A comparative study of three bacteriocins of Bacteroides fragilis

Three different bacteriocins produced by strains of Bacteroides fragilis were compared in terms of their production kinetics, physico-chemical nature, and action on macromolecular synthesis in a common indicator strain. Bacteriocin 78/438 was produced during the logarithmic growth phase, was thermolabile and stable between pH 5 and 9. It was susceptible to trypsin and pepsin, and affected DNA, RNA and protein syntheses in susceptible cells. Bacteriocin A49 was produced during the stationary growth phase, was thermolabile and stable between pH 7 and 9. This bacteriocin was also susceptible to trypsin and pepsin, but only RNA synthesis was affected in the indicator strain. Bacteriocin A55 differed markedly from both 78/438 and A49, and was found to be predominantly cell-bound, resistant to inactivation by high temperatures and stable over a wide pH range of 2 to 12. It was susceptible to trypsin but resistant to pepsin. A55 had a delayed effect on macromolecular synthesis with DNA synthesis being inhibited after 60 min. With all three bacteriocins, killing of the indicator strain followed single hit kinetics with the interaction of bacteriocin and target cell occurring in two stages. Killing by bacteriocin A55 was much slower than the other two and this may be related to its effect on macromolecular synthesis. The killing action of all three bacteriocins was dependent on the growth phase of the susceptible cells.     

Molecular basis of bacteriocins production and activity

Bacteriocins--bacterial proteins or peptides--are envisaged as candidates for the next generation of effective antimicrobials. Analysis of characteristics of natural and genetically engineered bacteriocins with regard to the molecular basis of their production and activity has been performed. Most bacteriocins have narrow spectrum of the inhibitory activity. Some of the broad-spectrum bacteriocins have circular molecular structure (C- and N-terminals of the aminoacid chain are joined by a peptide sequence). Fixed position of molecules' ends possibly accounts for the ability of the proteins to bind with various receptors on the surface of the target cells. Genes encoding bacteriocins and functionally associated proteins can be expressed in heterologous cells including eukaryotic cells. Also there were reports of changing bacteriocin characteristics by the use of site-specific mutagenesis.     

Microbial production of bacteriocins: Latest research development and applications

Bacteriocins are low molecular weight peptides secreted by the predator bacterial cells to kill sensitive cells present in the same ecosystem competing for food and other nutrients. Exceptionally few bacteriocins along with their native antibacterial property also exhibit additional anti-viral and anti-fungal properties. Bacteriocins are generally produced by Gm+, Gm– and archaea bacteria. Bacteriocins from Gm?+?bacteria especially from lactic acid bacteria (LAB) have been thoroughly investigated considering their great biosafety and broad industrial applications. LAB expressing bacteriocins were isolated from fermented milk and milk products, rumen of animals and soil using deferred antagonism assay. Nisin is the only bacteriocin that has got FDA approval for application as a food preservative, which is produced by Lactococcus lactis subsp. Lactis. Its crystal structure explains that its antimicrobial properties are due to the binding of NH₂ terminal to lipid II molecule inhibiting the peptidoglycan synthesis and carboxy terminal forming pores in bacterial cell membrane leading to cell lysis. The hinge region connecting NH₂ and carboxy terminus has been mutated to generate mutant variants with higher antimicrobial activity. In a 50 ton fermentation of the mutant strain 3807 derived from L. lactis subsp. lactis ATCC 11454, 9,960?IU/mL of nisin was produced. Currently, high purity of nisin (>99%) is very expensive and hardly commercially available. Development of more advanced tools for cost-effective separation and purification of nisin would be commercially attractive. Chemical synthesis and heterologous expression of bacteriocins ended in low yields of pure proteins. At present, bacteriocins are almost solely applied in food industries, but they have a great potential to be used in other fields such as feeds, organic fertilizers, environmental protection and personal care products. The future of bacteriocins is largely dependent on getting FDA approval for use of other bacteriocins in addition to nisin to promote the research and applications.     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

Pediocins: The bacteriocins of Pediococci. Sources, production, properties and applications

Class IIa bacteriocins from lactic acid bacteria are small, cationic proteins with antilisterial activity. Within this class, the pediocins are those bacteriocins that share a highly conserved hydrophilic and charged N-terminal part harboring the consensus sequence -YGNGV- and a more variable hydrophobic and/or amphiphilic C-terminal part. Several pediocins have been isolated and characterized. Despite the structural similarities, their molecular weight varies, as well as their spectrum of antimicrobial activity. They exhibit important technological properties, e.g. thermostability and retaining of activity at a wide pH range, which along with the bactericidal action against Gram-positive food spoilage and pathogenic bacteria, make them an important class of biopreservatives. Much new information regarding the pediocins has emerged during the last years. In this review, we summarize and discuss all the available information regarding the sources of pediocins, the characteristics of their biosynthesis and production in fermentation systems, the characteristics of the known pediocin molecules, and their antibacterial action. The advances made by genetic engineering in improving the features of pediocins are also discussed, as well as their perspectives for future applications.     

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.     

Hollow fibre bioreactor for ethanol production: Application to the conversion of lactose by Kluyveromyces fragilis

Kluyveromyces fragilis cells have been packed into the shell side of an industrial size hollow fibre module. The feed was pumped through the tube side under pressure. During continuous, single-pass operation with a synthetic lactose medium containing 50 g l^?1lactose, ethanol productivity was 30–60 g l^?1h^?1at dilution rates of 1–4 h^?1. With 150 g l^?1lactose concentration, the productivity was 100–135 g l^?1h^?1. Productivity was generally lower when cottage cheese whey permeate (45 g l^?1lactose) was used as the feed. Long-term stability of the hollow fibre bioreactor was good, provided adequate care was taken to bleed the gas generated and restrict cell concentration in the shell side.     

Bacteriocin production and sensitivity

Using an overlay test the production of bacteriocin-like activity and resistance was found in 6 of the total of 7 isolates (5 enterococcal and 2 streptococcal). The majority of strains were sensitive to all bacteriocin producers tested. After acetone precipitation, bacteriocin precipitates were tested for thermal stability. They exhibited high stability at 37 degrees C and some of them were active even after a treatment at 95 degrees C.     

Simultaneous ethanol production from lactose byKluyveromyces fragilis andZymomonas mobilis

Ethanol production from lactose byKluyveromyces fragilis NRRL 665 in monoculture and coculture with strains ofZymomonas mobilis was studied. One of the strains,Z. mobilis NRRL 1960, when cocultured withK. fragilis, produed 55.2 g/l of ethanol, whereasK. fragilis in monoculture procuded only 36 g/l ethanol from 200 g/l lactose medium. Increased Qp (g ethanol produced/g biomass/h) and Qs (g substrate consumed/g biomass/h) were observed in coculture than in monoculture. However, the residual sugar concentration increased in coculture; this increase might be due to the slow utilization rate of galactose.     

Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126

Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.     

Succinic acid production from Bacteroides fragilis: process optimization and scale up in a bioreactor

We report the effect of different physiological and nutritional parameters on succinic acid production from Bacteroides fragilis. This strain initially produced 0.70gL(-1) of succinic acid in 60h. However, when process optimization was employed, 5.4gL(-1) of succinic acid was produced in medium consisting of glucose (1.5%); tryptone (2.5%); Na(2)CO(3) (1.5%), at pH 7.0, when inoculated with 4% inoculum and incubated at 37 degrees C, 100rpm for 48h. A marked enhancement in succinic acid production was observed when the optimized conditions were employed in a 10L bioreactor. A total of 12.5gL(-1) of succinic acid was produced in 30h. This is approximately 12-fold increase in succinic acid production when compared to the initial un-optimized medium production. This enhancement in succinic acid production may be due to the control of CO(2) supply and the impeller speed. This is also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Trends in utilization of agro-industrial byproducts for production of bacteriocins and their biopreservative applications

Bacteriocins are proteinaceous, ribosomally synthesized bio-molecules having major roles in food preservation due to their antimicrobial action against food spoilage microorganisms. These have gained importance in the last decades because of increasing interest in natural products and their applications in the field of biopreservation, pharmaceutical, aquaculture, livestock, etc. Their production is quite expensive which includes the cost of synthetic media and downstream processing of which 30% of the total production cost relies on synthetic media and nutritional supplements used for growth of microorganisms. The low cost agro-industrial by-products, rich in nutritional supplements, can act as a good substitute for high valued synthetic media. This review provides comprehensive information on the use of cost effective, renewable agro-industrial by-products as substrates for the production of bacteriocins and their application in food as biopreservatives.     

Spermicidal bacteriocins: lacticin 3147 and subtilosin A

Spermicidal compounds that also exhibit antimicrobial properties would be extremely attractive agents as they could be used to not only prevent unwanted pregnancy but also to combat the growing prevalence of sexually transmitted infections (STI). One class of compounds that are potential candidates for development of dual-acting contraceptive products are antimicrobial peptides (AMPs). Herein, we report preliminary studies carried out to investigate the spermicidal activity of two bacteriocins, lacticin 3147 and subtilosin A, on bovine, horse/pony, boar and rat sperm.     

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor

BackgroundThe scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria.MethodsL. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches.ResultsThe mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain.ConclusionsOur results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins.General significanceThis is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.