Leon M. T. Dicks

Characterization of pentocin TV35b, a bacteriocin‐like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans

Lactobacillus pentosus TV35b, isolated from the posterior fornix secretions of the vagina of a prenatal patient, produced a bacteriocin‐like peptide (pentocin TV35b), which is inhibitory to Clostridium sporogenes, Cl. tyrobutyricum, Lact. curvatus, Lact. fermentum, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp. and Candida albicans. The mechanism of activity of pentocin TV35b is bactericidal, as shown by a decrease in the viable cell numbers of Lact. sake from approximately 4 × 108 to less than 10 cfu ml−1 over a period of 4 h. Pentocin TV35b added to the growth medium of C. albicans stimulated the formation of pseudohyphae during the first 36 h, followed by a slight repression in cell growth. Production of pentocin TV35b was at its maximum towards the end of the logarithmic growth phase of strain TV35b. The peptide was purified by ammonium sulphate precipitation, followed by SP‐Sepharose cation exchange chromatography. The molecular size of pentocin TV35b was estimated to be between 2·35 and 3·4 kDa, according to tricine‐SDS PAGE. However, results obtained by electrospray ionization mass spectroscopy indicated that the peptide is 3930·2 Da in size. Amino acid analysis performed by using the Pico‐Tag® method and a Nova‐Pak C18 HPLC column indicated that pentocin TV35b consists of 33 amino acids with a total mass of 3929·63 Da. Pentocin TV35b is inactivated when treated with papain and Proteinase K, but remains active after incubation at pH 1–10 for 2 h at 25 °C, and when heat‐treated for 30 min at 100 °C.

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces.

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by α‐chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS‐PAGE, was approximately 3·4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.

Boza, a natural source of probiotic lactic acid bacteria

Aims:  To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals.

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

ABSTRACT The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A andent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type ofE. faecalis bacteriocin.

Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis

Elongation factor Tu (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are surface-bound proteins with a role in adhesion of Lactobacillus plantarum 423 to Caco-2 cells. Removal of surface-bound proteins from L. plantarum 423 (treated with 4M guanidine-HCl) reduced adhesion to Caco-2 cells by 40%. In a competitive exclusion experiment where all three strains were given an equal chance to adhere to Caco-2 cells, L. plantarum 423 prevented 71% of cells of Clostridium sporogenes LMG 13570 and 89% of cells of Enterococcus faecalis LMG 13566 from adhering. Cells of L. plantarum 423, from which surface-bound proteins were removed, prevented 49% of cells of C. sporogenes LMG 13570 and 70% of cells of E. faecalis LMG 13566 from adhering to Caco-2 cells, suggesting that factors other than surface-bound proteins are involved in adhesion. Colonization of L. plantarum 423 to Caco-2 cells prevented adhesion of 74% of cells of C. sporogenes LMG 13570 and 62% of cells of E. faecalis LMG 13566. Furthermore, L. plantarum 423 displaced 81% of cells of C. sporogenes LMG 13570 and 91% of cells of E. faecalis LMG 13566 from Caco-2 cells. L. plantarum 423 is a potential probiotic strain.

Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.

Taxonomy of Leuconostoc Species, Particularly Leuconostoc oenos, as Revealed by Numerical Analysis of Total Soluble Cell Protein Patterns, DNA Base Compositions, and DNA-DNA Hybridizations

The levels of genotypic relatedness among Leuconostoc oenos (53 strains), Leuconostoc mesenteroides subsp. mesenteroides (7 strains), Leuconostoc mesenteroides subsp. dextranicum (5 strains), Leuconostoc paramesenteroides (3 strains), Leuconostoc lactis (3 strains), Leuconostoc sp. (3 strains), and Pediococcus acidilactici (1 strain) were determined. L. oenos is genotypically homogeneous and forms a distinct species. Leuconostoc sp. strains ATCC 21435, ATCC 21436, and ATCC 21437 are genotypically not closely related to any Leuconostoc sp. L. paramesenteroides and L. lactis are regarded as genotypically heterogeneous collections of strains. P. acidilactici ATCC 12697 and L. mesenteroides NCDO 530 should be reclassified as L. paramesenteroides and L. oenos, respectively.

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.

The Development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end‐products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast‐based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1·0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA‐1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA‐1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA‐1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P‐MFα1S‐pedA‐ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1‐encoded pediocin PA‐1 was directed by the yeast mating pheromone α‐factors secretion signal (MFα1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4·6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria. Copyright

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii.

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.