Nucleotide sequence and taxonomical distribution of the bacteriocin gene lin cloned from Brevibacterium linens M18.

Linocin M18 is an antilisterial bacteriocin produced by the red smear cheese bacterium Brevibacterium linens M18. Oligonucleotide probes based on the N-terminal amino acid sequence were used to locate its single copy gene, lin, on the chromosomal DNA. The amino acid composition, N-terminal sequence, and molecular mass derived from the nucleotide sequence of an open reading frame of 798 nucleotides coding for 266 amino acids found on a 3-kb BamHI restriction fragment correspond closely to those obtained from the purified protein (N. Valdés-Stauber and S. Scherer, Appl. Environ. Microbiol. 60:3809-3814, 1994). No sequence homology to any protein or nucleotide sequences deposited in databases was found. Comparison of the nucleotide sequence and the N-terminal amino acid sequence derived from the protein suggests that B. linens M18 produces an N-formyl-methionyl-CAC tRNA. A wide taxonomical distribution of the gene within coryneform bacteria has been demonstrated by PCR amplification. The structural gene from linocin M18 is present at least in three Brevibacterium species, five Arthrobacter species, and five Corynebacterium species.     

嗜酸乳杆菌产细菌素生物学特性的研究

嗜酸乳杆菌在MRS培养基中培养得到的细菌素经30、60、90、121℃处理20min后,活性几乎不变,对以金黄色葡萄球菌为代表的革兰阳性菌、大肠埃希菌为代表的革兰阴性菌有明显的抑制作用,Tricine—SDS—PAGE和不同pH值抑菌实验测定结果证明,嗜酸乳杆菌细菌素是一组低分子量,并在pH2—4时有抑菌作用的活性物质。     

Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.     

Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.     

Bacteriocin production by Pseudomonas syringae pv. ciccaronei NCPPB2355. Isolation and partial characterization of the antimicrobial compound

Pseudomonas syringae pv. ciccaronei strain NCPPB2355 was found to produce a bacteriocin inhibitory against strains of Ps. syringae subsp. savastanoi , the causal agent of olive knot disease. Treatments with mitomycin C did not substantially increase the bacteriocin titre in culture. The purification of the bacteriocin obtained by ammonium sulphate precipitation of culture supernatant fluid, membrane ultrafiltration, gel filtration and preparative PAGE, led to the isolation of a high molecular weight proteinaceous substance. The bacteriocin analysed by SDS-PAGE revealed three protein bands with molecular weights of 76, 63 and 45 kDa, respectively. The bacteriocin was sensitive to heat and proteolytic enzymes, was resistant to non-polar organic solvents and was active between pH 5·0–7·0. Plasmid-DNA analysis of Ps. syringae ciccaronei revealed the presence of 18 plasmids; bacteriocin-negative variants could not be obtained by cure experiments.     

Antimicrobial activity of lactic acid bacteria isolated from goat's milk and artisanal cheeses: characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL105

A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis , one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by α-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.     

Growth reduction of Listeria spp. caused by undefined industrial red smear cheese cultures and bacteriocin-producing Brevibacterium lines as evaluated in situ on soft cheese.

The undefined microbial floras derived from the surface of ripe cheese which are used for the ripening of commercial red smear cheeses have a strong impact on the growth of Listeria spp. In some cases, these microbial consortia inhibit Listeria almost completely. From such undefined industrial cheese-ripening floras, linocin M18-producing (lin+) (N. Valdés-Stauber and S. Scherer, Appl. Environ. Microbiol. 60:3809-3814, 1994) and -nonproducing Brevibacterium linens strains were isolated and used as single-strain starter cultures on model red smear cheeses to evaluate their potential inhibitory effects on Listeria strains in situ. On cheeses ripened with lin+ strains, a growth reduction of L. ivanovii and L. monocytogenes of 1 to 2 log units was observed compared to cheeses ripened with lin strains. Linocin M18 activity was detected in cheeses ripened with lin+ strains but was not found in those ripened with lin strains. We suggest that production of linocin M18 contributes to the growth reduction of Listeria observed on model red smear cheeses but is unsufficient to explain the almost complete inhibition of Listeria caused by some undefined microbial floras derived from the surface of ripe cheeses.     

Production and Characterization of Nisin-Like Peptide Produced by a Strain of Lactococcus lactis Isolated from Fermented Milk

An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited several related species of lactic acid bacteria and other Gram-positive bacteria. The inhibitory activity of the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.     

Detection, purification and efficacy of warnerin produced by Staphylococcus warneri

Summary Three strains of Staphylococcus warneri (FM10, FM20 and FM30) isolated from meat samples were investigated for their ability to synthesize bacteriocin. All the tested strains produced warnerin, a new peptide bacteriocin; which inhibits the growth of a large number of Gram-positive and Gram-negative bacteria. The inhibitory effect of warnerin produced by the FM20 isolate was high when compared to the other isolates. The results on the effect of carbon sources, nitrogen sources, pH, temperature, incubation time and surfactant (tween 80) inferred that the bacteriocin production was high in medium supplemented with 1% glucose (12,800 AU/ml), 1% urea (6800 AU/ml), and 0.5% Tween 80 (25,600 AU/ml). The higher productivity of bacteriocin was registered during 12 h of incubation in the medium pH 6.5 at 37 °C temperature. Among the various indicator strains tested, Staphylococcus aureus was more sensitive to the bacteriocin activity. Partially purified warnerin exhibited a single band on SDS-PAGE with an apparent molecular weight of 2500 Da. Warnerin, the antibacterial compound was determined as a proteinaceous substance, since it lost its activity when pepsin was added.     

Plantacin B, a bacteriocin produced by Lactobacillus plantarum NCDO 1193

Abstract Strains of Lactobacillus plantarum and Leuconostoc mesenteroides were tested for bacteriocin production against each other and a range of closely related bacteria. L. plantarum 1193 was found to produce an inhibitory substance active against L. plantarum 340 and 1752, L. mesenteroides 8015 and Pediococcus damnosus 1832. This substance is a potential bacteriocin and has been named plantacin B.     

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.     

In vitro study on bacteriocin production of Enterococci associated with chickens

In recent years, the approach of using innovative strategies such as probiotics or bacteriocins for the prevention or treatment of bacterial infections has come into focus. The present study was undertaken to check in vitro ability of Enterococci-isolated from the gastrointestinal tract of chickens-to produce a bacteriocin-like substance and to describe some further probiotic properties in five selected Enterococcus faecium strains. All strains (n=17) were found to produce bacteriocin-like substances against 14 out of 20 indicator bacteria of animal, food or environmental origin. Selected E. faecium strains expressed sufficient survival by pH 3.0 after 3h, in the presence of 1% bile after 24h and they were sensitive to most of antimicrobials tested. All tested strains adhere to the human, canine and porcine intestinal mucus (between 1.5% and 9.2%). However, better adhesion ability was observed for the canine mucus. PCR detection of enterocin structural genes determined presence of enterocins A and P genes in all selected strains. Characterization of bacteriocin substance in detail was performed in E. faecium EF55. The EF55 strain produced a bacteriocin-like substance (during the late logarithmic and early stationary growth phase) with inhibitory activity mostly against Gram-positive bacteria (100-51,200 AU/mL) including Listeria monocytogenes. Proteinaceous character of the bacteriocin substance was confirmed (its inhibitory activity was lost after its treatment with proteases), it was found to be stable after heating (100 degrees C 10 min) and during 12 months storage at -20 degrees C. The highest inhibitory activity of bacteriocin produced by EF55 strain (growing in MRS) broth was achieved between pH 7.0 and 9.0.     

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Antibacterial activity of Lactobacillus sake isolated from meat

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Characterization of bacteriocin produced by Pediococcus pentosaceus from wine

A.M. STRASSER DE SAAD AND M.C. MANCA DE NADRA. 1993. Twenty strains of Pediococcus pentosaceus isolated from wine were examined for production of bacteriocins. Only two of them showed inhibitory activity, Ped. pentosaceus N4p against the indicator strains of the same species and N5p against 19 strains of the three genera of lactic acid bacteria from wine. The antimicrobial substance from N5p strains was removed by membrane (0.2 μm) filtration, destroyed by organic solvents and proteolytic enzymes. It was stable for 60 min at 100C. The bacteriocin was produced early in the growth cycle and its production was maximum after 48 h of culture in tomato juice medium at an initial pH of 6.5. The bactericidal effect was observed.     

Partial characterization and cloning of leuconocin J, a bacteriocin produced by Leuconostoc sp. J2 isolated from the Korean fermented vegetable Kimchi

Leuconostoc sp. J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J. This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food-borne pathogens. The antimicrobial substance was secreted into the medium during the late log phase. It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat-stable. The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2.5-3.5 kDa. Leuconostoc sp. J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5 alpha. Phenotypic expression of the bacteriocin production was detected in transformants harboring pULBJ5.5. Finally, Southern blotting with the 2.3 kb insert as a probe against plasmid digests of Leuconostoc sp. J2 revealed that the cloned foreign DNA originated from Leuconostoc sp. J2.     

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.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

Enterolysin A,a cell wall-degrading bacteriocin from Enterococcus faecalis LMG 2333

A novel antimicrobial protein, designated enterolysin A, was purified from an Enterococcus faecalis LMG 2333 culture. Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli. Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35 degrees C), the bacteriocin activity was increased to 5,120 bacteriocin units ml(-1). Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases. Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action. Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing. This bacteriocin is translated as a 343-amino-acid preprotein with an sec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein. Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24. The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures. These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family. The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins.     

Vibrio sp. strain NM 10, isolated from the intestine of a Japanese coastal fish, has an inhibitory effect against Pasteurella piscicida.

Vibrio sp. strain NM 10 with an inhibitory activity against Pasteurella piscicida K-III was isolated from the intestine of a spotnape ponyfish (Leiognathus nuchalis). This bacterium efficiently produced an antibacterial substance after growth at 20 degrees C for 24 h on 1/5 PYBG agar prepared with 50% seawater at pHs of 7.5 to 9.0. The antibacterial substance was heat labile and proteinaceous, with a molecular mass of less than 5 kDa, possibly a bacteriocin or a bacteriocin-like substance.