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In view of the current threat of antibiotic resistance, new antimicrobials with low risk of resistance development are demanded. Lcn972 is a lactococcal bacteriocin that inhibits septum formation by binding to the cell wall precursor lipid II in Lactococcus. It has a species-specific spectrum of activity, making Lcn972 an attractive template to develop or improve existing antibiotics. The aim of this work was to identify mutations present in the Lcn972-resistant clone Lactococcus cremoris D1-20, previously evolved from the sensitive strain L. cremoris MG1614. Whole-genome sequencing and comparison over the reference genome L. cremoris MG1363 identified several unexpected mutations in the parental strain MG1614, likely selected during in-house propagation. In the Lcn972R clone, two previously identified mutations were mapped and confirmed. Additionally, another transposition event deregulating cellobiose uptake was identified along with three point mutations of unknown consequences for Lcn972 resistance. Two new independent evolution experiments exposing L. cremoris MG1614 to Lcn972 revealed transposition of IS981 into the LLMG_RS12285 locus as the predominant mutation selected by Lcn972. This event occurs early during evolution and was found in 100% of the evolved clones, while other mutations were not selected. Therefore, activation of LLMG_RS12285 coding for a putative anti-ECF (extra-cytoplasmic function) sigma factor is regarded as the main Lcn972 resistance factor in L. cremoris MG1614., This work was funded by grant PID2020-119697RB-I00 funded by MCIN/AEI/10.13039/501100011033 and grant AYUD/2021/52120 (Program of Science, Technology and Innovation 2018–2022, Principado de Asturias, FICYT, FEDER-UE). Ö.C.O. Umu, D.B. Diep and B. Martínez were recipients of a mobility grant Spain-Norway, supported by EEA Coordinated Mobility of Researchers NILS Science and Sustainability Project 017-CM-01-2013.

The rise of antibiotic-resistant bacteria is among the biggest challenges in human and veterinary medicine. One of the major factors that contributes to resistance is use of frontline clinical antibiotics in veterinary practices. To avoid this problem, searching for antimicrobials aimed at veterinary applications is becoming especially important. Thiopeptide micrococcin P1 and leaderless peptide EntEJ97s are two different bacteriocins that are very active against many gram-positive bacteria; however, sensitive bacteria can rapidly develop resistance towards those bacteriocins. To overcome this problem, we searched for synergy between those bacteriocins and conventional antibiotics against methicillin-resistant Staphylococcus pseudintermedius (MRSP): a common pathogen in animal skin infections. The two bacteriocins acted synergistically with each other and with penicillin G against MRSP clinical isolates in both planktonic and biofilm assays; they also prevented resistance development. The therapeutic potential was further validated in a murine skin infection model that showed that a combination of micrococcin P1, EntEJ97s and penicillin G reduced cell-forming units of MRSP by 2-log10 CFU/g. Taken together, our data show that a combination of bacteriocins with conventional antibiotics can not only prevent resistance development but also pave the way to revitalize some old, less useful antibiotics, such as penicillin, which by itself has no effect on methicillin-resistant pathogens.

Background The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. Results We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin. Conclusions We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.

Resistance to nonribosomally synthesized peptide antibiotics affecting the cell envelope is well studied and mostly associated with the action of peptide-sensing and detoxification (PSD) modules, which consist of a two-component system (TCS) and an ATP-binding cassette (ABC) transporter. In contrast, the mechanisms of resistance to ribosomally synthesized bacterial toxic peptides (bacteriocins), which also affect the cell envelope, are studied to a lesser extent, and the possible cross-resistance between them and antibiotics is still poorly understood. In the present study, we investigated the development of resistance of Lactococcus lactis to aureocin A53- and enterocin L50-like bacteriocins and cross-resistance with antibiotics. First, 19 spontaneous mutants resistant to their representatives were selected and also displayed changes in sensitivity to peptide antibiotics acting on the cell envelope (bacitracin, daptomycin, and gramicidin). Sequencing of their genomes revealed mutations in genes encoding the ABC transporter YsaCB and the TCS KinG-LlrG, the emergence of which induced the upregulation of the dltABCD and ysaDCB operons. The ysaB mutations were either nonsense or frameshift mutations and led to the generation of truncated YsaB but with the conserved N-terminal FtsX domain intact. Deletions of ysaCB or llrG had a minor effect on the resistance of the obtained mutants to the tested bacteriocins, daptomycin, and gramicidin, indicating that the development of resistance is dependent on the modification of the protein rather than its absence. In further corroboration of the above-mentioned conclusion, we show that the FtsX domain, which functions effectively when YsaB is lacking its central and C-terminal parts, is critical for resistance to these antimicrobials.

Bovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption. IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

Bacteriocins are antimicrobial peptides produced by bacteria to compete with other bacteria for nutrients and ecological niches. The antimicrobial effect of these peptides on the bacterial populations in the gut is likely dynamic as the survival of the microbes in this environment depends on both competition and cooperation. In this study, we evaluated four different bacteriocins from lactic acid bacteria (LAB): nisin, enterocin A (EntA), enterocin K1 (EntK1), and garvicin ML (GarML), which have different inhibition spectra and physicochemical properties. The bacteriocins were tested in vitro using fecal slurry batch cultures from infants. The abundances of some bacterial populations in the cultures were determined using quantitative PCR (qPCR) and the metabolic activity of the gut microbiota was assessed by measuring the production of short-chain fatty acids (SCFA) using gas chromatography. The effects of the bacteriocins correlated well with their antimicrobial spectra and the administered concentrations. Nisin and GarML, with broad antimicrobial spectra, shifted the abundance of several intestinal bacterial groups, while EntA and EntK1, with relative narrower inhibition spectra, showed no or little effect. Moreover, the results from the SCFA analysis were consistent with changes obtained in the bacterial composition. In particular, a reduction in acetate concentration was observed in the samples with low abundance of Bifidobacterium, which is a well-known acetate producer. The variability imposed on the intestinal bacterial populations by the different bacteriocins tested suggests that this type of antimicrobials have great potential to modulate the gut microbiota for medical purposes., OCOU was supported by a strategic scholarship program to food science research, from Norwegian University of Life Sciences (NMBU) (project 1205051025), and she has currently a postdoctoral position funded by Research Council of Norway. NS was the recipient of a “Clarín” postdoctoral contract (Marie Curie European CoFund Program) cofounded by the “Plan Regional de Investigación” of Principado de Asturias and she is currently the recipient of a postdoctoral contract awarded by the Fundación para la Investigación Biosanitaria de Asturias (FINBA). Travels and stays in Spain and Norway for this study were supported by EEA Coordinated Mobility of Researchers NILS Science and Sustainability Project 017-CM-01-2013.

Superficial infections in chronic wounds can prevent the wound healing process by the development of persistent infections and drug-resistant biofilms. Topically applied antimicrobial formulations with stabilized and controlled release offer significant benefits for the effective treatment of wound infections. Bacteriocins are the antimicrobial peptides (AMPs) produced by bacteria that are viable alternatives to antibiotics owing to their natural origin and low propensity for resistance development. Herein, we developed a hybrid hydrogel composed of Pluronic F127 (PF127), ethylenediaminetetraacetic acid (EDTA) loaded liposomes, glutathione (GSH), and the bacteriocin Garvicin KS (GarKS) referred to as "GarKS gel". The GarKS gel exhibited suitable viscosity and rheological properties along with controlled release behavior (up to 9 days) for effective peptide delivery following topical application. Potent in vitro antibacterial and anti-biofilm effects of GarKS gel were evident against the Gram-positive bacterium Staphylococcus aureus. The in vivo treatment of methicillin resistant S. aureus (MRSA) infected mouse wounds suggested potent antibacterial effects of the GarKS gel following multiple applications of once-a-day application for three consecutive days. Altogether, these results provide proof-of-concept for the successful development of AMP loaded topical formulation for effective treatment of wound infections.

The membrane-bound protease Eep is an important virulence factor in pathogenic enterococci. The protein is involved in stress response via the RIP pathway which is crucial for pathogenic enterococci to evade host immune attacks during infection. Eep serves also as a receptor for the bacteriocins enterocin K1 and enterocin EJ97. The bacteriocins kill Enterococcus faecium and E. faecalis, respectively, and their antibiotic resistant derivatives including vancomycin resistant enterococci (VRE). This functional duality of Eep makes these two enterocins very promising as options in the prospective treatment of enterococcal infections because wildtype enterococcal cells (with an intact Eep) are sensitive to the bacteriocins while bacteriocin-resistant-mutants (without a functional Eep) become less virulent. As a first step to explore their therapeutic potential in the treatment of systemic enterococcal infections, we investigated the compatibility of the bacteriocins with human blood, and the phenotypic changes of eep-mutants toward different stress conditions. We found that the bacteriocins were compatible with blood, as they did not cause haemolysis and that the bacteriocins retained most of their antibacterial effect when incubated in blood. The bacteriocins were autoclavable which is a crucial criterium for the development of parenteral administration. Eep-mutants, which became resistant to the bacteriocin were, as expected, less capable to withstand stress conditions such as exposure to lysozyme and desiccation. Further, their ability to chain, a trait implicated in niche adaptation as well as being necessary for genetic transfer via conjugation, was also severely affected. Together, these results indicate that the bacteriocins are promising for treatment of VRE infection.

Bacteriocins are ribosomally-synthesized antimicrobial peptides, showing great potential as novel treatment options for multidrug-resistant pathogens. In this study, we designed a novel hybrid bacteriocin, Hybrid 1 (H1), by combing the N-terminal part and the C-terminal part of the related bacteriocins enterocin K1 (K1) and enterocin EJ97 (EJ97), respectively. Like the parental bacteriocins, H1 used the membrane-bound protease RseP as receptor, however, it differed from the others in the inhibition spectrum. Most notably, H1 showed a superior antimicrobial effect towards Staphylococcus haemolyticus—an important nosocomial pathogen. To avoid strain-dependency, we further evaluated H1 against 27 clinical and commensal S. haemolyticus strains, with H1 indeed showing high activity towards all strains. To curtail the rise of resistant mutants and further explore the potential of H1 as a therapeutic agent, we designed a bacteriocin-based formulation where H1 was used in combination with the broad-spectrum bacteriocins micrococcin P1 and garvicin KS. Unlike the individual bacteriocins, the three-component combination was highly effective against planktonic cells and completely eradicated biofilm-associated S. haemolyticus cells in vitro. Most importantly, the formulation efficiently prevented development of resistant mutants as well. These findings indicate the potential of a bacteriocins-based formulation as a treatment option for S. haemolyticus.

Antibiotic-resistant bacterial pathogens have become a serious threat worldwide. One of these pathogens is methicillin-resistant Staphylococcus aureus (MRSA), a major cause of skin and soft tissue infections. In this study we identified a strain of Staphylococcus equorum producing a substance with high antimicrobial activity against many Gram-positive bacteria, including MRSA. By mass spectrometry and whole genome sequencing the antimicrobial substance was identified as the thiopeptide bacteriocin micrococcin P1 (MP1). Based on its properties we developed a one-step purification protocol resulting in high yield (15 mg/L) and high purity (98%) of MP1. For shorter incubation times (5-7 h) MP1 was very potent against MRSA but the inhibitory effect was overshadowed by resistance development during longer incubation time (24h or more). To overcome this problem a synergy study was performed with a number of commercially available antibiotics. Among the antibiotics tested, the combination of MP1 and rifampicin gave the best synergistic effect, with MIC values 25 and 60 times lower than for the individual drugs, respectively. To assess the therapeutic potential of the MP1-rifampicin combination, we used a murine skin infection model based on the use of the multidrug-resistant luciferase-tagged MRSA strain Xen31. As expected, neither of the single antimicrobials (MP1 or rifampicin) could eradicate Xen31 from the wounds. By contrary, the MP1-rifampicin combination was efficient not only to eradicate but also to prevent the recurrence of Xen31 infection. Furthermore, compared to fucidin cream, which is commonly used in skin infection treatments, MP1-rifampicin combination was superior in terms of preventing resistance development. Our results show that combining MP1, and probably other thiopeptides, with antibiotics can be a promising strategy to treat SSTIs caused by MRSA and likely many other Gram-positive bacteria.

The emergence of antibiotic-resistant pathogens has caused a serious worldwide problem in infection treatment in recent years. One of the pathogens is methicillin-resistant Staphylococcus aureus (MRSA), which is a major cause of skin and soft tissue infections. Alternative strategies and novel sources of antimicrobials to solve antibiotic resistance problems are urgently needed. In this study, we explored the potential of two broad-spectrum bacteriocins, garvicin KS and micrococcin P1, in skin infection treatments., The emergence of antibiotic-resistant pathogens has caused a serious worldwide problem in infection treatment in recent years. One of the pathogens is methicillin-resistant Staphylococcus aureus (MRSA), which is a major cause of skin and soft tissue infections. Alternative strategies and novel sources of antimicrobials to solve antibiotic resistance problems are urgently needed. In this study, we explored the potential of two broad-spectrum bacteriocins, garvicin KS and micrococcin P1, in skin infection treatments. The two bacteriocins acted synergistically with each other and with penicillin G in killing MRSA in vitro. The MICs of the antimicrobials in the three-component mixture were 40 ng/ml for micrococcin P1 and 2 μg/ml for garvicin KS and penicillin G, which were 62, 16, and at least 1,250 times lower than their MICs when assessed individually. To assess its therapeutic potential further, we challenged the three-component formulation in a murine skin infection model with the multidrug-resistant luciferase-tagged MRSA Xen31, a strain derived from the clinical isolate S. aureus ATCC 33591. Using the tagged-luciferase activity as a reporter for the presence of Xen31 in wounds, we demonstrated that the three-component formulation was efficient in eradicating the pathogen from treated wounds. Furthermore, compared to Fucidin cream, which is an antibiotic commonly used in skin infection treatments, our formulation was also superior in terms of preventing resistance development.

Antibiotic-resistant and biofilm-associated infections brought about by methicillin-resistant Staphylococcus aureus (MRSA) strains is a pressing issue both inside as well as outside nosocomial environments worldwide. Here, we show that a combination of two bacteriocins with distinct structural and functional characteristics, garvicin KS, and micrococcin P1, showed a synergetic antibacterial activity against biofilms produced in vitro by S. aureus, including several MRSA strains. In addition, this bacteriocin-based antimicrobial combination showed the ability to restore the sensitivity of the highly resilient MRSA strain ATCC 33591 to the β-lactam antibiotic penicillin G. By using a combination of bacterial cell metabolic assays, confocal and scanning electron microscopy, we show that the combination between garvicin KS, micrococcin P1, and penicillin G potently inhibit cell viability within S. aureus biofilms by causing severe cell damage. Together these data indicate that bacteriocins can be valuable therapeutic tools in the fight against biofilm-associated MRSA infections.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 is, however, relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30°C). To improve the production, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes, a new medium formulation was made that increased GarKS production about 60-fold compared to that achieved in GM17. The new medium was composed of pasteurized milk and tryptone (PM-T). GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing the gene dose of the bacteriocin gene cluster (gak) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and cultivation at a constant pH 6 and a 50–60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto.

Staphylococci, like Staphylococcus aureus and S. epidermidis, are common colonizers of the human microbiota. While being harmless in many cases, many virulence factors result in them being opportunistic pathogens and one of the major causes of hospital-acquired infections worldwide. One of these virulence factors is the ability to form biofilms—three-dimensional communities of microorganisms embedded in an extracellular polymeric matrix (EPS). The EPS is composed of polysaccharides, proteins and extracellular DNA, and is finely regulated in response to environmental conditions. This structured environment protects the embedded bacteria from the human immune system and decreases their susceptibility to antimicrobials, making infections caused by staphylococci particularly difficult to treat. With the rise of antibiotic-resistant staphylococci, together with difficulty in removing biofilms, there is a great need for new treatment strategies. The purpose of this review is to provide an overview of our current knowledge of the stages of biofilm development and what difficulties may arise when trying to eradicate staphylococcal biofilms. Furthermore, we look into promising targets and therapeutic methods, including bacteriocins and phage-derived antibiofilm approaches.

Many enterococcal strains produce bacteriocins, which could be useful as natural food preservatives through inhibition of pathogenic and spoilage microorganisms. There is little knowledge of the distribution and spectrum of bacteriocin activity and the distribution of bacteriocin-encoding genes in enterococci isolated from dogs. Therefore, we subjected 160 enterococcal isolates (E. faecium n=92, E. faecalis n=35, E. hirae n=28, E. casseliflavus n=3, E. mundtii n=2) from 105 samples of dog faeces to polymerase chain reaction (PCR) detection of genes for enterocin A, P, B, L50A, L50B, AS-48, and bac31 and to screening for bacteriocin activity. The results showed the presence of at least one of the tested genes in 54/160 isolates, with E. faecium the most common gene-possessing species. The most frequently occurring gene for production of enterocin A was observed in combination with enterocin P and B. Bacteriocin activity was observed in 76/160 isolates against at least one of 5 indicator bacteria from the genus Listeria, Enterococcus, Streptococcus and Staphylococcus. Four selected strains (IK25, Bri, I/Dz, P10) were active mostly against different species of Enterococcus (in the range 400-25 600 AU/mL) and Listeria sp. (800-12 800 AU/mL) but no Gram-negative bacteria were inhibited. Protein character, thermostability (up to 121°C) and stability at different pH values (3.0–10.0) were confirmed for crude bacteriocins of these four strains. The antimicrobial substance of E. faecium IK25 strain was identified as enterocin B using molecular weight detection and the presence of genes.

The present study was undertaken to investigate the microbial quality of raw milk from dairy farms in Kosovo with emphasis on the potential pathogen Lactococcus garvieae . Based on selective media and growth profiles approximately 770 isolates were obtained from raw milk collected from 221 farms and 119 of these isolates were shown to belong to L . garviae based on 16S rRNA gene sequencing. The 119 L . garvieae isolates were derived from 43 farms. It also appeared that L . garviae was established as a persistent bacterium in these farms since it was found in most samples collected over a period of 6 months. The L . garvieae -containing farms were not geographically related as isolates of this bacterium were found across the country. Genetic diversity assessed by rep-PCR fingerprinting revealed 8 major groups and the largest group contained isolates from 13 farms. Isolates within the individual groups 1, 2, 3, 4, and 5 showed strong genetic relatedness while the isolates from the remaining groups 6, 7 and 8, did not give a clear picture of relatedness. Twenty one isolates showed weak alpha haemolytic activity and antibiotic resistance was demonstrated in 39 of the 43 isolates. Diversity in the fermentation profiles was also observed among the 43 L . garvieae isolates that could be divided into 5 separate fermentation groups.

A novel antimicrobial peptide designated enterocin O16 was purified fromEnterococcus faecalis. Mass spectrometry showed a monoisotopic mass of 7,231 Da, and N-terminal Edman degradation identified a 29-amino-acid sequence corresponding to residues 90 to 119 of the EF_1097 protein. Bioinformatic analysis showed that enterocin O16 is composed of the 68 most C-terminal residues of the EF_1097 protein. Introduction of an in-frame isogenic deletion in theef1097gene abolished the production of enterocin O16. Enterocin O16 has a narrow inhibitory spectrum, as it inhibits mostly lactobacilli. Apparently,E. faecalisis intrinsically resistant to the antimicrobial peptide, as no immunity connected to the production of enterocin O16 could be identified.ef1097has previously been identified as one of three loci regulated by thefsrquorum-sensing system. The introduction of a nonsense mutation intofsrBconsistently impaired enterocin O16 production, but externally added gelatinase biosynthesis-activating pheromone restored the antimicrobial activity. Functional genetic analysis showed that the EF_1097 proprotein is processed extracellularly into enterocin O16 by the metalloprotease GelE. Thus, it is evident that thefsrquorum-sensing system constitutes the regulatory unit that controls the expression of the EF_1097 precursor protein and the protease GelE and that the latter is required for the formation of enterocin O16. On the basis of these results, this study identified antibacterial antagonism as a novel aspect related to the function offsrand provides a rationale for whyef1097is part of thefsrregulon.IMPORTANCEThefsrquorum-sensing system modulates important physiological functions inE. faecalisvia the activity of GelE. The present study presents a new facet offsrsignaling. The system controls the expression of three primary target operons (fsrABCD,gelE-sprE, andef1097-ef1097b). We demonstrate that the concerted expression of these operons constitutes the elements necessary for the production of a bacteriocin-type peptide and that antimicrobial antagonism is an intrinsic function offsr. The bacteriocin enterocin O16 consists of the 68 most C-terminal residues of the EF_1097 secreted proprotein. The GelE protease processes the EF_1097 proprotein into enterocin O16. In this manner,fsrsignaling enablesE. faecalispopulations to express antimicrobial activity in a cell density-dependent manner.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes . Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides.

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB ; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis , also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.

Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.

Mannose phosphotransferase system (Man-PTS) is the main mannose permease in bacteria but it is also a known receptor for subclass IIa bacteriocins (pediocin-like group) as well as subclass IId lactococcin A (LcnA) and lactococcin B (LcnB) (LcnA-like group). Subclass IIa bacteriocins exhibit a strong activity against Listeria spp. but they are not against Lactococcus spp. In contrast, the LcnA-like bacteriocins act only against Lactococcus lactis strains. Garvicin Q (GarQ) is a subclass IId bacteriocin with minor similarity to LcnA-like bacteriocins and a relatively broad antimicrobial spectrum including, among others, Listeria and Lactococcus spp. To identify the GarQ receptor, we obtained GarQ-resistant mutants of Lactococcus garvieae IBB3403 and L. lactis IL1403 and sequenced their genomes that revealed mutations in genes encoding the membrane-bound Man-PTS IIC or IID subunits encoded by ptnCD in L. lactis and manCD in L. garvieae. This is the first time that a bacteriocin outside the pediocin- and LcnA-like groups is shown to target Man-PTS. The interaction between GarQ and Man-PTS may occur through a new binding pattern involving specific amino acids highly conserved among the GarQ-sensitive bacterial species located in the N-terminal part and extracellular loops of subunit IID and in transmembrane region of IIC.

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97) and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium, including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP, a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates ( L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus . All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus , Bacillus , Listeria , and Enterococcus . IMPORTANCE Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine.

Introduction: Milk is considered to be a healthy, nutritious food product. Microbiological quality is an important aspect in evaluating the quality of milk. Methodology: A total of 603 bulk tank milk samples from 221 farms distributed across ten different regions were collected for milk quality assessment. Quality was judged by total viable count, and the prevalence of two foodborne pathogens (Listeria monocytogenes and Staphylococcus aureus) by using selective media and 16S rRNA gene sequencing. The presence of virulence genes was detected by polymerase chain reaction (PCR) using specific primers. Results: Milk from only 7% (15/221) of farms were found to comply with the European Union standard. Interestingly, the microbiological quality of milk from the larger herd size farms (more than 10 cows) was better than in smaller herds. L. monocytogenes was found in 2.7% (6/221) of farms, and all the examined L. monocytogenes isolates were positive with respect to the virulence genes prfA, actA, and hlyA. S. aureus was found in 39.8% (88/221) of the farms. In total, 30.7% (27/88) of the staphylococci were positive for enterotoxin production. The enterotoxins identified were toxin B (40.7%), toxin D (33.4%), toxin C (18.5%), and toxin A (7.4%). Conclusions: The total number of bacteria in milk was very high. The presence of two foodborne pathogens in raw milk represents a great health risk to consumers. To improve the microbial quality of milk in Kosovo, important measures to improve hygiene, including better information, guidance, and control, are needed.

The present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging of Enterococcus faecalis . To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains of E. faecalis were constructed. pSL101 harbors the luxABCDE operon from pPL2 lux and the pREG696 broad-host-range replicon and axe - txe toxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly ( R 2 > 0.98) with the viable-cell count. We employed lux -tagged E. faecalis strains to monitor growth in real time in milk and urine in vitro . Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in the Galleria mellonella model system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI of E. faecalis and an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.

The aim of the project was to isolate and characterize bacteriocin-producing enterococci, as well as determine the prevalence of enterocin structural genes in 187 enterococcal clinical isolates from the northwest of Iran. The isolates were screened for antibacterial activity against 15 different indicator strains. The proteinaceous nature of the antimicrobial substances was confirmed by sensitivity to proteinase K; their stability to heat treatment was tested at 60 °C and 100 °C for 20 and 10 min, respectively. The PCR method was applied to detect previously identified enterocin genes. Our results showed that 38 (20.3%) of the enterococcal isolates were considered to be potential bacteriocinogenic strains. Furthermore, genes encoding diverse bacteriocin are highly distributed among clinical enterococci, and the strains with multi-bacteriocin genes displayed high antimicrobial activity. Enterocin A, enterolysin A, and enterocin L50A/B were the most abundant structural genes detected in bacteriocinogenic strains. This work is the first survey on the prevalence of bacteriocin genes among clinical enterococci in Iran that has isolated a strain with high antimicrobial activity and sensitivity to clinically relevant antibiotics.

Due to their very potent antimicrobial activity against diverse food-spoiling bacteria and pathogens and their favourable biochemical properties, peptide bacteriocins from Gram-positive bacteria have long been considered promising for applications in food preservation or medical treatment. To take advantage of bacteriocins in different applications, it is crucial to have detailed knowledge on the molecular mechanisms by which these peptides recognize and kill target cells, how producer cells protect themselves from their own bacteriocin (self-immunity) and how target cells may develop resistance. In this review we discuss some important recent progress in these areas for the non-lantibiotic (class II) bacteriocins. We also discuss some examples of how the current wealth of genome sequences provides an invaluable source in the search for novel class II bacteriocins.

Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive Listeria monocytogenes ( mpt ) and the nonsensitive Lactococcus lactis ( ptn ) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins.

The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota.

Introduction: Mastitis is one of the most frequent and costly disease in cattle. We studied milk samples from cattle with mastitis from farms in Kosovo to identify mastitis-causing pathogens and possible effective antibiotics. Our ultimate goal is to help implement adequate antibiotic management and treatment practices in Kosovo Methodology: A total of 152 milk samples were collected from cows with clinical mastitis from different farms in Kosovo. After identification of microorganisms, antibiotic susceptibility and the occurrence of enterotoxins was investigated. Results: Staphylococci were found in 89 samples, of which 58 were coagulase negative and 31 coagulase positive. S. aureus was isolated from 27 samples, S. epidermidis from 25, and S. chromogenes from 15, while other species of staphylococci were isolated from the remaining 22 isolates. Interestingly, the bacterial diversity was different between cows in different periods of lactation and among different breeds. Most of the isolates (76/89) were resistant to two or more antibiotics. The highest resistance was to penicillin and ampicillin (> 65%), followed by tetracycline, oxacillin, streptomycin, chloramphenicol (> 23%), and less than 3% to erythromycin. Of the 89 isolates, 40 produced enterotoxins that were most frequently typed as A and C. Conclusions: We detected human bacterial pathogens in the cultures of milk samples from cows with mastitis. The isolates demonstrated resistance to two or more antibiotics, some of which are frequently used to treat animal and human infections. We recommend increased control and more stringent use of antibiotics in veterinary as well as human medicine.

Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning., OCOU was supported by a strategic scholarship program to food science research, from Norwegian University of Life Sciences (NMBU) (project 1205051025). CB and part of this work was supported by a contract financed by the Spanish Ministerio de Economia y Competitividad (AGL2013-47420-R). PBP is supported by a grant from the European Research Council (336355-MicroDE). Travels and stays in Spain and Norway for this study were supported by EEA Coordinated Mobility of Researchers NILS Science and Sustainability Project 017-CM-01-2013. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Staphylococcus aureus is known as a frequent colonizer of the skin and mucosa. Among bacterial factors involved in colonization are adhesins such as the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Serine aspartate repeat containing protein D (SdrD) is involved in adhesion to human squamous cells isolated from the nose. Here, we identify Desmoglein 1 (Dsg1) as a novel interaction partner for SdrD. Genetic deletion of sdrD in S. aureus NCTC8325-4 through allelic replacement resulted in decreased bacterial adherence to Dsg1- expressing HaCaT cells in vitro. Complementary gain-of-function was demonstrated by heterologous expression of SdrD in Lactococcus lactis, which increased adherence to HaCaT cells. Also ectopic expression of Dsg1 in HEK293 cells resulted in increased adherence of S. aureus NCTC8325-4 in vitro. Increased adherence of NCTC8325-4, compared to NCTC8325-4ΔsdrD, to the recombinant immobilized Dsg1 demonstrated direct interaction between SdrD and Dsg1. Specificity of SdrD interaction with Dsg1 was further verified using flow cytometry and confirmed binding of recombinant SdrD to HaCaT cells expressing Dsg1 on their surface. These data demonstrate that Dsg1 is a host ligand for SdrD.

The primary structure of a bacteriocin produced by Enterococcus hirae DCH5 was determined by combined amino acid and DNA sequencing. Nucleotide analysis of a 2838-bp DNA fragment of E. hirae DCH5 revealed five putative ORFs. The first orf (hirJM79) encodes a 74-amino-acid peptide containing an N-terminal signal peptide of 30 amino acids, followed by the amino acid sequence of the mature bacteriocin, hiracin JM79 (HirJM79), of 44 amino acids. The second orf (hiriJM79) encodes the putative immunity protein of HirJM79. Contiguous ORFs encode a putative mobilization protein (orfC), a relaxase/mobilization nuclease domain (orfD), and a hypothetical protein (orfE). The production and functional expression of HirJM79 by heterologous hosts suggest that hirJM79 is the minimum requirement for production of biologically active HirJM79, that HirJM79 is most likely externalized by the general secretory pathway or sec-dependent pathway, and that HiriJM79 is the immunity protein for HirJM79.

Expression of the five (pln) operons involved in the bacteriocin production of Lactobacillus plantarum C11 is regulated by a so-called pheromone-based signal-transducing network, in which the peptide pheromone (PlnA) induces bacteriocin production through the action of a histidine protein kinase (PlnB) and two antagonizing response regulators (PlnC as an activator and PlnD as a negative regulator). All pln-regulated promoters contain a conserved pair of direct repeats that serve as binding sites for PlnC and PlnD. In the present work, we show that the five PlnA-responsive operons are differentially expressed with regard to both timing and strength, and that the pheromone triggers a strong autoactivating loop of the regulatory unit (plnABCD) during an early stage of induction that gradually leads to enhanced activation of the other operons. The transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins, is also expressed relatively early upon induction, but is quickly turned off soon after peak expression. Further investigation of the various promoters revealed that, although subtle differences within the promoter regions could account for the observed differential regulation, the presence of a downstream promoter-proximal sequence in one promoter was found to cause delayed peak activity. How phosphorylation regulates the activity of the pln response regulators was also accessed by direct mutagenesis at their phosphorylation sites. It was found that the two response regulators exert activity at two different levels: a low level when they are not phosphorylated and an elevated level when they are phosphorylated. The present data demonstrate that bacteriocin production in L. plantarum C11 is a highly regulated process, in which different regulatory mechanisms are applied to fine tune the timing and strength of expression of the five pln operons.

The honey bee (Apis mellifera) is a domestic insect of high value to human societies, as a crop pollinator in agriculture and a model animal in scientific research. The honey bee, however, has experienced massive mortality worldwide due to the phenomenon Colony Collapse Disorder (CCD), resulting in alarming prospects for crop failure in Europe and the USA. The reasons for CCD are complex and much debated, but several honey bee pathogens are believed to be involved. Paratransgenesis is a Trojan horse strategy, where endogenous microorganisms are used to express effector molecules that antagonise pathogen development. For use in honey bees, paratransgenesis must rely on a set of criteria that the candidate paratransgenic microorganism must fulfil in order to obtain a successful outcome: (1) the candidate must be genetically modifiable to express effector molecules; (2) the modified organism should have no adverse effects on honey bee health upon reintroduction; and (3) it must survive together with other non-pathogenic bee-associated microorganisms. Lactic acid bacteria (LAB) are common gut bacteria in vertebrates and invertebrates, and some have naturally beneficial properties in their host. In the present work we aimed to find a potential paratransgenic candidate within this bacterial group for use in honey bees. Among isolated LAB associated with bee gut microbiota, we found the fructophilic Lactobacillus kunkeei to be the most predominant species during foraging seasons. Four genetically different strains of L. kunkeei were selected for further assessment. We demonstrated (1) that L. kunkeei is transformable; (2) that the transformed cells had no obvious adverse effect on honey bee survival; and (3) that transformed cells survived well in the gut environment of bees upon reintroduction. Our study demonstrates that L. kunkeei fulfils the three criteria for paratransgenesis and can be a suitable candidate for further research on this strategy in honey bees.

Bacteriocins produced by lactic acid bacteria (LAB) attract considerable interest as natural and nontoxic food preservatives and as therapeutics whereas the bacteriocin-producing LAB are considered potential probiotics for food, human and veterinary applications, and in the animal production field. Within LAB the lactobacilli are increasingly used as starter cultures for food preservation and as probiotics. The lactobacilli are also natural inhabitants of the gastrointestinal (GI) tract and attractive vectors for delivery of therapeutic peptides and proteins, and for production of bioactive peptides. Research efforts for production of bacteriocins in heterologous hosts should be performed if the use of bacteriocins and the LAB bacteriocin-producers is ever to meet the high expectations deposited in these antimicrobial peptides. The recombinant production and functional expression of bacteriocins by lactobacilli would have an additive effect on their probiotic functionality. The heterologous production of the bacteriocin enterocin A (EntA) was evaluated in different Lactobacillus spp. after fusion of the versatile Sec-dependent signal peptide (SP usp45 ) to mature EntA plus the EntA immunity gene (entA + entiA) (fragment UAI), and their cloning into plasmid vectors that permitted their inducible (pSIP409 and pSIP411) or constitutive (pMG36c) production. The amount, antimicrobial activity (AA) and specific antimicrobial activity (SAA) of the EntA produced by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 transformed with the recombinant plasmids pSIP409UAI, pSIP411UAI and pMGUAI varied depending of the expression vector and the host strain. The Lb. casei CECT475 recombinant strains produced the largest amounts of EntA, with the highest AA and SAA. Supernatants from Lb. casei CECT (pSIP411UAI) showed a 4.9-fold higher production of EntA with a 22.8-fold higher AA and 4.7-fold higher SAA than those from Enterococcus faecium T136, the natural producer of EntA. Moreover, supernatants from Lb. casei CECT475 (pSIP411UAI) showed a 15.7- to 59.2-fold higher AA against Listeria spp. than those from E. faecium T136. Lb. casei CECT457 (pSIP411UAI) may be considered a promising recombinant host and cell factory for the production and functional expression of the antilisterial bacteriocin EntA.

Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being the main topic of this review. During the past decade many of these bacteriocins have been isolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have been unravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closely related bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for these narrow inhibitory spectra by producing several bacteriocins belonging to different classes and having different inhibitory spectra. The latter may also help in counteracting the possible development of resistance mechanisms in target organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using a secreted peptide-pheromone for quorum-sensing. The sensing of its own growth, which is likely to be comparable to that of related species, enables the producing organism to switch on bacteriocin production at times when competition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocins and the regulation of their production, several fundamental questions remain to be solved. These include questions regarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity.

Garvicin ML (GarML) is a circular bacteriocin produced by Lactococcus garvieae DCC43. The recently published draft genome of this strain allowed determination of the genetic background for bacteriocin production. Bioinformatic analysis identified a gene cluster consisting of nine open reading frames likely involved in the production of and immunity to GarML. The garA gene encodes the bacteriocin precursor, garX a large transmembrane protein, garBCDE a putative immunity protein (garB) followed by an ATPase and two transmembrane proteins, and garFGH a putative ABC transporter complex. Functional genetic analysis revealed that deletion of garFGH had no effect on sensitivity to or production of GarML. In contrast, deletion of garBCDE or inactivation of garX resulted in high-level sensitivity to GarML and completely abolished production of active bacteriocin. Mass spectrometry of culture supernatants revealed that wild-type cultures contained the mature circular form as well as the linear forms of the bacteriocin, both with and without the three-amino-acid leader sequence, while bacteriocin-negative mutants contained only the linear forms. These results indicate that cleavage of the leader peptide precedes circularization and is likely performed by a functional entity separate from the GarML gene cluster. To our knowledge, this is the first conclusive evidence for these processes being separated in time. Loss of immunity and antimicrobial activity in addition to our inability to detect the circular bacteriocin in the ΔgarBCDE and garX::pCG47 mutants demonstrate that both these units are indispensable for GarML biosynthesis as well as immunity. Furthermore, the results indicate that these genes are implicated in the circularization of the bacteriocin and that their functions are probably interlinked.

The regulatory operon (plnABCD) involved in bacteriocin production in Lactobacillus plantarum C11 encodes four different proteins: a cationic prepeptide (PlnA); a histidine protein kinase (PlnB); and two highly homologous response regulators (PlnC and PlnD; over 75% sequence similarity). The mature product of PlnA, plantaricin A, serves as an extracellular pheromone that induces bacteriocin production. The exact roles of plnBCD in bacteriocin production have not been established experimentally. A reporter system containing the gusA gene fused with the plnA promoter was used to study plnABCD. We demonstrated that the plnABCD operon codes for an autoregulatory unit capable of activating its own promoter. Deletion analyses, performed in a heterologous expression host to define the roles of the individual genes, confirmed that both the inducer gene (plnA) and the kinase gene (plnB) are required for autoactivation. Apparently, the latter gene encodes a protein that serves as a receptor for the pheromone peptide. It was also demonstrated conclusively that the two regulators PlnC and PlnD, which have been shown previously to bind specifically to the DNA regulatory repeats of the plnA promoter, possess differential activities on the plnA promoter, with PlnC being much more active than PlnD. The functions of the response regulators were investigated further in the bacteriocin producer strain C11 in order to reveal their roles in bacteriocin production. Surprisingly, the two response regulators display totally opposite functions: although overexpression of plnC activated transcription and bacteriocin production, the overexpression of plnD repressed both processes, thus strongly suggesting that PlnD plays a role in the downregulation of bacteriocin synthesis. To our knowledge, this is the first evidence for a protein involved directly in negative regulation of bacteriocin production, and also it was shown for the first time that two highly homologous response regulators, with opposite functions, are encoded by genes located on the same operon.

Aeromonas salmonicida containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of aerA led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by A. salmonicida .

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25 degreesC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo , we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P 16S , cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation ( R 2 > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10 9 CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.

The microorganisms living in our gut have been a black box to us for a long time. However, with the recent advances in high throughput DNA sequencing technologies, it is now possible to assess virtually all microorganisms in our gut including non-culturable ones. With the use of powerful bioinformatics tools to deal with multivariate analyses of huge amounts of data from metagenomics, metatranscriptomics, metabolomics, we now start to gain some important insights into these tiny gut inhabitants. Our knowledge is increasing about who they are, to some extent, what they do and how they affect our health. Gut microbiota have a broad spectrum of possible effects on health, from preventing serious diseases, improving immune system and gut health to stimulating the brain centers responsible for appetite and food intake control. Further, we may be on the verge of being capable of manipulating the gut microbiota by diet control to possibly improve our health. Diets consisting of different components that are fermentable by microbiota are substrates for different kinds of microbes in the gut. Thus, diet control can be used to favor the growth of some selected gut inhabitants. Nowadays, the gut microbiota is taken into account as a separate organ in human body and their activities and metabolites in gut have many physiological and neurological effects. In this mini-review, we discuss the diversity of gut microbiota, the technologies used to assess them, factors that affect microbial composition and metabolites that affect human physiology, and their potential applications in satiety control via the gut-brain axis.

A large number of new bacteriocins in lactic acid bacteria (LAB) has been characterized in recent years. Most of the new bacteriocins belong to the class II bacteriocins which are small (30-100 amino acids) heat- stable and commonly not post-translationally modified. While most bacteriocin producers synthesize only one bacteriocin, it has been shown that several LAB produce multiple bacteriocins (2-3 bacteriocins). Based on common features, some of the class II bacteriocins can be divided into separate groups such as the pediocin-like and strong anti-listeria bacteriocins, the two-peptide bacteriocins, and bacteriocins with a sec-dependent signal sequence. With the exception of the very few bacteriocins containing a sec-dependent signal sequence, class II bacteriocins are synthesized in a preform containing an N-terminal double-glycine leader. The double-glycine leader-containing bacteriocins are processed concomitant with externalization by a dedicated ABC-transporter which has been shown to possess an N-terminal proteolytic domain. The production of some class II bacteriocins (plantaricins of Lactobacillus plantarum C11 and sakacin P of Lactobacillus sake) have been shown to be transcriptionally regulated through a signal transduction system which consists of three components: an induction factor (IF), histidine protein kinase (HK) and a response regulator (RR). An identical regulatory system is probably regulating the transcription of the sakacin A and carnobacteriocin B2 operons. The regulation of bacteriocin production is unique, since the IF is a bacteriocin-like peptide with a double-glycine leader processed and externalized most probably by the dedicated ABC-transporter associated with the bacteriocin. However, IF is not constituting the bacteriocin activity of the bacterium, IF is only activating the transcription of the regulated class II bacteriocin gene(s). The present review discusses recent findings concerning biosynthesis, genetics, and regulation of class II bacteriocins.