Interactions between pairs of bacteriocins from lactic bacteria

Activity of pairs of crude extracts of lactic acid bacteria (LAB) containing different bacteriocins (nisin, pediocin AcH, lacticin 481, lactacin F, and lactacin B) was measured against 10 different indicator strains. Experiments were carried out both in liquid and on solid media. Both synergisms and antagonisms were observed. Lacticin 481 produced mainly antagonistic effects whereas pediocin AcH produced mainly synergistic effects. The use of more than one LAB bacteriocin as a combination biopreservative might be envisaged.     

Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content

Three out of 297 Lactobacillus strains isolated from pig faeces were selected for a feeding trial on account of their high bile-salt hydrolase (BSH) activity, bile-salt resistance, low pH tolerance and the production of antimicrobial substances. Two strains were identified as Lactobacillus johnsonii and one as Lactobacillus reuteri by DNA-DNA hybridisation. L. johnsoniii BFE 1061 produced a bacteriocin active against a range of lactic acid bacteria (LAB) and nonrelated bacteria including Clostridium perfringens. Six minipigs were maintained on a high-fat, high-cholesterol ('Western Style') diet for 17 weeks after which the diet was supplemented with the 'probiotic mixture' containing the above mentioned three Lactobacillus strains at 2 x 10(12) CFU per pig per day for five weeks. The mixture was given as a resuspended lyophilisate. During a two week follow-up period the minipigs received only the 'Western-style' diet without probiotic supplementation. A lowering effect on serum cholesterol levels was indicated after three weeks probiotic feeding, concomitant with an increase in the moisture content of the faeces and Lactobacillus cell numbers. Triglycerides, pH and number of lactic acid bacteria in faeces were not significantly influenced by probiotic supplementation.     

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin

A total of 663,533 colonies from 72 dairy and meat sources showed a detection rate of 0.2% for bacteriocin producers using direct plating techniques. A further 83,000 colonies from 40 fish and vegetable sources showed a detection rate of 3.4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus, Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.     

Cultured human ankle and knee cartilage differ in susceptibility to damage mediated by fibronectin fragments

A culture of the psychotrophic strain FloraCarn L-2 of Lactobacillus alimentarius was added to ground beef (pH 5.4) inoculated with two isolates of Listeria monocytogenes able to grow in refrigerated ground beef. The ground beef was vacuum-packaged and stored for 9 weeks at 4 degrees C. Populations of inoculated L. monocytogenes initially were 6.3 to 6.4 log10 CFU/g and increased to 7.4 log10 CFU/g in ground beef with no added lactobacilli. Addition of L. alimentarius L-2 or its antibiotic-resistant mutant SRL-2 reduced the final populations of L. monocytogenes to 4.3 or 4.1 log10 CFU/g, respectively. L. alimentarius L-2 did not produce bacteriocins or hydrogen peroxide in vitro. The antilisterial effect of L. alimentarius observed in laboratory media and ground beef is attributed to lactic acid (ca. 50 mM) produced by growing cultures.     

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.     

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.     

Sociality and the rate of rDNA sequence evolution in wasps (Vespidae) and honeybees (Apis)

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially clostridium difficile.     

Genetic analysis of the bacteriocin-encoding plasmids pRJ6 and pRJ9 of Staphylococcus aureus by transposon mutagenesis and cloning of genes involved in bacteriocin production

pRJ6 and pRJ9, small Staphylococcus aureus plasmids which code for bacteriocins, exhibited a bactericidal activity against several lactic acid bacteria and strains of Listeria monocytogenes, an important food-borne pathogen. Filter-mating experiments using plasmid derivatives tagged with either Tn551 or Tn917-lac showed that pRJ6, but not pRJ9, could be mobilized by staphylococcal conjugative plasmids. Transposon mutagenesis of both plasmids was also performed. The bacteriocin and immunity structural genes of pRJ6 are part of the same operon, which is located around co-ordinate 4.0, being transcribed from right to left. However, gene cloning experiments using a staphylococcal vector showed some evidence for the involvement of additional functions of pRJ6 in bacteriocin expression. One function involved in pRJ6 mobilization mapped around co-ordinate 5.2, and it appears to be transcribed from left to right. The bactericidal action exerted by strains harbouring pRJ9 appears to reflect the activity of at least two bacteriocins, whose combined action results in a broader spectrum of activity and in a higher antagonistic activity. Gene cloning experiments also supported these assumptions.     

Genetic characterization and heterologous expression of brochocin-C, an antibotulinal, two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754

Brochocin-C, produced by Brochothrix campestris ATCC 43754, is active against many strains of the closely related meat spoilage organism Brochothrix thermosphacta and a wide range of other gram-positive bacteria, including spores of Clostridium botulinum. Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins. They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively. Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded downstream of brcB functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream of brcI, designated brcT and brcD, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.     

Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A

Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by Lactobacillus plantarum C11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that L. plantarum C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 10(3) times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN.     

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum

Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 degrees C, but loses 50% of its activity after 60 min at 100 degrees C and 75% of its activity after autoclaving (121 degrees C, 15 min). Plantaricin 423 remains active after incubation at pH 1-10 and is inactivated when treated with pepsin, papain, alpha-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3.5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1.0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.     

Partial characterisation of pediocin PO2 and comparison with nisin for biopreservation of meat products

A plasmid associated bacteriocin (pediocin PO2) was isolated by ammonium sulphate precipitation from cell-free growth media and subsequent studies showed that the partially purified pediocin PO2 was most likely identical (molecular mass approximately 3200 daltons in size by SDS-PAGE, stable to low pH and heat at 121 degrees C for 15 min, inactivated by various proteolytic enzymes and resistant to treatment with a range of solvents, except 10% formaldehyde) to other pediocins (PA-1 and AcH) previously reported. The antagonistic spectrum of activity of pediocin PO2 was compared with nisin and showed a narrower host-range, but a much greater activity against Listeria species including strains of Listeria monocytogences, than did nisin. A rapid method of reflectance colorimetry was used to quantitate growth and acid production (as determined by the colour change in bromcresol purple) of Lactobacillus curvatus, added to a meat product model system. The combined effects of refrigeration temperature, microbial load and bacteriocin concentration were determined in the model over 15 days storage. Both nisin and pediocin demonstrated inhibitory activity against Lactobacillus curvatus in the model system. However, when bacteriocins were incorporated into a manufactured cooked meat product only low nisin activity and no pediocin activity was detected, after challenge of vacuum packaged slices of product with Lactobacillus curvatus, over a 21 day storage trial under refrigeration temperatures.     

Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained

P1 lysogens of Escherichia coli carry the prophage as a stable low copy number plasmid. The frequency with which viable cells cured of prophage are produced is about 10(-5) per cell per generation. Here we show that a significant part of this remarkable stability can be attributed to a plasmid-encoded mechanism that causes death of cells that have lost P1. In other words, the lysogenic cells appear to be addicted to the presence of the prophage. The plasmid withdrawal response depends on a gene named doc (death on curing), encoding a 126 amino acid protein. Expression of doc is not SOS-inducing and killing by Doc is recA-independent. In cells that retain P1 the killing is prevented by the product of a gene named phd (prevent host death), encoding a 73 amino acid protein. The genes phd and doc have been cloned and expressed from a 0.7 kb segment of P1 DNA. The two genes constitute an operon and the synthesis of Doc appears to be translationally coupled to that of Phd. Homologs of the P1 addiction genes are found elsewhere, but phd and doc are unrelated to previously described genes of other plasmids that also cause an apparent increase in plasmid stability by post-segregational killing.     

Bacteriocins: nature, function and structure

Bacteriocins are extracellular substances produced by different types of bacteria, including both Gram positive and Gram negative species. They can be produced spontaneously or induced by certain chemicals such as mitomycin C. They are biologically one of the important substances, and have been found to be useful in membrane studies and also in typing pathogenic microorganisms causing serious nosocomial infections. Bacteriocins are a heterogeneous group of particles with different morphological and biochemical entities. They range from a simple protein to a high molecular weight complex: the active moiety of each molecule in all cases seems to be protein in nature. The genetic determinants of most of the bacteriocins are located on the plasmids, apart from few which are chromosomally encoded. These bactericidal particles are species specific. They exert their lethal activity through adsorption to specific receptors located on the external surface of sensitive bacteria, followed by metabolic, biological and morphological changes resulting in the killing of such bacteria. This review summarises the classification, biochemical nature, morphology and mode of action of bacteriocins as well as their genetic determinants and the microbiological relevance of these bactericidal agents.     

The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA.