A large scale prediction of bacteriocin gene blocks suggests a wide functional spectrum for bacteriocins

Bacteriocins are peptide-derived molecules produced by bacteria, whose recently-discovered functions include virulence factors and signalling molecules as well as their better known roles as antibiotics. To date, close to five hundred bacteriocins have been identified and classified. Recent discoveries have shown that bacteriocins are highly diverse and widely distributed among bacterial species. Given the heterogeneity of bacteriocin compounds, many tools struggle with identifying novel bacteriocins due to their vast sequence and structural diversity. Many bacteriocins undergo post-translational processing or modifications necessary for the biosynthesis of the final mature form. Enzymatic modification of bacteriocins as well as their export is achieved by proteins whose genes are often located in a discrete gene cluster proximal to the bacteriocin precursor gene, referred to as \textit{context genes} in this study. Although bacteriocins themselves are structurally diverse, context genes have been shown to be largely conserved across unrelated species. Using this knowledge, we set out to identify new candidates for context genes which may clarify how bacteriocins are synthesized, and identify new candidates for bacteriocins that bear no sequence similarity to known toxins. To achieve these goals, we have developed a software tool, Bacteriocin Operon and gene block Associator (BOA) that can identify homologous bacteriocin associated gene clusters and predict novel ones. We discover that several phyla have a strong preference for bactericon genes, suggesting distinct functions for this group of molecules. Availability: https://github.com/idoerg/BOAComment: Accepted for publication in BMC Bioinformatic

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

peer-reviewedThe bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers

Use of Carnobacterium piscicola to limit the growth of Listeria monocytogenes in mussel products : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Bacteria were screened in order to find an organism antagonistic to Listeria monocytogenes which could be applied to mussel products and enhance their safety, especially when temperature-abused. A Listeria monocytogenes isolate from the seafood industry was selected as the target organism. Strains of Lactobacillus reuteri and Enterococcus fecium were screened on plates incubated at 35°C and 10°C for anti-listerial compounds, but none were found. A non-bacteriocinogenic strain of Carnobacterium piscicola, A9b- was selected as the antagonist for detailed examination of growth in broth, agar and mussel systems at 10°C. This temperature was chosen to represent temperature abuse of refrigerated products. To distinguish between the growth of the Carnobacterium piscicola strain and wild-type Listeria monocytogenes a "semi-selective" agar was developed using phenol-red indicator, and mannitol as the sole carbohydrate source. Growth rates of Carnobacterium piscicola and Listeria monocytogenes were compared when grown alone and as a co-culture in agar and broth. Growth rates of Listeria monocytogenes when grown alone, and in the presence of Carnobacterium piscicola, were determined on mussels. Regression analyses were done for the inhibition of Listeria monocytogenes by Carnobacterium piscicola. In all cases Carnobacterium piscicola significantly inhibited the growth of Listeria monocytogenes (P broth = 0.018, P agar <0.001, P mussels < 0.001). Growth of both organisms was faster in broth, than on mussels or agar. The greatest inhibition of Listeria monocytogenes was observed in broth reaching log₁₀4.8 at 41 hours of incubation, prior to decreasing after this time. In agar and mussels the inhibition lasted longer and had not decreased at the end of the trial. The log₁₀ reduction in growth of Listeria monocytogenes in agar was measured at 3.4 and in mussels measured at 1.6. These results were statistically significant (P<0.001 for all). Inhibition of wild type Listeria monocytogenes was also shown in broth when a much lower concentration of Carnobacterium piscicola was used. These results should be considered as preliminary and further confirmatory work should be done. However, Carnobacterium piscicola A9b- shows promise as an antagonistic organism to assist in the control of Listeria monocytogenes in mussel products along with industry-accepted good hygienic practices

Antimicrobial antagonists against food pathogens; a bacteriocin perspective

peer-reviewedEfforts are continuing to find novel bacteriocins with enhanced specificity and potency. Traditional plating techniques are still being used for bacteriocin screening studies, however, the availability of ever more bacterial genome sequences and the use of in silico gene mining tools have revealed novel bacteriocin gene clusters that would otherwise have been overlooked. Furthermore, synthetic biology and bioengineering-based approaches are allowing scientists to harness existing and novel bacteriocin gene clusters through expression in different hosts and by enhancing functionalities. The same principles apply to bacteriocin producing probiotic cultures and their application to control pathogens in the gut. We can expect that the recent developments on bacteriocins from Lactic Acid Bacteria (LAB) described here will contribute greatly to increased commercialisation of bacteriocins in food systems.This work was funded by the Alimentary Pharmabiotic Centre, a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI (grant no. 12/RC/2273

Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?

peer-reviewedBacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings.KE, DF, CH, PC, MR, RR are supported by the Irish Government under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries and Food, Ireland (DAFM 13/F/462) to PC and MR, a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA 14/TIDA/2286) to DF, SFI-PI funding (11/PI/1137) to PDC and the APC Microbiome Insitute under Grant Number SFI/12/RC/2273

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;Pectobacterium atrosepticum&lt;/i&gt; with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that &lt;i&gt;Pectobacterium spp.&lt;/i&gt; carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;atrosepticum&lt;/i&gt; that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells

Immobilization of Bacteriocins from Lactic Acid Bacteria and Possibilities for Application in Food Biopreservation

Bacteriocins are biologically active compounds produced by a large number of bacteria, including lactic acid bacteria (LAB), which exhibit antimicrobial activity against various saprophytic and pathogenic microorganisms. In recent decades, bacteriocins are increasingly becoming more important in different branches of the industry due to their broad antibacterial and antifungal spectrum - in the food industry for natural food preservation and expiry date extension; in the health sector for preparation of probiotic foods and beverages; in the clinical practice as alternatives of conventional antibiotics; in the agriculture as biocontrol agents of plant pathogens and alternatives of chemical pesticides for plant protection. The broad antimicrobial spectrum of bacteriocins has stimulated the research attention on their application mainly in the food industry as natural preservatives. Most scientific achievements concerning the application food biopreservation are related to bacteriocins produced by LAB. The lactic acid bacteria bacteriocins can be produced in the food substrate during its natural fermentation or can be added in the food products after obtaining by in vitro fermentations under optimal physical and chemical conditions. Moreover, the immobilization of LAB bacteriocins on different matrices of organic and inorganic origin has been proposed as an advanced approach in the natural food preservation for their specific antimicrobial activity, anti-biofilm properties and potential use as tools for pathogen detection

Bioengineering Lantibiotics for Therapeutic Success

peer-reviewedSeveral examples of highly modified antimicrobial peptides have been described. While many such peptides are non-ribosomally synthesized, ribosomally synthesized equivalents are being discovered with increased frequency. Of the latter group, the lantibiotics continue to attract most attention. In the present review, we discuss the implementation of in vivo and in vitro engineering systems to alter, and even enhance, the antimicrobial activity, antibacterial spectrum and physico-chemical properties, including heat stability, solubility, diffusion and protease resistance, of these compounds. Additionally, we discuss the potential applications of these lantibiotics for use as therapeutics.DF,CH,PC,RR are supported by the Irish Government under the National Development Plan, through a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA14/TIDA/2286) to DF, a SFI Investigator awards to CH and RR (10/IN.1/B3027),SFI-PIfunding(11/PI/1137) to PDC and the Alimentary Pharmabiotic Centre under Grant Number SFI/12/RC/2273

The role of the N-acetylglucosamine phosphoenolpyruvate phosphotransferase system from Lactobacillus plantarum 8014 in the mechanism of action of glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand

The rise in antibiotic-resistant bacteria is becoming a severe public health problem because of the shortage of new antibiotics to combat existing resistant bacterial pathogens. Should this trend of increasing bacterial drug resistance continue, the previously treatable conditions may once again become fatal. Using broad-spectrum antibiotics causes collateral damage to the commensal microbiota of the host leading to complications and a greater susceptibility to opportunistic pathogenic infection. As a result, narrow spectrum antibacterials effective against specific pathogens, are becoming increasingly sought after. Among the many alternative classes of narrow-spectrum antibiotics, is a diverse group of ribosomally-synthesised antimicrobial peptides known as bacteriocins. Glycocin F (GccF), a rare and uniquely diglycosylated bacteriocin produced by Lactobacillus plantarum KW80, appears to target a specific N-acetylglucosamine (GlcNAc) phosphotransferase system (PTS) and causes almost instant bacteriostasis by an as yet unknown mechanism. This thesis demonstrates how the GlcNAc-PTS is involved in the GccF mechanism of action and that the gccH gene provides immunity to GccF. Using transgenic and gene editing techniques, regions of the GlcNAc-PTS were either removed or altered to prevent normal function before being tested in vivo. The results demonstrated that only the EIIC domain of the GlcNAc-PTS is required in the GccF mechanism of action and that it acts like a "lure" that attracts the bacteriocin to the main target that is as yet unknown. Furthermore, the immunity gene was discovered, and using PTS knockout cell lines the immunity mechanism was shown to act independently of the GlcNAc-PTS. This work will form the foundation for the work needed to unravel the bacteriostatic mechanism of action of GccF, which may lead to the development a novel antimicrobial agent

Biopreservation of Fresh Strawberries by Carboxymethyl Cellulose Edible Coatings Enriched with a Bacteriocin of Bacillus methylotrophicus BM47

Bacteriocins are a large group of antimicrobial compoundsthat aresynthesized byrepresentatives ofthegenus Bacillusand lactic acid bacteria (LAB). Bacteriocinsare used extensively in thefood industry as biopreservatives. Incorporatedin the composition of edible coatings, bacteriocins canreduce microbial growthand decay incidencein perishable fruits, thusimproving productshelf-life and commercial appearance. The present study aims to investigatethe effect of edible coatings of0.5% carboxymethyl cellulose (CMC) enriched with a purified bacteriocin fromBacillus methylotrophicusBM47 on the shelf-life extension of fresh strawberries. During storage at 4\ub0Cand 75 % relative humidity(RH) for 16 days, measurements of massloss, decay percentage, total soluble solids(TSS), titratable acidity(TA),pH, organic acids, total phenolic and anthocyaninscontents and antioxidant activity were taken.The results demonstrated that the application of 0.5% CMC and 0.5% CMC+bacteriocin (CMC+B) edible coatings led to a significant decrease of massloss in treated strawberriescompared to the uncoated fruit. After the 8-th day of storage, significant reductions in decay percentage along with the absence of fungal growth in CMC+B-coated fruit wereobserved in comparison to the CMC-coated and control strawberries. During the second half of the storage period, CMC and CMC+B treatmentsreduced TSS levels inthe coated fruitcompared to the control, but did not affectincreasedTA and loweredpH valuesthat arenormally associated with post-harvest changes. The CMC and CMC+B coatings wereineffectiveagainst thedecrease inascorbic acid, total phenolics and anthocyanins contentduringcold storage. The application of CMC and CMC+B coatingsexhibited a significant inhibitory effect on decreasing antioxidant activity throughout the storage period and maintained the antioxidant levels inboth treatmentsclose to the initial value of 76.8 mmol TE/100 g of fm