The thnI gene is not required for thurincin H biosynthesis or immunity.

Thurincin H is a bacteriocin produced by Bacillus thuringiensis, it is encoded in a group of ten genes, most of which have been characterized experimentally or by homology. However, the activity of the thnI gene encoding a 95 amino acid ORF remains unknown. In this work, the thnI gene was cloned under the regulation of two promoters and transformed into a sensitive strain to determine if it acts as an immunity protein. In addition, a deletion mutant without the thnI gene was used to test whether thnI is required or not for the biosynthesis of thurincin H. It was concluded that thnI does not provide immunity and is not required to produce thurincin H.

Biosynthesis of Gut-Microbiota-Derived Lantibiotics Reveals a Subgroup of S8 Family Proteases for Class III Leader Removal.

Lanthipeptides are a group of ribosomally synthesized and post-translationally modified peptides with diverse structural features and bioactivities. Gut-microbiota-derived lanthipeptides play important roles in gut homeostasis of the host. Herein, we report the discovery and biosynthesis of class III lantibiotics named amylopeptins, which are derived from the gut microbiota of Sprague-Dawley rats and display a narrow antimicrobial spectrum. In contrast to known class III lanthipeptides, the biosynthesis of amylopeptins employs AmyP, which belongs to a subgroup of S8 family serine proteases, to remove the leader of corresponding precursor peptides in a site-specific manner during the last step of their maturation. Overall, this study shows for the first time that S8 family proteases participate in the biosynthesis of class III lanthipeptides.

Evolution of ColE1-like plasmids across γ-Proteobacteria: From bacteriocin production to antimicrobial resistance.

Antimicrobial resistance is one of the major threats to Public Health worldwide. Understanding the transfer and maintenance of antimicrobial resistance genes mediated by mobile genetic elements is thus urgent. In this work, we focus on the ColE1-like plasmid family, whose distinctive replication and multicopy nature has given rise to key discoveries and tools in molecular biology. Despite being massively used, the hosts, functions, and evolutionary history of these plasmids remain poorly known. Here, we built specific Hidden Markov Model (HMM) profiles to search ColE1 replicons within genomes. We identified 1,035 ColE1 plasmids in five Orders of γ-Proteobacteria, several of which are described here for the first time. The phylogenetic analysis of these replicons and their characteristic MOBP5/HEN relaxases suggest that ColE1 plasmids have diverged apart, with little transfer across orders, but frequent transfer across families. Additionally, ColE1 plasmids show a functional shift over the last decades, losing their characteristic bacteriocin production while gaining several antimicrobial resistance genes, mainly enzymatic determinants and including several extended-spectrum betalactamases and carbapenemases. Furthermore, ColE1 plasmids facilitate the intragenomic mobilization of these determinants, as various replicons were identified co-integrated with large non-ColE1 plasmids, mostly via transposases. These results illustrate how families of plasmids evolve and adapt their gene repertoires to bacterial adaptive requirements.

Identification of Potential Probiotics Producing Bacteriocins Active against Listeria monocytogenes by a Combination of Screening Tools.

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow's milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis.

Production of a Class IIb Bacteriocin with Broad-spectrum Antimicrobial Activity in Lactiplantibacillus plantarum RUB1.

Bacteriocins produced by lactic acid bacteria have potential use as natural food preservatives, which may alleviate current problems associated with the overuse of antibiotics and emerging multi-drug-resistant microbes. In this work, Lactiplantibacillus plantarum RUB1 was found to produce a class IIb bacteriocin with strong antibacterial activity. Except for plnXY encoding putative proteins, L. plantarum RUB1 contains most genes in five operons (plnABCD, plnGHSTUVW, plnMNOP, plnIEF, and plnRLJK) related to bacteriocin synthesis. Adding low (100 and 500 ng/mL) and medium (1 µg/mL) concentrations of PlnA to broth promoted bacteriocin production and upregulated bacteriocin gene plnA, while high concentrations (50 and 200 µg/mL) inhibited expression of these genes. Co-culturing L. plantarum RUB1 with Enterococcus hirae 1003, Enterococcus hirae LWS, Limosilactobacillus fermentum RC4, L. plantarum B6, and even Listeria monocytogenes ATCC 19111 and Staphylococcus aureus ATCC 6538 enhanced bacteriocin activity and expression of bacteriocin-related genes. This study verifies that PlnA can indeed upregulate the expression of bacteriocin genes, and also bacteriocin production can be induced by co-culture with some specific bacteria or their cell-free supernatants. Bacteriocin production by L. plantarum RUB1 is mediated by a quorum sensing mechanism, directly influenced by autoinducing peptide or specific strains. The findings provide new methods and insight into bacteriocin production mechanisms.

Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in Bacillus subtilis.

A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo. The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex.

Comparative genomics analysis of Pediococcus acidilactici species.

Pediococcus acidilactici is a reliable bacteriocin producer and a promising probiotic species with wide application in the food and health industry. However, the underlying genetic features of this species have not been analyzed. In this study, we performed a comprehensive comparative genomic analysis of 41 P. acidilactici strains from various ecological niches. The bacteriocin production of 41 strains were predicted and three kinds of bacteriocin encoding genes were identified in 11 P. acidilactici strains, namely pediocin PA-1, enterolysin A, and colicin-B. Moreover, whole-genome analysis showed a high genetic diversity within the population, mainly related to a large proportion of variable genomes, mobile elements, and hypothetical genes obtained through horizontal gene transfer. In addition, comparative genomics also facilitated the genetic explanation of the adaptation for host environment, which specify the protection mechanism against the invasion of foreign DNA (i.e. CRISPR/Cas locus), as well as carbohydrate fermentation. The 41 strains of P. acidilactici can metabolize a variety of carbon sources, which enhances the adaptability of this species and survival in different environments. This study evaluated the antibacterial ability, genome evolution, and ecological flexibility of P. acidilactici from the perspective of genetics and provides strong supporting evidence for its industrial development and application.

Acetate Activates Lactobacillus Bacteriocin Synthesis by Controlling Quorum Sensing.

Bacteriocins are useful for controlling the composition of microorganisms in fermented food. Bacteriocin synthesis is regulated by quorum sensing mediated by autoinducing peptides. In addition, short-chain fatty acids, especially acetic acid, reportedly regulate bacteriocin synthesis. Five histidine kinases that regulated the synthesis of bacteriocins were selected to verify their interactions with acetate. Acetate activated the kinase activity of PlnB, SppK, and HpK3 in vitro and increased the yield of their cognate bacteriocins plantaricin EF, sakacin A, and rhamnosin B in vivo. The antimicrobial activity against Staphylococcus aureus of the fermentation supernatants of Lactobacillus plantarum, Lactobacillus sakei, and Lactobacillus rhamnosus with addition of acetate increased to 298%, 198%, and 289%, respectively, compared with that in the absence of acetate. Our study elucidated the activation activity of acetate in bacteriocin synthesis, and it might provide a potential strategy to increase the production of bacteriocin produced by Lactobacillus. IMPORTANCE Bacteriocins produced by lactic acid bacteria (LAB) are particularly useful in food preservation and food safety. Bacteriocins might increase bacterial competitive advantage against the indigenous microbiota of the intestines; at the same time, bacteriocins could limit the growth of undesired microorganisms in yogurt and other dairy products. This study confirmed that three kinds of histidine kinases were activated by acetate and upregulated bacteriocin synthesis both in vitro and in vivo. The increasing yield of bacteriocins reduced the number of pathogens and increased the number of probiotics in milk. Bacteriocin synthesis activation by acetate may have a broad application in the preservation of dairy products and forage silage.

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum.

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production.

Plant-produced bacteriocins inhibit plant pathogens and confer disease resistance in tomato.

Bacteriocins are a diverse group of bacterial antimicrobial peptides (AMPs) that represent potential replacements for current antibiotics due to their novel modes of action. At present, production costs are a key constraint to the use of bacteriocins and other AMPs. Here, we report the production of bacteriocins in planta - a potentially scalable and cost-effective approach for AMP production. Nine bacteriocin genes with three different modes of action and minimal or no post-translational modifications were synthesized, cloned and used to transform Arabidopsis thaliana. To confirm bacteriocin functionality and the potential to use these plants as biofactories, Arabidopsis T3 crude leaf extracts were subjected to inhibition assays against the bacterial pathogens Clavibacter michiganensis subsp. michiganensis (Cmm) and Pseudomonas syringae pv. tomato DC3000 (Pst). Six and seven of nine extracts significantly inhibited Cmm and Pst, respectively. Three bacteriocin genes (plantaricin, enteriocin, and leucocin) were then selected for over-expression in tomato (Solanum lycopersicum). In vitro plant pathogen inhibition assays of T0, T1 and T2 transgenic tomato leaf extracts confirmed antimicrobial activity against both pathogens for all three generations of plants, indicating their potential use as stable biopesticide biofactories. Plantaricin and leucocin-expressing T2 tomato plants were resistant to Cmm, and leucocin-expressing T2 plants were resistant to Pst. This study highlights that plants can be used as biofactories for AMP production and that the expression of bacteriocins in planta may offer new opportunities for disease control in agriculture.

Bacteriocinogenic Potential of Bacillus amyloliquefaciens Isolated from Kimchi, a Traditional Korean Fermented Cabbage.

Bacteriocin production is considered a favorable property for various beneficial cultures. In addition to their potential as biopreservatives, bacteriocins are also promising alternatives for the control of multidrug-resistant pathogens and the inhibition of some viruses and cancer cells. The objective of this study was to screen and characterize a bacteriocin-producing strain with the aim of its future application for control of Listeria monocytogenes, an important food-borne pathogen. A total of 22 potentially bacteriocinogenic strains active against L. monocytogenes ATCC15313 were isolated from locally produced kimchi through a three-level approach. Pure cultures were obtained according to good microbiological practices and differentiated through RAPD-PCR using the primers OPL01, OPL09, and OPL11. Altogether, 5 strains were selected for further study. Specific focus was given to strain ST05DL based on its specific inhibitory activity against L. monocytogenes ATCC15313, while not affecting different strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella, most of which are beneficial microorganisms. The strain ST05DL was identified as Bacillus amyloliquefaciens based on its sugar fermentation profile obtained through API50CHB analysis and 16S rRNA partial sequencing. The antimicrobial compound produced by B. amyloliquefaciens ST05DL was found to be sensitive to pepsin and α-chymotrypsin, evidence of its proteinaceous nature. The presence of skim milk, NaCl, Tween 80, glycerol, and SDS did not affect the antimicrobial activity. The addition of 20% cell-free supernatant (CFS) obtained from a 24-h culture of B. amyloliquefaciens ST05DL to an exponentially growing culture of L. monocytogenes ATCC15313 successfully inhibited the test microorganisms during the monitored 10-h incubation. Optimal bacteriocin production by B. amyloliquefaciens ST05DL was observed during the stationary phase at 12 h (800 AU/mL) and remained stable for the next 15 h. The ratio between live and dead cells during this period was 74.37% and 25.66%, respectively, as determined by flow cytometry. The presence of the virulence genes hblA, hblB, hblC, nheA, nheB, and nheC was not detected in the total DNA of B. amyloliquefaciens ST05DL, and the strain was resistant only to ampicillin out of 10 tested antibiotics. Future evaluation of expressed bacteriocin/s by B. amyloliquefaciens ST05DL (amino acid sequence, molecular mass, cytotoxicity, detailed mode of action, etc.), will be the next step in the characterization and its potential application as biopreservative and/or pharmaceutical product.

Optimized Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed To Test the Roles of gcc Cluster Genes in Bacteriocin Production.

The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino-acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting the growth of the most susceptible bacterial strains at picomolar concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here, we report the development of pRV610gcc, an easily modifiable 11.2-kbp plasmid that enables the production of GccF in L. plantarum NC8. gcc gene expression relies on native promoters in the cloned cluster, and NC8(pRV610gcc) produces mature GccF at levels similar to KW30. Key findings are that the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence but glycosylation of the loop serine is both sequence and spatially specific, that glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation, that neither of the putative thioredoxin proteins is essential for peptide maturation, and that removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity.IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the Escherichia coli-lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.

Distribution and diversity of glycocin biosynthesis gene clusters beyond Firmicutes.

Glycocins are the ribosomally synthesized glycosylated bacteriocins discovered and characterized in Firmicutes, only. These peptides have antimicrobial activity against several pathogenic bacteria, including Streptococcus pyogenes , methicillin-resistant Staphylococcus aureus and food-spoilage bacteria Listeria monocytogenes. Glycocins exhibit immunostimulatory properties and make a promising source of new antibiotics and food preservatives akin to Nisin. Biochemical studies of Sublancin, Glycocin F, Pallidocin and ASM1 prove that the nested disulfide-bonds are essential for their bioactivities. Using in silico approach of genome mining coupled with manual curation, here we identify 220 new putative glycocin biosynthesis gene clusters (PGBCs) spread across 153 bacterial species belonging to seven different bacterial phyla. Based on gene composition, we have grouped these PGBCs into five distinct conserved cluster Types I-V. All experimentally identified glycocins belong to Type I PGBCs. From protein sequence based phylograms, tanglegrams, global similarity heat-maps and cumulative mutual information analysis, it appears that glycocins may have originated from closely related bacteriocins, whereas recruitment of cognate glycosyltransferases (GTs) might be an independent event. Analysis further suggests that GTs may have coevolved with glycocins in cluster-specific manner to define distinctive donor specificities of GTs or to contribute to glycocin diversity across these clusters. We further identify 162 hitherto unreported PGBCs wherein the corresponding product glycocins have three or less than three cysteines. Secondary structure predictions suggest that these putative glycocins may not form di-nested disulfide-bonds. Therefore, production of such glycocins in heterologous host Escherichia coli is feasible and may provide novel antimicrobial spectrum and or mechanism of action for varied applications.

Compatibility, Cytotoxicity, and Gastrointestinal Tenacity of Bacteriocin-Producing Bacteria Selected for a Consortium Probiotic Formulation to Be Used in Livestock Feed.

Bacteriocin-producing Escherichia coli ICVB442, E. coli ICVB443, Enterococcus faecalis ICVB497, E. faecalis ICVB501, and Pediococcus pentosaceus ICVB491 strains were examined for their pathogenic risks and compatibility and hence suitability as consortium probiotic bacteria. Except for E. coli ICVB442, all were inclined to form biofilm. All were gelatinase-negative, sensitive to most of the antibiotics tested and not cytotoxic to porcine intestinal epithelial cells (IPEC-1) when tested at a multiplicity of infection (MOI) of 1. P. pentosaceus ICVB491 stood apart by inhibiting the other four strains. Both E. coli strains and E. faecalis ICVB497 strain were ß-hemolytic. Survival in the TIM-1 dynamic model of the human digestive system was 139% for the tested E. coli ICVB443 strain, 46% for P. pentosaceus ICVB491, and 32% for the preferred E. faecalis ICVB501 strain. These three potential probiotics, which are bacteriocin-producing strains, will be considered for simultaneous use as consortium with synergistic interactions in vivo on animal model.

Biodiversity and Phylogenetic Relationships of Novel Bacteriocinogenic Strains Isolated from Animal's Droppings at the Zoological Garden of Lille, France.

This study aimed at exploring droppings of animals living in captivity in the zoological garden (Zoo) of Lille (France), as novel sources of bacteriocinogenic strains. A collection of 295 bacterial isolates was constituted from droppings of capybara, alpaca, muntjac, zebra, tapir, rhinoceros, binturong, armadillo, saki monkey and cockatoo. Of 295 isolates, 51 exhibited antagonism against a panel of pathogenic target bacteria like Escherichia coli MC4100, Clostridium perfringens DSM 756 and Salmonella enterica subsp. enterica Newport ATCC6962. Remarkably, within this collection, only 2 Gram-negative bacilli exhibited activity against E. coli MC4100 strain used as target organism. Then, the 16S rDNA sequencing revealed these thereafter cited species, Pediococcus pentosaceus, Weissella cibaria, E. coli, Lactobacillus reuteri, Enterococcus hirae and Enterococcus faecalis. Characterization of this antagonism has revealed 11 strains able producing extracellular protease-sensitive inhibitory compounds. These strains included E. coli ICVB442 and ICVB443, Ent. faecalis ICVB472, ICVB474, ICVB477 ICVB479, ICVB481, ICVB497 and ICVB501 and Ped. pentosaceus ICVB491 and ICVB492. The genomes of the 5 most promising bacteriocinogenic strains were sequenced and analysed with Bagel4 software. Afterwards, this bioinformatics analysis permitted to locate genes encoding bacteriocins like colicin Y (E. coli), enterocin 1071A, enterocin 107 B (Ent. faecalis) and penocin A (Ped. pentosaceus), associating the above-mentioned antibacterial activity of proteinaceous nature to possible production of bacteriocins. All these results enabled us to select different bacteriocinogenic strains for a further characterization in terms of beneficial traits.

Heterologous Expression of the Leuconostoc Bacteriocin Leucocin C in Probiotic Yeast Saccharomyces boulardii.

The yeast Saccharomyces boulardii is well known for its probiotic effects such as treating or preventing gastrointestinal diseases. Due to its ability to survive in stomach and intestine, S. boulardii could be applied as a vehicle for producing and delivering bioactive substances of interest to human gut. In this study, we cloned the gene lecC encoding the antilisterial peptide leucocin C from lactic acid bacterium Leuconostoc carnosum in S. boulardii. The constructed S. boulardii strain secreted a peptide, which had molecular weight corresponding to leucocin C in SDS-PAGE. The peptide band inhibited Listeria monocytogenes in gel overlay assay. Likewise, concentrated S. boulardii culture supernatant inhibited the growth of L. monocytogenes. The growth profile and acid tolerance of the leucocin C secreting S. boulardii were similar as those of the strain carrying the empty vector. We further demonstrated that the cells of the leucocin C producing S. boulardii efficiently killed L. monocytogenes, also without antibiotic selection pressure. These results showed that antilisterial activity could be added to the arsenal of probiotic activities of S. boulardii, demonstrating its potential as a carrier for therapeutics delivery.

Non-antibiotic antibacterial peptides and proteins of Escherichia coli: efficacy and potency of bacteriocins.

INTRODUCTION: The emergence and spread of antibiotic resistance among pathogenic bacteria drives the search for alternative antimicrobial therapies. Bacteriocins represent a potential alternative to antibiotic treatment. In contrast to antibiotics, bacteriocins are peptides or proteins that have relatively narrow spectra of antibacterial activities and are produced by a wide range of bacterial species. Bacteriocins of Escherichia coli are historically classified as microcins and colicins, and, until now, more than 30 different bacteriocin types have been identified and characterized. AREAS COVERED: We performed bibliographical searches of online databases to review the literature regarding bacteriocins produced by E. coli with respect to their occurrence, bacteriocin role in bacterial colonization and pathogenicity, and application of their antimicrobial effect. EXPERT OPINION: The potential use of bacteriocins for applications in human and animal medicine and the food industry includes (i) the use of bacteriocin-producing probiotic strains, (ii) recombinant production in plants and application in food, and (iii) application of purified bacteriocins.

Stimulation of Bovicin HC5 Production and Selection of Improved Bacteriocin-Producing Streptococcus equinus HC5 Variants.

Bovicin HC5 is a peptide that has inhibitory activity against various pathogenic microorganisms and food spoilage bacteria. Aiming to improve the productivity of this bacteriocin, we evaluated several potential factors that could stimulate the synthesis of bovicin HC5 and selected variants of Streptococcus equinus (Streptococcus bovis) HC5 with enhanced bacteriocin production by adaptive laboratory evolution (ALE). The highest production of the bacteriocin (1.5-fold) was observed when Strep. equinus HC5 was cultivated with lactic acid (100 mmol/L). For the ALE experiment, Strep. equinus HC5 cells were subjected to acid-shock (pH 3.0 for 2 h) and maintained in continuous culture for approximately 140 generations (40 days) in media with lactic acid (100 mmol/L) and pH-controlled at 5.5 ± 0.2. An adapted variant was selected showing a distinct phenotype (sedimentation, pigmentation) compared with the parental strain. Bacteriocin production increased 2-fold in this adapted Strep. equinus HC5 variant, which appears to be associated with changes in the cell envelope of the adapted variant and enhanced bacteriocin release into the culture media. In addition, the adapted variant showed higher levels of expression of all bovicin HC5 biosynthetic genes compared with the parental strain during the early and late stages of growth. Results presented here indicate that ALE is a promising strategy for selecting strains of lactic acid bacteria with increased production of bacteriocins.

Engineering Artificial Biodiversity of Lantibiotics to Expand Chemical Space of DNA-Encoded Antibiotics.

The discovery of antibiotics was one of the fundamental stages in the development of humanity, leading to a dramatic increase in the life expectancy of millions of people all over the world. The uncontrolled use of antibiotics resulted in the selection of resistant strains of bacteria, limiting the effectiveness of antimicrobial therapy nowadays. Antimicrobial peptides (AMPs) were considered promising candidates for next-generation antibiotics for a long time. However, the practical application of AMPs is restricted by their low therapeutic indices, impaired pharmacokinetics, and pharmacodynamics, which is predetermined by their peptide structure. Nevertheless, the DNA-encoded nature of AMPs enables creating broad repertoires of artificial biodiversity of antibiotics, making them versatile templates for the directed evolution of antibiotic activity. Lantibiotics are a unique class of AMPs with an expanded chemical space. A variety of post-translational modifications, mechanisms of action on bacterial membranes, and DNA-encoded nature make them a convenient molecular template for creating highly representative libraries of antimicrobial compounds. Isolation of new drug candidates from this synthetic biodiversity is extremely attractive but requires high-throughput screening of antibiotic activity. The combination of synthetic biology and ultrahigh-throughput microfluidics allows implementing the concept of directed evolution of lantibiotics for accelerated creation of new promising drug candidates.

Metagenomics-based approach for studying and selecting bioprotective strains from the bacterial community of artisanal cheeses.

A metagenome-based approach was used to assess the taxonomic affiliation and functional potential for bacteriocin production of the bacterial community in cow's milk artisanal cheeses from Northwestern Argentina. Three different samples were analyzed by high-throughput sequencing of the V4 region of the 16S rRNA gene and shotgun metagenomics. Taxonomic analysis showed that cheese A and C were quite similar whereas cheese B displayed a rather different bacterial composition. Overall, two families, Streptococceae and Enterococceae, dominated the artisanal cheese microbiota, being the former family prevalent in cheese B and the later family the most important in samples A and C. Besides the usual species associated to cheeses, a number of bacterial taxa that have not been previously found in Argentinean artisanal cheeses were reported in the present work such as Macrococcus caseolyticus and Streptococcus macedonicus Functional metagenomics analysis using the bacteriocin mining software BAGEL3, identified 2 ORFs encoding antimicrobial peptides in cheese B and 42 different peptides in sample C. The bacteriocin genes found showed good correlation with taxonomy. Based on the microbial diversity and functional features found through shotgun metagenomic sequencing, a culture-dependent approach was applied aiming to isolate bacteriocin-producing bacteria able to inhibit the growth of the foodborne pathogen Listeria monocytogenes. From 151 bacterial colonies derived from the cheese samples, 10 were associated to high anti-Listeria activity. Based on partial 16S rRNA gene sequencing and RAPD-PCR analysis, all bacteriocinogenic isolates were identified as Enterococcus faecium. Finally, we carried out a pilot experiment with L. monocytogenes-contaminated cheese using one of the enterococcal isolates as a bioprotective adjunct culture. The use of E. faecium CRL1879 during artisanal cheese manufacturing did not alter the main organoleptic properties of the cheese and ensured an efficient control of the foodborne pathogen up to 30 days. This finding supports the use of E. faecium CRL1879 as an adjunct culture in the cheese-making process with a combination of both safety and minimal processing.