Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH₂-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.     

Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX

Weissella paramesenteroides DX has been shown to produce a 4450-Da class IIa bacteriocin, weissellin A, composed of 43 amino acids with the sequence KNYGNGVYCNKHKCSVDWATFSANIANNSVAMAGLTGGNAGN. The bacteriocin shares 68% similarity with leucocin C from Leuconostoc mesenteroides. Computational analyses predict that the bacteriocin is a hydrophobic molecule with a beta-sheet type conformation. Weissellin A exhibited various levels of activity against all gram-positive bacteria tested, but was not active against Salmonella enterica Enteritidis. The antimicrobial activity was not associated with target-cell lysis. The bacteriocin retained activity after exposure to 121 °C for 60 min or to −20 °C for 6 months, and to pH 2.0-10.0. It was not sensitive to trypsin, α-chymotrypsin, pepsin and papain, but was inactivated by proteinase K. At a dissolved oxygen concentration of 50%, weissellin A was produced with growth-associated kinetics. The properties of weissellin A make this bacteriocin a potentially suitable agent for food and feed preservation.     

Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3

The structure of enterocin NKR-5-3C, an anti-listerial bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, was determined. Enterocin NKR-5-3C is a novel class IIa bacteriocin that possesses an YGNGL motif sequence and two disulfide bridges in its structure. It is encoded on gene ent53C together with an 18-amino-acid-residue double glycine leader peptide.     

一种新型IIa类细菌素的克隆表达和活性鉴定

NB-C1为一种潜在的IIa类细菌素基因,为实现其在大肠杆菌中的高效可溶表达,首先构建了NB-C1蛋白与绿色荧光蛋白 (GFP) 的融合表达载体pIVEX 2.4d-GFP-NB-C1,然后将构建的表达载体转化大肠杆菌BL21(DE3) pLysS,经诱导表达后,重组蛋白GFP-NB-C1以可溶的形式存在于细胞内。经Ni-NTA亲和层析柱分离纯化后,重组融合蛋白的纯度大于95％,产量达36.1 mg/L。抑菌试验表明,纯化后的重组蛋白对单核细胞增生李斯特氏菌具有明显的抑制作用。     

Characterization of a bacteriocin produced by Streptococcus thermophilus ST134

A pH-dependent adsorption/desorption technique was used to screen Streptococcus thermophilus strains for the production of bacteriocins. Agar-diffusion tests with S. thermophilus strains as targets identified 13 out of 41 strains as producers of antibacterial activity. Thermophilin A, the bacteriocin-like substance present in the culture supernatant of S.thermophilus ST134 was purified to homogeneity by ammonium sulfate precipitation and ion-exchange chromatography, followed by ultrafiltration. Thermophilin A is a relatively heat-stable and apparently glycosylated bacteriocin with a bactericidal mode of action against sensitive cells.     

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.     

Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

Mechanism of action of lactostrepcin 5, a bacteriocin produced by Streptococcus cremoris 202

The mechanism of bactericidal activity of lactostrepcin 5 (Las 5), a bacteriocin produced by Streptococcus cremoris 202, was investigated. Las 5 did not kill protoplasts of sensitive cells, and its activity was decreased about 10-fold after pretreatment of the cells with trypsin, suggesting the involvement of the cell wall in the activity of this bacteriocin. In susceptible cells, the bacteriocin slowed down and then stopped synthesis of DNA, RNA, and protein, although this did not appear to be the primary effect of Las 5 action. Las 5 also inhibited uridine transport in susceptible cells and induced leakage of K+ ions and ATP. Survival of cells treated with Las 5 in phosphate buffer was higher in the presence of K+, CA2+, or Mg2+ ions.     

Action of divergicin M35, a class IIa bacteriocin, on liposomes and Listeria

AIMS: The mode of action of divergicin M35, a class IIa bacteriocin, was studied against Listeria monocytogenes with sensitive (DivS) and resistant (DivM) phenotypes, as well as on synthetic phospholipid liposomes. METHODS AND RESULTS: Divergicin-induced release of 1,6-diphenyl-1,3,5-hexatriene (DPH) from zwitterionic (DMPC) and anionic (DMPC/DMPG, 4:1) liposomes, divergicin binding to liposomes, intracellular ATP concentration, cation efflux, cell affinity for hydrocarbons and cell lysis were measured and cell damage was visualized by fluorescence imaging and transmission electron microscopy. Divergicin M35 at 5 microg ml(-1) induced DPH efflux from anionic and zwitterionic liposomes at rates of about 2.58% and 1.61% per minute, respectively. DPH efflux rate from anionic liposomes was reduced by about 1.83% and 2.1% per minute in the presence of Li+ and Ca2+, respectively. Binding affinity of divergicin M35 to anionic and zwitterionic liposomes was about 86% and 63%, respectively. Intracellular ATP decreased in the sensitive and the resistant strains by 96.7% and 72.8%, respectively after 20 min of exposure to 5 microg ml(-1) divergicin M35. Lysis of the sensitive strain reached 57% in 18 h at a concentration of 5 microg ml(-1) when compared with the lysis of the divergicin-resistant strain (38.8%). The K+ and Na+ efflux from the divergicin-sensitive strain reached 87% and 80% of the total ion content within 5 min of exposure. This strain also showed higher affinity for hydrocarbons. CONCLUSIONS: The cell death of listerial strains upon addition of divergicin M35 could result from ATP depletion, K+ and Na+ efflux, and bacteriolysis. This triple biological effect was attenuated in the DivM strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to the understanding of the mode of action of divergicin M35, a pediocin-like bacteriocin.     

Mechanism of action of lactostrepcin 5, a bacteriocin produced by Streptococcus cremoris 202.

The mechanism of bactericidal activity of lactostrepcin 5 (Las 5), a bacteriocin produced by Streptococcus cremoris 202, was investigated. Las 5 did not kill protoplasts of sensitive cells, and its activity was decreased about 10-fold after pretreatment of the cells with trypsin, suggesting the involvement of the cell wall in the activity of this bacteriocin. In susceptible cells, the bacteriocin slowed down and then stopped synthesis of DNA, RNA, and protein, although this did not appear to be the primary effect of Las 5 action. Las 5 also inhibited uridine transport in susceptible cells and induced leakage of K+ ions and ATP. Survival of cells treated with Las 5 in phosphate buffer was higher in the presence of K+, CA2+, or Mg2+ ions.     

Purification and characterization of thermophilin T, a novel bacteriocin produced by Streptococcus thermophilus ACA-DC 0040

ACA-DC 0040 produced an antimicrobial agent, which was named thermophilin T, active against several lactic acid bacteria strains of different species and food spoilage bacteria, such as Clostridium sporogenes C22/10 and Cl. tyrobutyricum NCDO-1754. The crude antimicrobial compound is sensitive to proteolytic enzymes and α-amylase, heat-stable (100 °C for 30 min), resistant to pH exposure at pH 1–12 and demonstrates a bactericidal mode of action against the sensitive strain Lactococcus cremoris CNRZ-117. The production of bacteriocin was optimized approximately 10-fold in an aerobic fermenter held at constant pH 5·8 and 6·2. Ultrafiltration experiments with culture supernatant fluids containing the bacteriocin, and further estimation of molecular weight with gel filtration chromatography, revealed that bacteriocin in the native form has a molecular weight in excess of 300 kDa. SDS-gel electrophoresis of partially purified thermophilin T showed that bacteriocin activity was associated with a protein band of approximately 2·5 kDa molecular mass.     

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.     

Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus

Enterococcus faecium T8, isolated from vaginal secretions of children with human immunodeficiency virus, produces a class IIa sec-dependent bacteriocin that is structurally different from three other class IIa sec-dependent bacteriocins, i.e., enterocin P and an enterocin P-like bacteriocin, produced by Enterococcus faecium, and bacteriocin 31, produced by Enterococcus faecalis, and from a class III bacteriocin produced by E. faecalis. The genes encoding the bacteriocin, immunity protein, mobilization protein, and relaxase nuclease are located on a 7-kb plasmid. Bacteriocin T8 has a molecular mass of 5.1 kDa based on its DNA sequence, similar to the 5.0 kDa recorded for bacteriocin 31 but larger than the 4.6 kDa reported for enterocin P. At the amino acid level, bacteriocin T8 is 69% homologous to bacteriocin 31 and 47% homologous to enterocin P. Bacteriocin T8 is active against E. faecalis isolated from patients diagnosed with vaginosis, against Lactobacillus sakei, and against a Propionibacterium sp. The peptide is heat stable (60 min at 100 degrees C) and remains active in phosphate buffer from pH 4.0 to 10.0. The mode of activity is bactericidal, as determined with E. faecalis.     

A counterselection method for Lactococcus lactis genome editing based on class IIa bacteriocin sensitivity

Applied Microbiology and Biotechnology - In this paper, we present a new counterselection method for deleting fragments from Lactococcus lactis chromosome. The method uses a non-replicating plasmid...     

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.     

Heterologous expression and purification of active divercin V41, a class IIa bacteriocin encoded by a synthetic gene in Escherichia coli

Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 microg per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm.     

Molecular dynamics simulation study of interaction between a class IIa bacteriocin and its immunity protein

Molecular dynamics (MD) simulations of carnobacteriocin B2 (CbnB2), a structurally well-characterized class IIa bacteriocin, and its immunity protein (ImB2) in lipid bilayer environment have been conducted to explore the interaction between them. Six 30-ns simulations were conducted in DPPC or POPG bilayer systems. In these simulations, ImB2 was placed in the aqueous layer with different orientations facing CbnB2 to sample all the faces of ImB2. The MD results indicate that (i) while CbnB2 remained embedded in the bilayer, it tends to move toward the interface, and (ii) the presence of CbnB2 in the DPPC bilayer attracts ImB2 toward the bilayer. In one of the orientations in DPPC bilayer system (simulation 1), ImB2 penetrates the bilayer and interacts with CbnB2 by ion-pair interaction. At several instances toward the later half of the simulation (15-30 ns), ImB2 and CbnB2 were found to form salt-bridge between Arg95 of ImB2 and Glu24 of CbnB2. Simulation in POPG bilayer displayed strong interaction between the positively charged ImB2 and the negatively charged polar head groups of the POPG molecules at the lipid-water interface. However, ImB2 was not able to penetrate the bilayer thereby preventing any interaction between ImB2 and CbnB2.     

Activation of bovine plasminogen by Streptococcus uberis

Abstract Thiosulfate and tetrathionate oxidation activity of Thiobacillus ferrooxidans were found to be absent in iron-growth cell as well as in the cells grown anaerobically on elemental sulfur. While the thiosulfate oxidase activity was absent in the cell-free extract of the above cells, the activity of rhodanese was present irrespective of the culture condition of T. ferrooxidans . It is thus conceivable that rhodanese is not involved in thiosulfate metabolism. During growth in presence of ferrous sulfate plus elemental sulfur, the thiosulfate/tetrathionate oxidation activity was absent till the oxidation of ferrous iron was complete and the cells harvested only in the latter period acquired the thiosulfate/tetrathionate oxidation activity. Thus it becomes evident that the inhibition of thiosulfate and tetrathionate oxidation is solely due to presence of ferrous iron.     

Acidophilucin A, a new heat-labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060

Lactobacillus acidophilus LAPT 1060, isolated from infant faeces, produced an antimicrobial agent active against six strains of Lactobacillus delbrueckii subsp. bulgaricus and six strains of Lactobacillus helveticus . The agent was sensitive to proteolytic enzymes and heat (10 min at 60°C) and is a bacteriocin and designated acidophilucin A.