Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterial competition.

Antibiotic resistance is a key medical concern, with antibiotic use likely being an important cause. However, here we describe an alternative route to clinically relevant antibiotic resistance that occurs solely due to competitive interactions among bacterial cells. We consistently observe that isolates of Methicillin-resistant Staphylococcus aureus diversify spontaneously into two distinct, sequentially arising strains. The first evolved strain outgrows the parent strain via secretion of surfactants and a toxic bacteriocin. The second is resistant to the bacteriocin. Importantly, this second strain is also resistant to intermediate levels of vancomycin. This so-called VISA (vancomycin-intermediate S. aureus) phenotype is seen in many hard-to-treat clinical isolates. This strain diversification also occurs during in vivo infection in a mouse model, which is consistent with the fact that both coevolved phenotypes resemble strains commonly found in clinic. Our study shows how competition between coevolving bacterial strains can generate antibiotic resistance and recapitulate key clinical phenotypes.

Discovery, biosynthesis, and engineering of lantipeptides.

Aided by genome-mining strategies, knowledge of the prevalence and diversity of ribosomally synthesized natural products (RNPs) is rapidly increasing. Among these are the lantipeptides, posttranslationally modified peptides containing characteristic thioether cross-links imperative for bioactivity and stability. Though this family was once thought to be a limited class of antimicrobial compounds produced by gram-positive bacteria, new insights have revealed a much larger diversity of activity, structure, biosynthetic machinery, and producing organisms than previously appreciated. Detailed investigation of the enzymes responsible for installing the posttranslational modifications has resulted in improved in vivo and in vitro engineering systems focusing on enhancement of the therapeutic potential of these compounds. Although dozens of new lantipeptides have been isolated in recent years, bioinformatic analyses indicate that many hundreds more await discovery owing to the widespread frequency of lantipeptide biosynthetic machinery in bacterial genomes.

Commensal E. coli limits Salmonella gut invasion during inflammation by producing toxin-bound siderophores in a tonB-dependent manner.

The gastrointestinal tract is densely colonized by a polymicrobial community known as the microbiota which serves as primary line of defence against pathogen invasion. The microbiota can limit gut-luminal pathogen growth at different stages of infection. This can be traced to specific commensal strains exhibiting direct or indirect protective functions. Although these mechanisms hold the potential to develop new approaches to combat enteric pathogens, they remain far from being completely described. In this study, we investigated how a mouse commensal Escherichia coli can outcompete Salmonella enterica serovar Typhimurium (S. Tm). Using a salmonellosis mouse model, we found that the commensal E. coli 8178 strain relies on a trojan horse trap strategy to limit S. Tm expansion in the inflamed gut. Combining mutants and reporter tools, we demonstrated that inflammation triggers the expression of the E. coli 8178 antimicrobial microcin H47 toxin which, when fused to salmochelin siderophores, can specifically alter S. Tm growth. This protective function was compromised upon disruption of the E. coli 8178 tonB-dependent catecholate siderophore uptake system, highlighting a previously unappreciated crosstalk between iron intake and microcin H47 activity. By identifying the genetic determinants mediating S. Tm competition, our work not only provides a better mechanistic understanding of the protective function displayed by members of the gut microbiota but also further expands the general contribution of microcins in bacterial antagonistic relationships. Ultimately, such insights can open new avenues for developing microbiota-based approaches to better control intestinal infections.

Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison.

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.

Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome.

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

Klebicin E, a pore-forming bacteriocin of Klebsiella pneumoniae, exploits the porin OmpC and the Ton system for translocation.

Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting that it is a pore-forming toxin. In addition to a C-terminal toxic domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.

Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus.

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

The evolution of spectrum in antibiotics and bacteriocins.

A key property of many antibiotics is that they will kill or inhibit a diverse range of microbial species. This broad-spectrum of activity has its evolutionary roots in ecological competition, whereby bacteria and other microbes use antibiotics to suppress other strains and species. However, many bacteria also use narrow-spectrum toxins, such as bacteriocins, that principally target conspecifics. Why has such a diversity in spectrum evolved? Here, we develop an evolutionary model to understand antimicrobial spectrum. Our first model recapitulates the intuition that broad-spectrum is best, because it enables a microbe to kill a wider diversity of competitors. However, this model neglects an important property of antimicrobials: They are commonly bound, sequestered, or degraded by the cells they target. Incorporating this toxin loss reveals a major advantage to narrow-spectrum toxins: They target the strongest ecological competitor and avoid being used up on less important species. Why then would broad-spectrum toxins ever evolve? Our model predicts that broad-spectrum toxins will be favored by natural selection if a strain is highly abundant and can overpower both its key competitor and other species. We test this prediction by compiling and analyzing a database of the regulation and spectrum of toxins used in inter-bacterial competition. This analysis reveals a strong association between broad-spectrum toxins and density-dependent regulation, indicating that they are indeed used when strains are abundant. Our work provides a rationale for why bacteria commonly evolve narrow-spectrum toxins such as bacteriocins and suggests that the evolution of antibiotics proper is a signature of ecological dominance.

Pangenome analyses of Clostridium butyricum provide insights into its genetic characteristics and industrial application.

Clostridium butyricum is a Gram-positive anaerobic bacterium known for its ability to produce butyate. In this study, we conducted whole-genome sequencing and assembly of 14C. butyricum industrial strains collected from various parts of China. We performed a pan-genome comparative analysis of the 14 assembled strains and 139 strains downloaded from NCBI. We found that the genes related to critical industrial production pathways were primarily present in the core and soft-core gene categories. The phylogenetic analysis revealed that strains from the same clade of the phylogenetic tree possessed similar antibiotic resistance and virulence factors, with most of these genes present in the shell and cloud gene categories. Finally, we predicted the genes producing bacteriocins and botulinum toxins as well as CRISPR systems responsible for host defense. In conclusion, our research provides a desirable pan-genome database for the industrial production, food application, and genetic research of C. butyricum.

Genomic exploration of the fermented meat isolate Staphylococcus shinii IMDO-S216 with a focus on competitiveness-enhancing secondary metabolites.

BACKGROUND: Staphylococcus shinii appears as an umbrella species encompassing several strains of Staphylococcus pseudoxylosus and Staphylococcus xylosus. Given its phylogenetic closeness to S. xylosus, S. shinii can be found in similar ecological niches, including the microbiota of fermented meats where the species may contribute to colour and flavour development. In addition to these conventional functionalities, a biopreservation potential based on the production of antagonistic compounds may be available. Such potential, however, remains largely unexplored in contrast to the large body of research that is available on the biopreservative properties of lactic acid bacteria. The present study outlines the exploration of the genetic basis of competitiveness and antimicrobial activity of a fermented meat isolate, S. shinii IMDO-S216. To this end, its genome was sequenced, de novo assembled, and annotated. RESULTS: The genome contained a single circular chromosome and eight plasmid replicons. Focus of the genomic exploration was on secondary metabolite biosynthetic gene clusters coding for ribosomally synthesized and posttranslationally modified peptides. One complete cluster was coding for a bacteriocin, namely lactococcin 972; the genes coding for the pre-bacteriocin, the ATP-binding cassette transporter, and the immunity protein were also identified. Five other complete clusters were identified, possibly functioning as competitiveness factors. These clusters were found to be involved in various responses such as membrane fluidity, iron intake from the medium, a quorum sensing system, and decreased sensitivity to antimicrobial peptides and competing microorganisms. The presence of these clusters was equally studied among a selection of multiple Staphylococcus species to assess their prevalence in closely-related organisms. CONCLUSIONS: Such factors possibly translate in an improved adaptation and competitiveness of S. shinii IMDO-S216 which are, in turn, likely to improve its fitness in a fermented meat matrix.

Genome mining of Lactiplantibacillus plantarum PA21: insights into its antimicrobial potential.

BACKGROUND: The dramatic increase of antimicrobial resistance in the healthcare realm has become inexorably linked to the abuse of antibiotics over the years. Therefore, this study seeks to identify potential postbiotic metabolites derived from lactic acid bacteria such as Lactiplantibacillus plantarum that could exhibit antimicrobial properties against multi-drug resistant pathogens. RESULTS: In the present work, the genome sequence of Lactiplantibacillus plantarum PA21 consisting of three contigs was assembled to a size of 3,218,706 bp. Phylogenomic analysis and average nucleotide identity (ANI) revealed L. plantarum PA21 is closely related to genomes isolated from diverse niches such as dairy products, food, and animals. Genome mining through the BAGEL4 and antiSMASH database revealed four bacteriocins in a single cluster and four regions of biosynthetic gene clusters responsible for the production of bioactive compounds. The potential probiotic genes indirectly responsible for postbiotic metabolites production were also identified. Additionally, in vitro studies showed that the L. plantarum PA21 cell-free supernatant exhibited antimicrobial activity against all nine methicillin-resistant Staphylococcus aureus (MRSA) and three out of 13 Klebsiella pneumoniae clinical isolates tested. CONCLUSION: Results in this study demonstrates that L. plantarum PA21 postbiotic metabolites is a prolific source of antimicrobials against multi-drug resistant pathogens with potential antimicrobial properties.

Merging Flow Synthesis and Enzymatic Maturation to Expand the Chemical Space of Lasso Peptides.

Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.

Lasso Peptides: Exploring the Folding Landscape of Nature's Smallest Interlocked Motifs.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

Marinomonas mediterranea synthesizes an R-type bacteriocin.

Prophages integrated into bacterial genomes can become cryptic or defective prophages, which may evolve to provide various traits to bacterial cells. Previous research on Marinomonas mediterranea MMB-1 demonstrated the production of defective particles. In this study, an analysis of the genomes of three different strains (MMB-1, MMB-2, and MMB-3) revealed the presence of a region named MEDPRO1, spanning approximately 52 kb, coding for a defective prophage in strains MMB-1 and MMB-2. This prophage seems to have been lost in strain MMB-3, possibly due to the presence of spacers recognizing this region in an I-F CRISPR array in this strain. However, all three strains produce remarkably similar defective particles. Using strain MMB-1 as a model, mass spectrometry analyses indicated that the structural proteins of the defective particles are encoded by a second defective prophage situated within the MEDPRO2 region, spanning approximately 13 kb. This finding was further validated through the deletion of this second defective prophage. Genomic region analyses and the detection of antimicrobial activity of the defective prophage against other Marinomonas species suggest that it is an R-type bacteriocin. Marinomonas mediterranea synthesizes antimicrobial proteins with lysine oxidase activity, and the synthesis of an R-type bacteriocin constitutes an additional mechanism in microbial competition for the colonization of habitats such as the surface of marine plants.IMPORTANCEThe interactions between bacterial strains inhabiting the same environment determine the final composition of the microbiome. In this study, it is shown that some extracellular defective phage particles previously observed in Marinomonas mediterranea are in fact R-type bacteriocins showing antimicrobial activity against other Marinomonas strains. The operon coding for the R-type bacteriocin has been identified.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

Micrococcin cysteine-to-thiazole conversion through transient interactions between the scaffolding protein TclI and the modification enzymes TclJ and TclN.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCE: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

The two-component regulatory systems GraRS and SrrAB mediate Staphylococcus aureus susceptibility to Pep5 produced by clinical isolate of Staphylococcus epidermidis.

Staphylococcus aureus is a common bacterium on the skin and in the nose that sometimes causes severe illness. Bacteriocins, antimicrobial peptides, or proteins produced by bacteria are candidates for the treatment of S. aureus infection. In this study, we found that a clinical Staphylococcus epidermidis strain, KSE112, produced the lantibiotic Pep5, which showed anti-S. aureus activity. The complete nucleotide sequence of the Pep5-encoding plasmid was determined. Several S. aureus two-component regulatory systems (TCSs) are known to be involved in bacteriocin susceptibility. Therefore, susceptibility tests were performed using TCS-inactivated S. aureus mutants to determine which TCS is responsible for Pep5 susceptibility; the ΔgraRS mutant exhibited increased susceptibility to Pep5, while the ΔsrrAB mutant exhibited decreased susceptibility. GraRS is known to regulate dltABCD and mprF in concert with vraFG, and Pep5 susceptibility was significantly increased in the ΔdltABCD, ΔmprF, and ΔvraFG mutants. Regarding the ΔsrrAB mutant, cross-resistance to aminoglycosides was observed. As aminoglycoside activity is known to be affected by aerobic respiration, we focused on qoxABCD and cydAB, which are quinol oxidase genes that are necessary for aerobic respiration and have downregulated the expression in the ΔsrrAB mutant. We constructed ΔqoxABCD and ΔcydAB mutants and found that qoxABCD inactivation decreased susceptibility to Pep5 and aminoglycosides. These results indicate that reduced aerobic respiration due to the reduced qoxABCD expression in the ΔsrrAB mutant decreased Pep5 activity.IMPORTANCEThe emergence of drug-resistant bacteria, including MRSA, is a severe health problem worldwide. Thus, the development of novel antimicrobial agents, including bacteriocins, is needed. In this report, we found a Pep5-producing strain with anti-S. aureus activity. We determined the complete sequence of the Pep5-encoding plasmid for the first time. However, in S. aureus, GraRS and its effectors conferred decreased susceptibility to Pep5. We also revealed that another TCS, SrrAB, affects susceptibility Pep5 and other lantibiotics by controlling aerobic respiration. In our study, we investigated the efficacy of Pep5 against S. aureus and other Gram-positive bacteria and revealed that respiratory constancy regulated by TCS is required for the antimicrobial activity of nisin, nukacin, and Pep5. These findings provide important information for the clinical application of bacteriocins and suggest that they have different properties among similar pore-forming lantibiotics.

Ursoricin, a bacteriocin of Streptococcus ursoris, has potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.

The emergence of drug-resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has increased the need to discover novel antimicrobial agents that are effective against these species. Here, we describe the identification and purification of the mutacin BHT-B-like gene locus and bacteriocin peptide from Streptococcus ursoris, which is closely related to Streptococcus ratti; hence, we named this bacteriocin ursoricin. Ursoricin is a cationic, chromosome-encoded peptide that has potent antimicrobial effects against Gram-positive pathogens, including MRSA and VRE, with minimum inhibitory concentrations in the micromolar range. Ursoricin also inhibits the biofilm formation of high biofilm-forming S. aureus. Antibacterial activity was retained after treatment at 100°C for 60 min at a pH range of 3-9 and was partially reduced by treatment with proteinase K for 2 h (63% residual activity). The potent anti-MRSA, anti-VRE, and antibiofilm effects of ursoricin suggest that it is a possible candidate for the treatment of MRSA, VRE, and biofilm-associated infections. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has posed a significant public health threat and economic burdens that make the identification and development of novel antimicrobial agents urgent. Bacteriocins are promising new agents that exhibit antibacterial activity against a wide range of human pathogens. In this study, we report that the bacteriocin produced by Streptococcus ursoris showed good antibacterial activity against a wide range of Staphylococcus aureus and enterococcus strains, particularly methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and high biofilm-forming S. aureus. Interestingly, this bacteriocin had a stronger effect on S. aureus than on Staphylococcus epidermidis, which is a major commensal bacterium in human skin; this result is important when considering the disturbance of bacterial flora, especially on the skin, mediated by the application of antibacterial agents.