerR binding motif (cerR box). The present study demonstrated that CerR, as the first single-domain LuxR family transcriptional regulator, serves as a transcriptional activator in cerecidin biosynthesis and activates the cerecidin gene cluster, which was otherwise cryptic in B. cereusIMPORTANCE Lantibiotics with intriguing and prominent bioactivities are potential peptide antibiotics that could be applied in many areas, including food and pharmaceutical industries. The biosynthesis of lantibiotics is generally controlled by two-component regulatory systems consisting of histidine kinases and response regulators, while some unique and interesting regulatory systems are also revealed with the ever-increasing discovery of lantibiotic gene clusters among diverse microorganisms. Dissection of diverse lantibiotic regulation machineries would permit deep understanding of the biological functions of lantibiotics in different niches and even enable genetic activation of lantibiotic gene clusters that are otherwise cryptic. The significance of our study is to illuminate the regulatory mechanism of a special single-domain protein, CerR, in regulating cerecidin biosynthesis in Bacillus cereus, providing a possible novel approach to activate cryptic lantibiotic clusters.

Identification of the biosynthesis gene cluster for the novel lantibiotic paenilan from Paenibacillus polymyxa E681 and characterization of its product.

To analyse the function of a putative lantibiotic gene cluster of Paenibacillus polymyxa E681 and to characterize its product, paenilan. Comparative analysis of the lantibiotic gene cluster of E681 revealed that the cluster, consisting of 11 open reading frames, is involved in the biosynthesis of a class I lantibiotic. The pnlA gene encoding the prepeptide PnlA was identified and P. polymyxa strain EPT14 producing only paenilan was constructed by knockout of the other five antibiotic biosynthetic gene clusters of E681. Paenilan was purified from EPT14 culture by solvent partitioning, ODS silica gel chromatography and reversed-phase preparative HPLC. The molecular mass (2510·10Da) and structure of paenilan analysed by Nanoelectrospray ionization mass spectrometry (MS) and MS/MS showed that paenilan is a novel class I lantibiotic. Paenilan exhibited antimicrobial activity against Gram-positive bacteria such as Bacillus cereus, Micrococcus luteus and Paenibacillus durus. The paenilan gene is well-conserved in different Paenibacillus sp. isolated from globally distant places. The lantibiotic gene cluster of P. polymyxa E681 was analysed and its product, a novel and useful lantibiotic named paenilan that inhibits the growth of some Gram-positive bacteria, was characterized. Paenibacillus species are a good source of new lantibiotics, and the conservation of the paenilan gene among Paenibacillus sp. implies paenilan has an important function(s) for their survival.

Distribution of Suicin Gene Clusters in Streptococcus suis Serotype 2 Belonging to Sequence Types 25 and 28.

Recently, we reported the purification and characterization of three distinct lantibiotics (named suicin 90-1330, suicin 3908, and suicin 65) produced by Streptococcus suis. In this study, we investigated the distribution of the three suicin lantibiotic gene clusters among serotype 2 S. suis strains belonging to sequence type (ST) 25 and ST28, the two dominant STs identified in North America. The genomes of 102 strains were interrogated for the presence of suicin gene clusters encoding suicins 90-1330, 3908, and 65. The gene cluster encoding suicin 65 was the most prevalent and mainly found among ST25 strains. In contrast, none of the genes related to suicin 90-1330 production were identified in 51 ST25 strains nor in 35/51 ST28 strains. However, the complete suicin 90-1330 gene cluster was found in ten ST28 strains, although some genes in the cluster were truncated in three of these isolates. The vast majority (101/102) of S. suis strains did not possess any of the genes encoding suicin 3908. In conclusion, this study indicates heterogeneous distribution of suicin genes in S. suis.

Characterization of the Antimicrobial Peptide Penisin, a Class Ia Novel Lantibiotic from Paenibacillus sp. Strain A3.

Attempts to isolate novel antimicrobial peptides from microbial sources have been on the rise recently, despite their low efficacy in therapeutic applications. Here, we report identification and characterization of a new efficient antimicrobial peptide from a bacterial strain designated A3 that exhibited highest identity with Paenibacillus ehimensis. Upon purification and subsequent molecular characterization of the antimicrobial peptide, referred to as penisin, we found the peptide to be a bacteriocin-like peptide. Consistent with these results, RAST analysis of the entire genome sequence revealed the presence of a lantibiotic gene cluster containing genes necessary for synthesis and maturation of a lantibiotic. While circular dichroism and one-dimension nuclear magnetic resonance experiments confirmed a random coil structure of the peptide, similar to other known lantibiotics, additional biochemical evidence suggests posttranslational modifications of the core peptide yield six thioether cross-links. The deduced amino acid sequence of the putative biosynthetic gene penA showed approximately 74% similarity with elgicin A and 50% similarity with the lantibiotic paenicidin A. Penisin effectively killed methicillin-resistant Staphylococcus aureus (MRSA) and did not exhibit hemolysis activity. Unlike other lantibiotics, it effectively inhibited the growth of Gram-negative bacteria. Furthermore, 80 mg/kg of body weight of penisin significantly reduced bacterial burden in a mouse thigh infection model and protected BALB/c mice in a bacteremia model entailing infection with Staphylococcus aureus MTCC 96, suggesting that it could be a promising new antimicrobial peptide.

A new quorum-sensing system (TprA/PhrA) for Streptococcus pneumoniae D39 that regulates a lantibiotic biosynthesis gene cluster.

The Phr peptides of the Bacillus species mediate quorum sensing, but their identification and function in other species of bacteria have not been determined. We have identified a Phr peptide quorum-sensing system (TprA/PhrA) that controls the expression of a lantibiotic gene cluster in the Gram-positive human pathogen, Streptococcus pneumoniae. Lantibiotics are highly modified peptides that are part of the bacteriocin family of antimicrobial peptides. We have characterized the basic mechanism for a Phr-peptide signaling system in S. pneumoniae and found that it induces the expression of the lantibiotic genes when pneumococcal cells are at high density in the presence of galactose, a main sugar of the human nasopharynx, a highly competitive microbial environment. Activity of the Phr peptide system is not seen when pneumococcal cells are grown with glucose, the preferred carbon source and the most prevalent sugar encountered by S. pneumoniae during invasive disease. Thus, the lantibiotic genes are expressed under the control of both cell density signals via the Phr peptide system and nutritional signals from the carbon source present, suggesting that quorum sensing and the lantibiotic machinery may help pneumococcal cells compete for space and resources during colonization of the nasopharynx.

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides.

Lantibiotics are ribosomally synthesized antimicrobial peptides with substantial posttranslational modifications. They are characterized by the unique amino acids lanthionine and methyllanthionine, which are introduced by dehydration of Ser/Thr residues and linkage of the resulting dehydrated amino acids with Cys residues. BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database revealed a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Production of an antimicrobial substance with activity against Gram-positive bacteria was detectable in a cell wash extract of this strain. The substance was partially purified, and mass spectrometric analysis predicted a peptide of 2,786 Da in the active fraction. In order to characterize the putative lantibiotic further, heterologous expression of the predicted biosynthetic genes was performed in Escherichia coli. Coexpression of the prepeptide (PseA) along with the corresponding modification enzyme (PseM) resulted in the production of a modified peptide with the corresponding mass, carrying four out of eight possible dehydrations and supporting the presence of four thioether and one disulfide bridge. After the proteolytic removal of the leader, the core peptide exhibited antimicrobial activity. In conclusion, pseudomycoicidin is a novel lantibiotic with antimicrobial activity that was heterologously produced in E. coli.

ApnI, a transmembrane protein responsible for subtilomycin immunity, unveils a novel model for lantibiotic immunity.

Subtilomycin was detected from the plant endophytic strain Bacillus subtilis BSn5 and was first reported from B. subtilis strain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genes apnI and apnT downstream of apnAPBC, which may be involved in subtilomycin immunity. Through analysis of expression of the apnI and/or apnT genes in the subtilomycin-sensitive strain CU1065 and inactivation of apnI and apnT in the producer strain BSn5, we showed that the single gene apnI, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity in Bacillus and Paenibacillus. We cloned the paeI gene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinant B. subtilis expressing apnI interacted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common.

Characterization of Amylolysin, a Novel Lantibiotic from Bacillus amyloliquefaciens GA1

BackgroundLantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity.Methodology/Findings Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus.Conclusion/SignificanceApart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.

Bioinformatic evaluation of the secondary metabolism of antistaphylococcal environmental bacterial isolates

The increasing occurrence of drug-resistant Staphylococcus aureus is exacerbated with a declining rate of antibiotic discovery, particularly those with new mechanisms of action. The decline in antibiotic discovery from traditional sources, such as soil actinobacteria, necessitates examination of lesser studied microbes. Here, we present a strategy to select for organisms that may have a propensity to result in new antistaphylococcal agents by using S. aureus as a bait organism, and selecting organisms that have a natural lytic activity towards it. We have isolated over 80 environmental isolates and typed these organisms using 16S rDNA sequence comparison and deployed bioinformatics to assess the secondary metabolic potential of the isolated antistaphylococcal bacteria using genomic sequences. Bioinformatic analysis highlights the enriched and unique suite of potential antibiotic polyketides and nonribosomal peptides and lantibiotic gene clusters from these organisms. Profiling organic microbial extracts further showed that many of the organisms from the 10 staphylolytic genera secrete agents with antistaphylococcal activity and may serve as new sources for future antistaphylococcal drug discovery.

Heterologous Expression and Engineering Studies of Labyrinthopeptins, Class III Lantibiotics from Actinomadura namibiensis

Labyrinthopeptins are class III lantibiotics produced by the actinomycete Actinomadura namibiensis. The most characteristic structural feature is the posttranslationally installed triamino triacid labionin with a quaternary α-carbon. In addition to the unique structure, labyrinthopeptin A2 possess remarkable antiviral and antiallodynic biological activities. To harness the substrate tolerance of the biosynthetic machinery, we developed an efficient system for the generation of labyrinthopeptin analogs. Streptomyces lividans was used as a heterologous host since the natural producer Actinomadura namibiensis remained genetically intractable. Generation of a library of 39 mutants allowed identification of variable and invariable regions in the labyrinthopeptin structures. Additional data on the flexibility of the biosynthetic machinery were provided by in vitro experiments. This study is detailed investigation on the potential to generate analogs of class III lantibiotics by genetic engineering.

Lantibiotics from Geobacillus thermodenitrificans.

The lantibiotic nisin has been used as an effective food preservative to combat food-borne pathogens for over 40y. Despite this successful use, nisin's stability at pH7 is limited. Herein, we describe a nisin analog encoded on the genome of the thermophilic bacterium Geobacillus thermodenitrificans NG80-2. This analog termed geobacillinI was obtained by heterologous expression in Escherichia coli and subsequent purification. Extensive NMR characterization demonstrated that geobacillinI contains seven thioether cross-links, two more than the five cross-links found in nisin and the most cross-links found in any lantibiotic to date. The antimicrobial spectrum of geobacillinI was generally similar to that of nisin A, with increased activity against Streptococcus dysgalactiae, one of the causative agents of bovine mastitis. Geobacillin I demonstrated increased stability compared to nisin A. In addition to geobacillinI, the genome of G. thermodenitrificans NG80-2 also contains a classII lantibiotic biosynthetic gene cluster. The corresponding compound was produced in E. coli, and has a ring topology different than that of any known lantibiotic as determined by tandem mass spectrometry. Interestingly, geobacillinII only demonstrated antimicrobial activity against Bacillus strains. Seven Geobacillus strains were screened for production of the geobacillins using whole-cell MALDI-MS and five were shown to produce geobacillinI, but none produced geobacillinII.

Further Identification of Novel Lantibiotic Operons Using LanM-Based Genome Mining.

Lantibiotics are gene-encoded antimicrobial peptides that are distinguished by the presence of the unusual structures, lanthionine and β-methyllanthionine, which are introduced through enzyme-catalysed post-translational modification. Lantibiotics can be subdivided on the basis of the nature of the enzyme(s) which catalyse this reaction. Lantibiotic synthetases, generically designated LanM, which catalyse the dehydration of serines (and threonines) followed by the formation lanthionine (and β-methyllanthioine), are responsible for the synthesis of the largest subdivision, type 2. One can take advantage of the conserved nature of LanM proteins to screen for and bioinformatically characterize novel lantibiotic-encoding operons in genome-sequenced microorganisms. Having employed this strategy with success previously, here we update the investigation to reveal the existence of 124 LanM homologs encoded within genome-sequenced microbes. Further analysis focussed specifically on 9 novel lantibiotic gene clusters in Anaerocellum thermophilum DSM 6725, Anaerococcus tetradius ATCC 35098, Corynebacterium matruchotti ATCC 33806, Streptococcus suis 98HAH33, Geobacillus sp. G11MC16, Nostoc punctiforme PCC 73102 (× 2; one on plasmid and one on the chromosome) and Streptococcus pneumoniae CDC 0288-04 and TIGR4. Furthermore, screening of metagenomic datasets revealed 11 additional LanM-encoding genes from a variety of environments. The alignment of these LanM proteins facilitated a detailed investigation of conserved domains and led to the design of an improved set of degenerate primers which can be employed in the laboratory to identify strains containing type 2 lantibiotic gene clusters.

Gene cluster for biosynthesis of thermophilin 1277 - a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide

To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer-walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9-kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI-expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Thermophilin 1277 biosynthesis genes were encoded by a 9·9-kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.

In silico analysis highlights the frequency and diversity of type 1 lantibiotic gene clusters in genome sequenced bacteria.

BackgroundLantibiotics 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.ResultsIn 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.ConclusionsMultiple 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.

Production of the Novel Two-Peptide Lantibiotic Lichenicidin by Bacillus licheniformis DSM 13

BackgroundLantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. 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) are organized in biosynthetic gene clusters.Methodology/Principal FindingsSequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides.Conclusions/SignificanceIn conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.

Gene-encoded antibiotics made in bacteria.

Production of antimicrobial peptides and proteins is very common among bacteria and a variety of such substances has been described. In general Gram-negative bacteria produce protein bacteriocins (e.g. colicins) with narrow action spectra based on receptor-mediated activity. They produce comparatively few peptides, such as the post-translationally modified microcin B17. In contrast Gram-positive bacteria tend to produce peptide bacteriocins smaller than 10 kDa and of wider activity spectra. These show particular potential for application. They can be divided into unmodified peptides (e.g. lactococcins, lactacins, pediocins) and lanthionine-containing peptides (lantibiotics, e.g. nisin, epidermin, Pep5). The unmodified peptides are mostly hydrophobic or amphiphilic and act by disturbing the function of the cytoplasmic membrane. They are synthesized as prepeptides with a characteristic N-terminal leader peptide. In some cases genes for immunity peptides were found in close proximity to structural genes; furthermore, two-component response regulators seem to be involved in the regulation of their synthesis. The biosynthetic genes for lantibiotics are also organized in operons. Lantibiotic gene clusters include genes encoding the unique enzymes which dehydrate serine and threonine and form the characteristic thioether-bridged lanthionines. Three types of lantibiotics are currently distinguished on the basis of structural features and functional aspects: type A, which include elongated, amphiphilic, pore-forming peptides (e.g. nisin); type B, which are of globular shape and inhibit phospholipases (e.g. duramycins); and type C (e.g. actagardine) with intermediate features which act by inhibiting bacterial cell wall biosynthesis.

New gene cluster for lantibiotic streptin possibly involved in streptolysin S formation.

Streptolysin S (SLS) is a serum-extractable and oxygen-stable hemolysin produced by Group A Streptococcus. A SLS-deficient mutant in which transposon Tn 916 was inserted in a locus distinct from the sag gene cluster [Nizet et al. (2000) Infect. Immun. 68, 4245-4254] was obtained by filter mating of the transposon-harbouring Enterococcus faecalis strain and Streptococcus pyogenes BL(T). This mutant, N22, had completely lost the hemolytic activity, in consequence of insertion of a single Tn 916 into a hitherto-unknown lantibiotic gene cluster composed of 10 open reading frames. The arrangement and sequence of this lantibiotic gene cluster were similar to those of nisin and subtilin, and so we designated this new lantibiotic as streptin. The bactericidal activity of streptin was abolished on treatment with trypsin or proteinase K. The different host range and nucleotide sequence clearly distinguished streptin from streptococcins. Streptin was not hemolytic and its bacteriocin activity was independent of carrier oligonucleotides effective for SLS. The fact that N22 also lost the anti-bacterial activity against indicator streptococci reveals that the factor(s) required for lantibiotic formation plays an important role in SLS formation as well.