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Within the vaginal ecosystem, lactobacilli and Gardnerella spp. likely interact and influence each other's growth, yet the details of this interaction are not clearly defined. Using medium simulating vaginal fluid and a two-chamber co-culturing system to prevent cell-to-cell contact between the bacteria, we examined the possibility that Lactobacillus jensenii 62B (Lj 62B) and/or G. piotii (Gp) JCP8151B produce extracellular factors through which they influence each other's viability. By 24 h post-inoculation (hpi) in the co-culture system and under conditions similar to the vaginal environment - pH 5.0, 37 °C, and 5% CO 2 , Lj 62B viability was not affected but Gp JCP8151B had been eliminated. Cell-free supernatant harvested from Lj 62B cultures (Lj-CFS) at 20 hpi, but not 16 hpi, also eliminated Gp JCP8151B growth. Neither lactic acid nor H 2 O 2 production by Lj 62B was responsible for this effect. The Lj-CFS did not affect viability of three species of lactobacilli or eight species of Gram-positive and Gram-negative uropathogens but eliminated viability of eight different strains of Gardnerella spp. Activity of the inhibitory factor within Lj-CFS was abolished by protease treatment and reduced by heat treatment suggesting it is most likely a bacteriocin-like protein; fractionation revealed that the factor has a molecular weight within the 10-30 kDa range. These results suggest that, in medium mimicking vaginal fluid and growth conditions similar to the vaginal environment, Lj 62B produces a potential bacteriocin-like inhibitory substance (Lj-BLIS) that clearly targets Gardnerella spp. strains. Once fully characterized, Lj-BLIS may be a potential treatment for Gardnerella- related BV that does not alter the vaginal microflora.

Microcins are an understudied and poorly characterized class of antimicrobial peptides. Despite the existence of only 15 examples, all identified from the Enterobacteriaceae , microcins display diversity in sequence, structure, target cell uptake, cytotoxic mechanism of action and target specificity. Collectively, these features describe some of the unique means nature has contrived for molecules to cross the 'impermeable' barrier of the Gram-negative bacterial outer membrane and inflict cytotoxic effects. Microcins appear to be widely dispersed among different species and in different environments, where they function in regulating microbial communities in diverse ways, including through competition. Growing evidence suggests that microcins may be adapted for therapeutic uses such as antimicrobial drugs, microbiome modulators or facilitators of peptide uptake into cells. Advancing our biological, ecological and biochemical understanding of the roles of microcins in bacterial interactions, and learning how to regulate and modify microcin activity, is essential to enable such therapeutic applications.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii ( Xb-Sj ), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp 1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath ( xbpS1 ) or tail fibre ( xbpH1 ) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii ( Xsz-Sr ), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj . Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr . To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj , suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta , while in competition with the xenorhabdicin-deficient strain ( Xb-Sj:S1 ), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

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

Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

Post-segregational killing (PSK) is a phenotype determined by plasmids using a toxin and an antitoxin gene pair. Loss of the genes depletes the cell's reserve of antitoxin and allows the toxin to act upon the cell. PSK benefits mobile elements when it increases reproductive success relative to other mobile competitors. A side effect of PSK is that plasmids become refractory to displacement from the cell during growth as a monoculture. Most PSK systems use a cytoplasmic toxin, but the external toxins of bacteriocins also have a PSK-like effect. It may be that any toxin and antitoxin gene pair can demonstrate PSK when it is on a plasmid. The secreted ribonuclease barnase and its protein inhibitor barstar have features in common with PSK modules, though their native context is chromosomal. We hypothesized that their recruitment to a plasmid could produce an emergent PSK phenotype. Others had shown that secreted barnase could exert a lethal effect on susceptible bacteria similarly to bacteriocins. However, barnase toxicity did not occur under the conditions tested, suggesting that barnase is toxic to neighbouring cells only under very specific conditions. Bacteriocins are only produced under some conditions, and some conditionality on toxin function or release may be advantageous in general to PSKs with external toxins because it would prevent killing of potential plasmid-naive hosts. Too much conditionality, however, would limit how advantageous the gene pair was to mobile elements, making the genes unlikely to be recruited as a PSK system.

Bacteriocins represent a rather underutilized class of antimicrobials despite often displaying activity against many drug-resistant pathogens. Lantibiotics are a post-translationally modified class of bacteriocins, characterized by the presence of lanthionine and methyllanthionine bridges. In this study, a novel two-peptide lantibiotic was isolated and characterized. Formicin was isolated from Bacillus paralicheniformis APC 1576, an antimicrobial-producing strain originally isolated from the intestine of a mackerel. Genome sequencing allowed for the detection of the formicin operon and, from this, the formicin structural genes were identified, along with those involved in lantibiotic modification, transport and immunity. The identified bacteriocin was subsequently purified from the bacterial supernatant. Despite the degree of conservation seen amongst the entire class of two-peptide lantibiotics, the formicin peptides are unique in many respects. The formicin α peptide is far less hydrophobic than any of the equivalent lantibiotics, and with a charge of plus two, it is one of the most positively charged α peptides. The β peptide is unique in that it is the only such peptide with a negative charge due to the presence of an aspartic acid residue in the C-terminus, possibly indicating a slight variation to the mode of action of the bacteriocin. Formicin also displays a broad spectrum of inhibition against Gram-positive strains, inhibiting many clinically relevant pathogens such as Staphylococcus aureus, Clostridium difficile and Listeria monocytogenes. The range of inhibition displayed against many important pathogens indicates a potential therapeutic use against such strains where antibiotic resistance is such a growing concern.

Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria to acquire survival benefits during competitive inter- and intra-species interactions in complex ecosystems. In this study, an AMP-producing soil bacterial strain designated SKDU10 was isolated and identified as a member of the genus Brevibacillus. The AMP produced by strain SKDU10 identified as a class IId bacteriocin with 57.6 % homology to laterosporulin, a defensin-like class IId bacteriocin. However, substantial differences were observed in the antimicrobial activity spectrum of this bacteriocin named laterosporulin10 when compared to laterosporulin. Laterosporulin10 effectively inhibited the growth of Staphylococcus aureus and Mycobacterium tuberculosis (Mtb H37Rv) with LD50 values of 4.0 µM and 0.5 µM, respectively. Furthermore, laterosporulin10 inhibited the growth of Mtb H37Rv strain at about 20 times lower MIC value compared to S. aureus MTCC 1430 or M. smegmatis MC2 155 in vitro and ex vivo. Electron micrographs along with membrane permeabilization studies using FACS analysis revealed that laterosporulin10 is a membrane-permeabilizing peptide. Interestingly, laterosporulin10 was able to efficiently kill Mtb H37Rv strain residing inside the macrophages and did not show haemolysis up to 40 µM concentration.

Dental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-D-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.

Lactococcin Q is a two-peptide (Qα and Qβ) bacteriocin produced by Lactococcus lactis QU 4, which exhibits specific antimicrobial activity against L. lactis strains. The lactococcin Q gene cluster (approximately 4.5 kb) was sequenced and found to include genes encoding lactococcin Q immunity (laqC), an ATP-binding cassette transporter (laqD) and a transport accessory protein (laqE), downstream of the lactococcin Q structural genes (laqA and laqB). In addition, the gene cluster showed high sequence identity with that of a lactococcin Q homologue bacteriocin, lactococcin G. Heterologous expression studies showed that LaqD was responsible for lactococcin Q secretion in a manner dependent on LaqE expression, and that LaqC conferred self-immunity to lactococcin Q and cross-immunity to lactococcin G. Amino acid alignment of both lactococcin transporters revealed that LaqD contains an insertion (160-168 residues) that is essential for lactococcin Q secretion, as L. lactis cells that expressed LaqDΔ160-168 were devoid of this function. Additional experiments demonstrated that the LaqDΔ160-168 mutant was, however, able to secrete lactococcin G, suggesting that the insertion is necessary only for the lactococcin Q secretion by LaqD. This report demonstrates the biosynthetic mechanism of lactococcin Q/G-type bacteriocins and the complementarity of the genes responsible for the secretion of lactococcins Q and G.

Lacticin Q (LnqQ) produced by Lactococcus lactis QU 5 is an unmodified linear bacteriocin, which is synthesized without an N-terminal leader peptide. In vitro synthesis and in vivo expression of LnqQ have revealed the intracellular toxicity of this leaderless peptide, as well as the necessity of a dedicated secretion and self-immunity system of producer cells. Further DNA sequencing and analysis have discovered 11 putative orf genes at the LnqQ locus. None of the orf genes showed similarities to any of the bacteriocin biosynthetic genes characterized to date; however, six orf genes (orf2q-7q), not including the structural gene (lnqQ), were highly conserved at the lacticin Z locus (orf2z-7z), which is a LnqQ homologue produced by L. lactis QU 14. ORF2q (ORF2z), the gene of which is located upstream of the structural gene, is a putative transcriptional regulator, whereas ORF6q and ORF7q (ORF6z and ORF7z) form a putative ATP-binding cassette transporter. The ORF3q-5q (ORF3z-5z) are all predicted to be membrane proteins with no clear functions. Co-expression of LnqQ and ORF3q-7q in a heterologous host allowed the extracellular production of LnqQ; additionally, the expression of ORF3q-7q rendered the host cells immune to LnqQ. This self-immunity was facilitated possibly by two means; firstly, by secreting the active LnqQ peptides, thus reducing the intracellular toxicity, and secondly, by protecting the host cells from extracellularly released LnqQ. This is the first report, to our knowledge, that describes intracellular toxicity of a leaderless bacteriocin and provides a rare example of biosynthetic genes that are required for bacteriocin secretion and immunity.

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.

Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) lung infections are responsible for much of the mortality in cystic fibrosis (CF). However, little is known about the ecological interactions between these two, often co-infecting, species. This study provides what is believed to be the first report of the intra- and interspecies bacteriocin-like inhibition potential of Pa and Bcc strains recovered from CF patients. A total of 66 strains were screened, and shown to possess bacteriocin-like inhibitory activity (97 % of Pa strains and 68 % of Bcc strains showed inhibitory activity), much of which acted across species boundaries. Further phenotypic and molecular-based assays revealed that the source of this inhibition differs for the two species. In Pa, much of the inhibitory activity is due to the well-known S and RF pyocins. In contrast, Bcc inhibition is due to unknown mechanisms, although RF-like toxins were implicated in some strains. These data suggest that bacteriocin-based inhibition may play a role in governing Pa and Bcc interactions in the CF lung and may, therefore, offer a novel approach to mediating these often fatal infections.

Membrane-located proteins (IIC and IID) of the mannose-phosphotransferase system (man-PTS) have previously been shown to serve as target receptors for several bacteriocins. Although many bacteria contain at least one such man-PTS in their genome, most bacteriocins display a narrow inhibitory spectrum, targeting predominantly bacteria closely related to the producers. In the present study we have analysed the receptor spectrum of one-peptide bacteriocins of class II. A phylogenetic analysis of 86 man-PTSs from a wide range of bacterial genera grouped the man-PTSs into three main clusters (groups I-III). Fourteen man-PTSs distributed across the phylogenetic tree were selected for experimental analysis in a heterologous host. Only members of group I could serve as receptors for class IIa bacteriocins, and the receptor efficiencies varied in a pattern directly related to their phylogenetic position. A multiple sequence alignment of IIC and IID proteins revealed three sequence regions (two in IIC and one in IID) that distinguish members of the bacteriocin-susceptible group from those of the other groups, suggesting that these amino acid regions confer the specific bacteriocin receptor function. Moreover, we demonstrated that variation in sensitivity might also exist within the same species due to differential expression levels of the receptor, since three strains of Lactobacillus sakei harbouring identical man-PTSs were shown to display different expression levels of a man-PTS gene that corresponded to the variation in bacteriocin sensitivity. Together, the results of our study show that the level of bacteriocin susceptibility for a bacterial cell is primarily determined by differences in its man-PTS proteins, although the expression levels of the corresponding genes also play an important role.

In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains of Lactobacillus, Enterococcus, Streptococcus, Bacillus and Listeria. Strain 925A, identified as Lactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01-04, and have molecular sizes of 1815, 3524, 8881 and 65 037 bp, respectively. We obtained bacteriocin non-producing derivatives by treatment of strain 925A with novobiocin. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed that breB is the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in both Escherichia coli and LAB such as Lactobacillus plantarum, Lb. brevis, Lactobacillus helveticus, Lactobacillus hilgardii and Enterococcus hirae. To determine the function of gene breE, which displays no significant similarity to any other sequences in the blast search database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.

Streptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibiotic mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high-phosphate brain heart infusion agar medium that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones for which we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups, including central metabolism, surface binding and sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when the gene is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.

Nisin-producing Lactococcus lactis protects its own cell membrane against the bacteriocin with the ABC transporter NisFEG, and the immunity lipoprotein NisI. In this study, in order to localize a site for specific nisin interaction in NisI, a C-terminal deletion series of NisI was constructed, and the C-terminally truncated NisI proteins were expressed in L. lactis. The shortest deletion (5 aa) decreased the nisin immunity capacity considerably in the nisin-negative strain MG1614, resulting in approximately 78% loss of immunity function compared with native NisI. A deletion of 21 aa decreased the immunity level even more, but longer deletions, up to 74 aa, provided the same level of nisin immunity as the 21 aa deletion, i.e. approximately 14% of the immunity provided by native NisI. Similar to native NisI, all the C-terminally truncated NisI proteins provided higher immunity to nisin in the NisFEG-expressing strain NZ9840 than in MG1614, i.e. approximately 40-50% of the immunity capacity of native NisI. Then, it was determined whether the NisI C-terminal 21 aa fragment could protect cells against nisin. To target the 21 aa fragment to its natural location, 21 C-terminal amino acids from the subtilin-specific immunity lipoprotein SpaI were replaced by 21 C-terminal amino acids from NisI. The expression of the SpaI'-'NisI fusion in L. lactis strains significantly increased their nisin immunity. This is the first time the immunity function of a lantibiotic immunity protein has been transferred to another protein. However, unlike native NisI, and the C-terminally truncated NisI fragments, the increase in nisin immunity conferred by the SpaI'-'NisI fusion was the same in both the NisFEG strain NZ9840 and MG1614. In conclusion, the SpaI'-'NisI fusion could not enhance nisin immunity by interacting with NisFEG, whereas the C-terminally truncated NisI fragments and native NisI were able to enhance nisin immunity, probably by co-operation with NisFEG. The results made it evident that the C terminus of NisI is involved in specific interaction with nisin, and that it confers specificity for the NisI immunity lipoprotein.

The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system. The gene cluster has an operon-like structure consisting of a putative pediocin-like bacteriocin gene (termed penA) and a potential immunity gene (termed peiA). Genetic determinants involved in bacteriocin transport and regulation are also found in proximity to penA and peiA but the so-called accessory gene involved in transport and the inducer gene involved in regulation are missing. Consequently, this bacterium is a poor bacteriocin producer. To analyse the potency of the putative bacteriocin operon, the two genes penA-peiA were heterologously expressed in a Lactobacillus sakei host that contains the complete apparatus for gene activation, maturation and externalization of bacteriocins. It was demonstrated that the heterologous host expressing penA and peiA produced a strong bacteriocin activity; in addition, the host became immune to its own bacteriocin, identifying the gene pair penA-peiA as a potent bacteriocin system. The novel pediocin-like bacteriocin, termed penocin A, has an isotopic mass [M+H]+ of 4684.6 Da as determined by mass spectrometry; this value corresponds well to the expected size of the mature 42 aa peptide containing a disulfide bridge. The bacteriocin is heat-stable but protease-sensitive and has a calculated pI of 9.45. Penocin A has a relatively broad inhibition spectrum, including pathogenic Listeria and Clostridium species. Immediately upstream of the regulatory genes reside some features that resemble remnants of a disrupted inducer gene. This degenerate gene was restored and shown to encode a double-glycine leader-containing peptide. Furthermore, expression of the restored gene triggered high bacteriocin production in P. pentosaceus ATCC 25745, thus confirming its role as an inducer in the pen regulon.

Genes encoding pediocin-like bacteriocins are usually co-transcribed with a gene encoding a cognate immunity protein. To investigate the functionality and specificity of immunity proteins, immunity genes belonging to the bacteriocins curvacin A, enterocin A, enterocin P, leucocin A, pediocin PA-1 and sakacin P, as well as a putative immunity gene, orfY, were expressed in three bacteriocin-sensitive lactic acid bacteria (Lactobacillus sake, Carnobacterium piscicola and Enterococcus faecalis). The transformed indicator strains, each containing one of the immunity genes, were tested for sensitivity towards seven different purified bacteriocins (curvacin A, enterocin A, enterocin P, leucocin A, leucocin C, pediocin PA-1 and sakacin P). Cross-immunity was observed almost exclusively in situations where either the bacteriocins or the immunity proteins belonged to the same sequence-based subgroup. In a few cases, the functionality of immunity proteins was strain-dependent; e.g. the leucocin A immunity gene provided immunity to enterocin A, pediocin PA-1 and leucocin A in Ent. faecalis, whereas in the other two indicators, this gene provided immunity to leucocin A only. The orfY gene, which is transcribed without a cognate bacteriocin, was shown to encode a functional immunity protein that expands the bacteriocin resistance of the strain possessing this gene. The results show that the bacteriocin sensitivity of a lactic acid bacterium strain can depend on (1) the presence of immunity genes in connection with its own bacteriocin production, (2) the presence of extra immunity genes and (3) more general properties of the strain such as the membrane composition or the presence of receptors.

The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N- and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the C-terminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may - apparently via the initial electrostatic binding of the bacteriocin to target cells - influence the target-cell specificity.

The sigma(54) RNA polymerase subunit has a prominent role in susceptibility of Listeria monocytogenes and Enterococcus faecalis to mesentericin Y105, a class IIa bacteriocin. Consequently, sigma(54)-dependent genes as well as specific activators also required for expression of these genes were sought. Five putative sigma(54)-associated activators were detected in the genome of E. faecalis V583, and all but one could activate the transcription of permease genes belonging to sugar phosphotransferase systems (PTSs). Interestingly, these activators display a helicase signature not yet reported in this activator family, which could explain the ATP-dependent mechanism of DNA unwinding preceding the start of transcription. To find which activator is linked to susceptibility of E. faecalis to mesentericin Y105, their respective genes were subsequently interrupted. Among them, only mptR gene interruption led to a resistance phenotype. Immediately downstream from mptR, a putative sigma(54)-dependent operon was found to encode a mannose PTS permease, namely EII(t)(Man). Moreover, in liquid culture, glucose and mannose induced the sensitivity of E. faecalis to mesentericin Y105. Since sugars have previously been reported to induce PTS permease expression, it appears that EII(t)(Man) expression, presumably induced in the presence of glucose and mannose, leads to an enhanced sensitivity of E. faecalis to the bacteriocin. Additional information was gained from knockouts within the permease operon. Interruption of the distal mptD gene, which encodes the IID subunit of EII(t)(Man), strikingly led to resistance to mesentericin Y105. Moreover, MptD appears to be a peculiar membrane subunit, bearing an additional domain compared to most known IID subunits. According to these results, EII(t)(Man) is clearly involved in susceptibility to mesentericin Y105 and could even be its receptor at the E. faecalis surface. Finally, it is hypothesized that MptD could be responsible for the targeting specificity, via an interaction between its additional domain and mesentericin Y105.

Addition of lactococcin 972 to exponentially growing sensitive cultures of Lactococcus lactis resulted in cell elongation and widening. Thin sections revealed that septum invagination was blocked. Cell growth progressed until most cells showed equatorial constriction and even initial deposition of material at the septum ring, although cell division did not proceed any further. The increase in the incorporation of labelled precursors into the cell wall shifted from an exponential to a linear mode in treated cultures, subsequently being arrested. Gross degeneration of the cells was observed prior to cell death, followed by slow lysis of the culture. In contrast, stationary-phase cultures remained unaffected.

Carnobacteriocin A (CbnA) is a regulated bacteriocin produced by Carnobacterium piscicola LV17A that is encoded on a 72 kb plasmid. A 10.0 kb fragment from this plasmid that contained information necessary for bacteriocin production and immunity was cloned and sequenced. Genetic analysis showed the presence of the previously sequenced structural gene for CbnA, as well as genes encoding proteins homologous to dedicated bacteriocin transport proteins and proteins of three-component signal transduction systems. The induction factor (CbnX) was chemically synthesized and induced CbnA production at 10(-11) M or higher in a C. piscicola LV17A culture that had lost the ability to produce bacteriocin as a result of dilution. The gene cbiA for the immunity protein is not located in typical close proximity to the structural gene for CbnA and is encoded in the opposite orientation. CbiA has homology with EniB, the immunity protein for enterocin B that is also encoded in the opposite orientation to the bacteriocin gene. CbiA and EniB cross-protected against the corresponding bacteriocins.

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli and clones were screened for a bacteriocin 28b insensitive phenotype. One clone was found that showed partial resistance to bacteriocin 28b. By using Tn5tac1 insertions it was shown that this phenotype was due to the expression in E. coli of an outer-membrane protein of 17 kDa (Omp4). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 515 bp. The deduced amino acid sequence has 172 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues and, when compared to other enterobacterial outer-membrane proteins, most closely resembles a family of small outer-membrane proteins of Enterobacteriaceae whose known functions appear to be related with virulence. Immunoblotting experiments showed that Omp4 is present in 15 biotypes of S. marcescens. The bacteriocin 28b resistance phenotype conferred on E. coli by Omp4 appears to be pleiotropic since overexpression of the Omp4-encoding gene leads to a decrease in the amount of OmpA, OmpF and/or OmpC; OmpA and OmpF are the receptors for bacteriocin 28b in E. coli.