Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat. The nonconjugative plasmids pCP40 and pCP49 associated with bacteriocin production in Carnobacterium piscicola LV17, a lactic acid bacterium isolated from meat, were mobilized by the wide host range conjugative plasmid pAMβ1 by two stage conjugation. At the first stage, pAMβ1 was conjugally transferred into C. piscicola LV17 containing the two plasmids associated with bacteriocin production and a cryptic plasmid. Mobilization of the two bacteriocin plasmids by pAMβ1 was done by the second stage conjugation between the pAMβ1-containing C. piscicola LV17 and chloramphenicol (Cm)-resistant Bac^- mutant of C. piscicola LV17. The transconjugants had either partial bacteriocin activity associated with acquisition of pCP40 or pCP49, or complete bacteriocin activity associated with acquisition of all three of the resident plasmids from C. piscicola LV17 or an 89 MDa cointegrated plasmid derived from pCP40 and pCP49. Further manipulation of the transconjugants and a mutant strain of C. piscicola LV17 resulted in separate strains with only pCP40 or pCP49 which produce different bacteriocins. The bacteriocin gene from pCP49 was cloned into pCaT, a chloramphenicol resistance-encoding vector, and electrotransformed into another bacteriocin-producing strain of C. piscicola , enhancing the antagonistic spectrum of the recipient strain.     

Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287-2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.     

Factors affecting production of an antilisterial bacteriocin by Carnobacterium piscicola strain A9b in laboratory media and model fish systems

AIMS: To investigate factors influencing bacteriocin production and bacteriocin stability of the bioprotective culture Carnobacterium piscicola strain A9b. METHODS AND RESULTS: Maximum activity was obtained in MRS7 broth (MRS adjusted to pH 7.2), with or without glucose. No bacteriocin was produced in APT broth when a low inoculum level (0.001%) was used. In contrast, inoculum level did not influence bacteriocin production in BHI and MRS7 without glucose. Bacteriocin production in APT was induced by the presence of an extracellular compound present in the sterile, filtered, cell-free supernatant fluid of a stationary-phase culture. Increasing concentrations of NaCl (2-7%) reduced bacteriocin production and maximum cell density of C. piscicola A9b when grown in cooked fish juice at 4 degrees C. CONCLUSION: Media composition, inoculum level and sodium chloride concentration affected production. SIGNIFICANCE AND IMPACT OF THE STUDY: The influence of NaCl on bacteriocin production may negate the inhibitory effect of C. piscicola A9b against Listeria monocytogenes in salty foods.     

Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.     

Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat.

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.     

Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1.

Cloning of a 16-kb DNA fragment from the 61-kb plasmid of Carnobacterium piscicola LV17B into plasmidless C. piscicola LV17C restores the production of the plasmid-encoded carnobacteriocin B2 and the chromosomally-encoded carnobacteriocin BM1 and restores the immune phenotype. This fragment also has sufficient genetic information to allow the expression of carnobacteriocin B2 and its immunity in a heterologous host. The gene locus (cbiB2) responsible for immunity to carnobacteriocin B2 is located downstream of the structural gene for carnobacteriocin B2 and encodes a protein of 111 amino acids (CbiB2). CbiB2 was expressed in Escherichia coli as a fusion of the maltose-binding protein and CbiB2. The fusion protein was purified on an amylose column and cleaved with factor Xa, and pure CbiB2 was isolated by high-performance liquid chromatography. The N-terminal amino acid sequence and mass spectrometry (molecular weight [mean +/- standard error], 12,662.2 +/- 3.4) of the purified protein agree with the information deduced from the nucleotide sequence of cbiB2. Western blot (immunoblot) analysis indicates that the majority of the intracellular pool of this immunity protein is in the cytoplasm and that a smaller proportion is associated with the membrane. CbiB2 confers immunity to carnobacteriocin B2, but not to carnobacteriocin BM1, when it is expressed in homologous or heterologous hosts. No protective effect is observed for sensitive cells growing in the presence of the bacteriocin when the immunity protein is added to the medium. The purified immunity protein does not show significant binding to microtiter plates coated with carnobacteriocin B2 and is not able to inactivate the bacteriocin in solution.     

The contribution of bacteriocin to inhibition of Listeria monocytogenes by Carnobacterium piscicola strains in cold-smoked salmon systems

AIMS: To study the importance of bacteriocin production for the antilisterial effect of a bacteriocinogenic Carnobacterium piscicola strain A9b on growth of Listeria monocytogenes in broth and cold-smoked salmon systems. METHODS AND RESULTS: Acriflavin treatment of strain A9b resulted in loss of bacteriocin production and of immunity to carnobacteriocin B2. Two plasmids present in the wild-type were lost in the variant that was also more sensitive to bavaricin and leucocin A than the wild-type indicating cross-resistance to class IIa bacteriocins. The growth rate of the bac- mutant was higher than that of the wild-type at 5 and 37 degrees C but not at 25 or 30 degrees C. In salmon juice the maximum cell density of L. monocytogenes was suppressed 3 and 6 log by co-culture with C. piscicola A9b bac- and bac+, respectively, as compared with the control. Sterile filtered cultures of C. piscicola A9b bac- caused a limited suppression of the maximum cell density of L. monocytogenes similar to that observed when sterile buffer was added in equal amounts. Semi-purified carnobacteriocin B2 caused a 3.5 log decline in viable cell count after 6 day of incubation in cold-smoked salmon juice at 5 degrees C. High resistance level to carnobacteriocin B2 was observed for L. monocytogenes cells exposed to semi-purified and in situ produced carnobacteriocin B2. CONCLUSIONS: The presence of bacteriocin production in C. piscicola enhances its inhibition of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the emergence of resistance, a bacteriocin negative lactic acid bacteria may be more suited for practical use as a bioprotective agent against L. monocytogenes in ready-to-eat foods.     

Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.     

A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens.

Divergicin A is a strongly hydrophobic, narrow-spectrum, nonlantibiotic bacteriocin produced by Carnobacterium divergens LV13. This strain of C. divergens contains a 3.4-kb plasmid that mediates production of, and immunity to, the bacteriocin. N-terminal amino acid sequencing of the purified divergicin A was used to locate the structural gene (dvnA). The structural gene encodes a prepeptide of 75 amino acids consisting of a 29-amino-acid N-terminal extension and a mature peptide of 46 amino acids. Directly downstream of dvnA there is a second open reading frame that encodes the immunity protein for divergicin A. Divergicin A has a calculated molecular mass of 4,223.89 Da. The molecular mass determined by mass spectrometry is 4,223.9 Da, indicating that there is no posttranslational modification of the peptide. The N-terminal extension of divergicin A has an Ala-Ser-Ala (positions -3 to -1) cleavage site and acts as a signal peptide that accesses the general export system of the cell (such as the sec pathway in Escherichia coli). This is the first bacteriocin of lactic acid bacteria to be reported that does not have dedicated maturation and secretion genes. Production of divergicin A was observed in heterologous hosts containing only the two genes associated with divergicin A production and immunity. Fusing alkaline phosphatase behind the signal peptide for divergicin resulted in the secretion of this enzyme in the periplasmic space and supernatant of E. coli.     

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.     

Role of acetate in production of an autoinducible class IIa bacteriocin in Carnobacterium piscicola A9b

Carnobacterium piscicola strain A9b isolated from cold smoked salmon inhibits growth of the food-borne pathogen Listeria monocytogenes partly due to the production of a proteinaceous compound (L. Nilsson, L. Gram, and H. H. Huss. J. Food Prot. 62:336-342, 1999). The purpose of the present study was to purify the compound and describe factors affecting its production, with particular emphasis on food-relevant factors. Amino acid sequencing showed that the compound is a class IIa bacteriocin with an N-terminal amino acid sequence identical to that of carnobacteriocin B2. The production of the bacteriocin was autoinducible, and the threshold level for induction was 9.6 x 10(-10) M. We also report, for the first time, that acetate acts as an induction factor, with a threshold concentration of 0.3 to 12 mM. Acetate could not act as an inducer during the late exponential phase of C. piscicola A9b. The induction of bacteriocin production showed a dose-dependent relationship at acetate concentrations of up to 10 to 20 mM (depending on the growth medium) and at a concentration of 1.9 x 10(-8) M for the bacteriocin itself; a saturation level of bacteriocin specific activity was reached at these concentrations of induction factors. The combined use of both inducers did not enhance the saturation level of bacteriocin production compared to that seen with the use of each inducer alone. Increasing NaCl and glucose concentrations negatively influenced the efficiency of acetate as an induction factor. Based on the results, carnobacteriocin B2 was used as an induction factor to manipulate the production of bacteriocin in cold smoked salmon juice and thus improve the ability to inhibit L. monocytogenes.     

Inhibition of Listeria monocytogenes by freeze-dried piscicocin CS526 fermentate in food

AIMS: The effectiveness of freeze-dried powder, fermented with bacteriocin producing Carnobacterium piscicola CS526, was evaluated for the inhibition of Listeria monocytogenes in a food model. METHODS AND RESULTS: A 10% solution of milk whey powder was fermented with a bacteriocinogenic C. piscicola CS526 Bac(+) or its nonbacteriocinogenic mutant strain CS526 Bac(-) at 30 degrees C for 12 h and freeze-dried. The freeze-dried piscicocin CS526 Bac(+) fermentate exhibited strong anti-listerial activity even at a concentration of 1% (w/v) in sterile water (pH 7), but the piscicocin CS526 Bac(-) fermentate and nonfermented whey powder had no anti-listerial activity. In the presence of 10% piscicocin CS526 Bac(+) fermentate, L. monocytogenes in ground meat rapidly decreased from 10(5) CFU g(-1) to less than the detection limit (3.0 x 10(3) CFU g(-1)) within 5 and 1 days at 4 and 12 degrees C, and was bacteriostatically inhibited for 25 and 4 days at 4 and 12 degrees C respectively. Furthermore, this inhibitory effect was enhanced at lower temperatures. CONCLUSIONS: Piscicocin CS526 Bac(+) fermentate was effective for the control of L. monocytogenes in a food model at refrigeration temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: A freeze-dried bioactive piscicocin CS526 Bac(+) powder can be a powerful tool to ensure food safety against L. monocytogenes contamination in refrigerated foods such as ready-to-eat products.     

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Purification of bacteriocin AS-48 from an Enterococcus faecium strain and analysis of the gene cluster involved in its production

The cyclic bacteriocin AS-48 has previously been shown to be produced by Enterococcus faecalis strains. A bacteriocin has been purified from an E. faecium strain (E. faecium 7C5), and it has been found to possess molecular mass, cyclization and amino acid sequence typical of bacteriocin AS-48. In addition to the structural gene as-48A, the sequence analysis of the AS-48 gene cluster present in E. faecium 7C5 has revealed the presence of several putative coding regions presumably involved in bacteriocin production and immunity. The results of DNA hybridization assays have indicated that the AS-48 gene cluster and the gene pd78 are present on the same plasmid, possibly the pPD1 plasmid, in E. faecium 7C5.     

Effect of sausage ingredients and additives on the production of enterocin A and B by Enterococcus faecium CTC492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages

Enterocin A and B in Enterococcus faecium CTC492 were co-induced by the different factors assayed in this study (r = 0.93) and followed primary metabolic kinetics. Enterocin production was significantly inhibited by sausage ingredients and additives, with the exception of nitrate. The addition of sodium chloride and pepper decreased production 16-fold. The temperature and pH influenced enterocin production, with optima between 25 and 35 degrees C, and from 6.0 to 7.5 of initial pH. The maximum activity was achieved, under favourable growth conditions, with MRS supplemented with sucrose (2%) plus glucose (0.25%) and Tween-80 (1%). MRS concentration, NaCl plus pepper addition, absence of Tween-80 in the growth medium, incubation at 45 degrees C and an initial pH under 5.5 were detrimental to bacteriocin production. Stress conditions did not favour enterocin production. Desadsorption was Tween-dependent. Enterocin A activity in the crude extracts stored at -80 degrees C was better preserved than enterocin B (when tested against their specific indicator strain), but anti-listerial activity remained intact. Applied as anti-listerial additives in dry fermented sausages, enterocins significantly diminished Listeria counts by 1. 13 log (P < 0.001), while Enterococcus faecium CTC492 added as starter culture did not significantly reduce Listeria counts (P > 0. 1) compared with the standard starter culture (Bac-). Enterocins A and B could be considered as extra biopreservative hurdles for listeria prevention in dry fermented sausages.     

Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.