Evidence for the Involvement of Pleckstrin Homology Domain-Containing Proteins in the Transport of Enterocin DD14 (EntDD14); a Leaderless Two-Peptide Bacteriocin.

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

Lactococcus lactis Resistance to Aureocin A53- and Enterocin L50-Like Bacteriocins and Membrane-Targeting Peptide Antibiotics Relies on the YsaCB-KinG-LlrG Four-Component System.

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.

Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens.

Lytic bacteriophages are expected as effective tools to control infectious bacteria in human and pathogenic or spoilage bacteria in foods. Leaderless bacteriocins (LLBs) are simple bacteriocins produced by Gram-positive bacteria. LLBs do not possess an N-terminal leader peptide in the precursor, which means that they are active immediately after translation. In this study, we constructed a novel antimicrobial agent, an LLB-producing phage (LLB-phage), by genetic engineering to introduce the LLB structural gene into the lytic phage genome. To this end, lnqQ (structure gene of an LLB, lacticin Q) and trxA, an essential gene for T7 phage genome replication, were integrated in tandem into T7 phage genome using homologous recombination in Escherichia coli host strain. The recombinant lnqQ-T7 phage was isolated by a screening method using ΔtrxA host strain. lnqQ-T7 phage formed a clear halo in agar plates containing both E. coli and lacticin Q-susceptible Bacillus coagulans, indicating that lnqQ-T7 phage could produce a significant amount of lacticin Q. Lacticin Q production did not exert a significant effect on the lytic cycle of T7 phage. In fact, the production of lacticin Q enhanced T7 phage lytic activity and helped to prevent the emergence of bacterial populations resistant against this phage. These results serve as a proof of principle for LLB-phages. There are different types of LLBs and phages, meaning that in the future, it may be possible to produce any number of LLB-phages which can be designed to efficiently control different types of bacterial contamination in different settings. IMPORTANCE We demonstrated that we could combine LLB and phage to construct promising novel antimicrobial agents, LLB-phage. The first LLB-phage, lnqQ-T7 phage, can control the growth of both the Gram-negative host strain and neighboring Gram-positive bacteria while preventing the emergence of phage resistance in the host strain. There are several different types of LLBs and phages, suggesting that we may be able to design a battery of LLB-phages by selecting novel combinations of LLBs and phages. These constructs could be tailored to control various bacterial contaminations and infectious diseases.

Over 2000-Fold Increased Production of the Leaderless Bacteriocin Garvicin KS by Increasing Gene Dose and Optimization of Culture Conditions.

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.

Circular and Leaderless Bacteriocins: Biosynthesis, Mode of Action, Applications, and Prospects.

Bacteriocins are a huge family of ribosomally synthesized peptides known to exhibit a range of bioactivities, most predominantly antibacterial activities. Bacteriocins from lactic acid bacteria are of particular interest due to the latter's association to food fermentation and the general notion of them to be safe. Among the family of bacteriocins, the groups known as circular bacteriocins and leaderless bacteriocins are gaining more attention due to their enormous potential for industrial application. Circular bacteriocins and leaderless bacteriocins, arguably the least understood groups of bacteriocins, possess distinctively peculiar characteristics in their structures and biosynthetic mechanisms. Circular bacteriocins have N-to-C- terminal covalent linkage forming a structurally distinct circular peptide backbone. The circular nature of their structures provides them superior stability against various stresses compared to most linear bacteriocins. The molecular mechanism of their biosynthesis, albeit has remained poorly understood, is believed to possesses huge application prospect as it can serve as scaffold in bioengineering other biologically important peptides. On the other hand, while most bacteriocins are synthesized as inactive precursor peptides, which possess an N-terminal leader peptide attached to a C-terminal propeptide, leaderless bacteriocins are atypical as they do not have an N-terminal leader peptide, hence the name. Leaderless bacteriocins are active right after translation as they do not undergo any post-translational processing common to other groups of bacteriocins. This "simplicity" in the biosynthesis of leaderless bacteriocins offers a huge commercial potential as scale-up production systems are considerably easier to assemble. In this review, we summarize the current studies of both circular and leaderless bacteriocins, highlighting the progress in understanding their biosynthesis, mode of action, application and their prospects.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

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.