Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide.

Lactococcin, a bacteriocin produced by Lactococcus lactis subsp. lactis ADRIA 85LO30, was purified as a 2.3-2.4 kDa peptide. Six non-bacteriocin-producing (Bac-) and non-immune (Imm-) strains were isolated after curing experiments. These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb). By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.     

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.     

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.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

Proteinase PI and lactococcin A genes are located on the largest plasmid in Lactococcus lactis subsp. lactis bv. diacetylactis S50

Lactococcus lactis subsp. lactis bv. diacetylactis S50 produces a lactococcin A-like bacteriocin named bacteriocin S50, and cell envelope-associated PI-type proteinase activity. This strain harbours 3 small size plasmids: pS6 (6.3 kb), pS7a (7.31 kb), and pS7b (7.27 kb). Plasmid curing using a combination of novobiocin treatment (10 microg.mL-1) and sublethal temperature (40 degrees C) resulted in a very low yield (0.17%) of Prt-, Bac-, Bacs derivatives, which retained all 3 small size resident plasmids. Pulsed-field gel electrophoresis of DNA isolated from the strain S50 and cured derivatives in combination with restriction enzyme analysis and DNA-DNA hybridization revealed that S50 contains 2 additional large plasmids: pS140 (140 kb) and pS80 (80 kb). Conjugation experiments using strain S50 as a donor and various lactococcal recipients resulted in Prt+, Bac+, Bacr transconjugants. Analysis of these transconjugants strongly indicated that plasmid pS140 harbours the prt and bac genes encoding proteinase and bacteriocin production, and immunity to bacteriocin, since each Prt+, Bac+, Bacr tranconjugant contained pS140. Accordingly, none of the Prt-,Bac-, Bacs transconjugants contained this plasmid. pS140 was a self-transmissible conjugative plasmid regardless of the host lactococcal recipient used in the test. Frequency of conjugation of plasmid pS140 did not depend on either the donor or recipient strain.     

Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources

AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.     

Protoplast-induced curing of bacteriocin plasmid in Lactococcus lactis subsp. lactis 484

Plasmid-specified traits like lactose metabolism and bacteriocin production could be eliminated from Lactococcus lactis subsp. lactis 484 culture during production and regeneration of protoplasts with lysozyme at the concentration of 300 μg/ml after 3 h treatment. Plasmid-free strains and cured derivatives harbouring only a single plasmid (2 MDa) were also obtained. Loss of high molecular weight (65 MDa) low copy number Lac plasmid occurred more frequently compared with low molecular weight (2 MDa) high copy number plasmid. Treatment of L. lactis subsp. lactis 484 cells with lysozyme at concentrations of 1000 μg/ml could produce a large number of Lac⁻ Bac⁻ variants at a very high frequency (94%). The curing data confirmed the linkage of Lac and Bac phenotypes to 65 and 2 MDa plasmids, respectively.     

A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB,a Class II Leaderless Bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.     

Development and application of a multiple typing system for Clostridium difficile

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Development and application of a multiple typing system for Clostridium difficile.

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Self-transferable plasmids determining the hemolysin and bacteriocin of Streptococcus faecalis var. zymogenes.

Strains of Streptococcus faecalis var. zymogenes, designated JH1 and JH3, produced a hemolysin and a bacteriocin. Hemolytic activity was lost from a low percentage of cells grown in broth at either 37 or 45 C. All nonhemolytic (Hly-) variants had lost bacteriocin activity (Ben-), and those from strain JH3 had also lost resistance to the bacteriocin (Bnr-). The majority of Hly-, Ben- variants from JH1 retained bacteriocin resistance (Bnrplus). Strains JH1 and JH3 contained a plasmid deoxyribonucleic acid species of molecular weight 38 times 10-6 (plasmids pJH2 and pJH3, respectively), and strain JH1 also contained a 50 times 10-6 molecular weight plasmid (pJH1) which has previously been shown to carry the genes determining resistance to the antibiotics kanamycin, neomycin, streptomycin, erythromycin, and tetracycline. Hly-, Bcn-, Bnr- variants of strain JH3 had completely lost plasmid pJH3. Hly-, Bcn-, Bnr- variants of strain JH1 had completely lost plasmid pJH2 and retained plasmid pJH1, but Hly-, Bcn-, Bnrplus variants had retained both plasmids pJH2 and pJH1. The Hlyplus, Bcnplus, Bnrplus traits from both parental strains were transferable to nonhemolytic S. faecalis strains during mixed incubation in broth at 37 C, and hemolytic recipient strains were found to have received plasmid pJH2 from strain JH1 and pJH3 from JH3. We conclude that the Hlyplus, Bnrplus traits are borne on plasmid pJH2 in strain JH1 and pJH3 in strain JH3 and that, in Hly-, Bcn-, Bnrplus variants of strain JH1, plasmic pJH2 has suffered a mutation affecting hemolysin and bacteriocin expression. We infer that the plasmids transfer by conjugation. Beta-hemolytic activity is the only property distinguishing the zymogenes variety from S. faecalis. Since we have shown that this activity is plasmid borne in strains JH1 and JH3, we endorse the view that the varietal status of zymogenes should be dropped.     

Plasmids in Bacillus stearothermophilus coding for bacteriocinogeny and temperature resistance.

Obligately thermophilic strains of Bacillus stearothermophilus were screened for the presence of plasmids by agarose gel electrophoresis. All strains in our collection contained large plasmids (20 x 10(6)-80 x 10(6)) and were divided into four groups with respect to their plasmid pattern and production of bacteriocins. The major plasmid species were designated pSE407 (38.7 x 10(6)), pSE409 (29.0 x 10(6)), pSE411 (21.5 x 10(6)), and pSE410 (23.5 x 10(6)). Their physical endonuclease maps were constructed, and by Southern blots and hybridizations it was shown that these plasmids were related. From curing experiments and electrotransformations (electroporations) we conclude that pSE407, pSE410, and pSE411 code for temperature resistance. In addition pSE410 codes for bacteriocin production and resistance. Plasmid pSE409 probably also codes for bacteriocin production and resistance.     

Characterization of Propionibacterium plasmids.

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.     

Characterization of Propionibacterium plasmids

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.     

Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene.

A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, lcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as lcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli lcnA did not cause an LCN-A+ phenotype. L. lactis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying lcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.     

Plasmid linkage of a bacteriocin-like substance in Streptococcus lactis subsp. diacetylactis strain WM4: transferability to Streptococcus lactis.

Streptococcus lactis subsp. diacetylactis strain WM4 transferred lactose-fermenting and bacteriocin-producing (Bac+) abilities to S. lactis LM2301, a lactose-negative, streptomycin-resistant (Lac- Strr), plasmid-cured derivative of S. lactis C2. Three types of transconjugants were obtained: Lac+ Bac+, Lac+ Bac-, and Lac-Bac+.S. diacetylactis WM4 possessed plasmids of 88, 33, 30, 5.5, 4.8, and 3.8 megadaltons (Mdal). In Lac+ Bac+ transconjugants, lactose-fermenting ability was linked to the 33-Mdal plasmid and bacteriocin-producing ability to the 88-Mdal plasmid. Curing the 33-Mdal plasmid from Lac+ Bac+ transconjugants resulted in loss of lactose-fermenting ability but not bacteriocin-producing ability (Lac- Bac+). These strains retained the 88-Mdal plasmid. Curing of both plasmids resulted in a Lac- Bac- phenotype. The Lac+ Bac- transconjugant phenotype was associated with a recombinant plasmid of 55 or 65 Mdal. When these transconjugants were used as donors in subsequent matings, the frequency of Lac transfer was about 2.0 X 10(-2) per recipient plated, whereas when Lac+ Bac+ transconjugants served as donors, the frequency of Lac transfer was about 2.0 X 10(-5) per recipient plated. Also, Lac- Bac+ transconjugants were found to contain the 88-Mdal plasmid. The data indicate that the ability of WM4 to produce bacteriocin is linked to an 88-Mdal conjugative plasmid and that lactose-fermenting ability resides on a 33-Mdal plasmid.     

Stability of plasmids in lactococci during extended incubation in growth media

The stability of plasmids in Lactococcus lactis ssp. lactis strains C2 and ML3, and L. lactis ssp. cremoris strains ML1 and SC607, was investigated by extended incubation of bacterial cells in low nutrient media under acidic conditions. Strains were grown overnight (16-18 h) in skim milk and unbuffered medium (M17-) at 32 degrees C and subsequently held at that temperature for extended periods (greater than or equal to 96 h). Lac- variants were obtained from each strain in milk and (M17-) broth. The plasmid profiles of Lac- variants when compared with their parental Lac+ strains showed loss of one or more plasmid bands. None of the Lac- mutants showed loss of smaller plasmids (less than 5 MDa) indicating that smaller plasmids in lactococci are more stable under these conditions than larger plasmids (greater than 10 MDa). Concomitant loss of the Lac+ phenotype and plasmids by the method used in the present investigation may have application for isolating mutants devoid of one or more plasmids.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid.

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

Cloning and heterologous production of Hiracin JM79, a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, in lactic acid bacteria and Pichia pastoris

Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis, Lactobacillus sakei, Enterococcus faecium, Enterococcus faecalis, and Pichia pastoris. For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene (hirJM79), with or without the HirJM79 immunity gene (hiriJM79), was cloned into the plasmid pMG36c under the control of the constitutive promoter P(32) and into the plasmid pNZ8048 under the control of the inducible P(NisA) promoter. For the production of HirJM79 in P. pastoris, the gene encoding the mature HirJM79 protein was cloned into the pPICZalphaA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris.