Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-1 by Lactococcus lactis.

The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides. Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martínez, J. M. Martínez, P. E. Hernández, M. J. Gasson, J. M. Rodríguez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818-823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host. Highest yields were achieved with L. lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain, Pediococcus acidilactici 347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L. lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.     

Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4.

The genes responsible for bacteriocin production and immunity in Lactococcus lactis subsp. lactis biovar diacetylactis WM4 were localized and characterized by DNA restriction fragment deletion, subcloning, and nucleotide sequence analysis. The nucleotide sequence of a 5.6-kb AvaII restriction fragment revealed a cluster with five complete open reading frames (ORFs) in the same orientation. DNA and protein homology analyses, combined with deletion and Tn5 insertion mutagenesis, implicated four of the ORFs in the production of and immunity to lactococcin A. The last two ORFs in the cluster were the lactococcin A structural and immunity genes, lcnA and lciA. The two ORFs immediately upstream of lcnA and lciA were designated lcnC and lcnD, and the proteins that they encoded showed similarities to proteins of signal sequence-independent secretion systems. lcnC encodes a protein of 716 amino acids that could belong to the HlyB family of ATP-dependent membrane translocators. LcnC contains an ATP binding domain in a conserved C-terminal stretch of approximately 200 amino acids and three putative hydrophobic segments in the N terminus. The lcnD product, LcnD, of 474 amino acids, is essential for lactococcin A expression and shows structural similarities to HlyD and its homologs. On the basis of these results, a secretion apparatus that is essential for the full expression of active lactococcin A is postulated.     

Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4.

The genes responsible for bacteriocin production and immunity in Lactococcus lactis subsp. lactis biovar diacetylactis WM4 were localized and characterized by DNA restriction fragment deletion, subcloning, and nucleotide sequence analysis. The nucleotide sequence of a 5.6-kb AvaII restriction fragment revealed a cluster with five complete open reading frames (ORFs) in the same orientation. DNA and protein homology analyses, combined with deletion and Tn5 insertion mutagenesis, implicated four of the ORFs in the production of and immunity to lactococcin A. The last two ORFs in the cluster were the lactococcin A structural and immunity genes, lcnA and lciA. The two ORFs immediately upstream of lcnA and lciA were designated lcnC and lcnD, and the proteins that they encoded showed similarities to proteins of signal sequence-independent secretion systems. lcnC encodes a protein of 716 amino acids that could belong to the HlyB family of ATP-dependent membrane translocators. LcnC contains an ATP binding domain in a conserved C-terminal stretch of approximately 200 amino acids and three putative hydrophobic segments in the N terminus. The lcnD product, LcnD, of 474 amino acids, is essential for lactococcin A expression and shows structural similarities to HlyD and its homologs. On the basis of these results, a secretion apparatus that is essential for the full expression of active lactococcin A is postulated.     

Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403.

A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.     

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.     

Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum.

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

     

Duplication of the pepF gene and shuffling of DNA fragments on the lactose plasmid of Lactococcus lactis.

The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF [V. Monnet, M. Nardi, A. Chopin, M.-C. Chopin, and J.-C. Gripon, J. Biol. Chem. 269:32070-32076, 1994]) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp. cremoris NCDO763. Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp. lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763. From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters. The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases. Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403. Traces of several recombination events are visible on the lactose-proteinase plasmid. This suggests that the duplication of pepF occurred by recombination from the chromosome of an L. lactis subsp. lactis strain followed by gene transfer. We discuss the possible functions of PepF and the role of its amplification.     

Lactococcin Q, a novel two-peptide bacteriocin produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, alpha and beta, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qalpha plus Qbeta) and lactococcin G (Galpha plus Gbeta) clarified that hybrid combinations (Qalpha plus Gbeta and Galpha plus Qbeta) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qalpha plus Gbeta was 32 times higher than that of Qalpha plus Qbeta, suggesting that the difference in beta peptides was important for the intensity of antibacterial activity.     

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.     

Production of Pediocin PA-1 by Lactococcus lactis Using the Lactococcin A Secretory Apparatus

The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.Bacteriocins of lactic acid bacteria have received considerable attention in recent years due to their potential application in the food industry as natural preservatives. Most interest has focused on lantibiotics (class I bacteriocins), e.g., nisin, and small heat-stable non-lanthionine-containing bacteriocins (class II) (22, 23). A major subgroup of class II bacteriocins (IIa) has been given the generic name of pediocin family (28) after its most extensively studied member, pediocin PA-1. Members of this class have a number of features in common, including a very strong antimicrobial activity against Listeria species (28). The food-borne pathogen Listeria monocytogenes is a major concern in the dairy industry since it can grow in a variety of dairy products at low temperature and pH (13). Although a pediocin PA-1-producing Lactobacillus plantarum strain has recently been isolated (12), this bacteriocin is generally produced by Pediococcus acidilactici strains of meat origin (3, 16, 18, 29, 31). Because of its antilisterial activity, the expression of pediocin PA-1 in strains of dairy origin would be highly desirable.Pediocin PA-1 production, immunity, and secretion are determined by an operon containing four genes (26). The structural gene, pedA, encodes the pediocin PA-1 precursor, pedB specifies immunity, and the pedC and pedD gene products are membrane-bound proteins required for secretion of the active peptide (39). Homologs of these genes have been described for related peptides. Biosynthesis of the well-characterized class II bacteriocin, lactococcin A, produced by strains of Lactococcus lactis also involves four genes (20, 36, 40). In addition to the structural gene (lcnA) and immunity gene (lciA), there are two genes (lcnC and lcnD) whose products together form a transport system dedicated to the translocation of lactococcin through the host membrane. The LcnC protein belongs to the family of ATP-binding cassette transporter proteins (40), and LcnD acts as an accessory protein (14). These two proteins have considerable homology to PedD and PedC, respectively (39), suggesting that the latter proteins play a similar role in the transport of active pediocin. The two bacteriocins also share the double glycine-processing site found in many lactic acid bacteria class II bacteriocins, some lantibiotics, and the Escherichia coli bacteriocin, colicin V (17).Van Belkum et al. (38) have recently investigated the role of leader sequences of the class II bacteriocins in the recognition of the precursor peptide by the dedicated translocation machinery of the host organism. By constructing hybrid genes, they demonstrated that the leader peptides of leucocin A, lactococcin A, and colicin V, which are cleaved at the Gly-Gly (positions −2 and −1) site, can direct the secretion of the nonrelated bacteriocin divergicin A. Our studies have focused on the class II bacteriocins pediocin PA-1 and lactococcin A. Since these peptides have a number of features in common, it might be expected that a pediocin PA-1 precursor could be secreted and processed by using the lactococcin A translocation machinery. L. lactis IL1403 is a plasmid-free strain that does not produce bacteriocin but contains chromosomal copies of genes analogous to lcnC and lcnD (33, 40). In addition, the natural resistance of this strain to pediocin PA-1 (8) makes it an ideal candidate for a production host to investigate the expression of pediocin PA-1 in lactococci.This paper describes the development of an expression system geared to the production of heterologous peptides in L. lactis. Testing the system with pediocin PA-1 involved the construction of a vector containing an in-frame fusion between sequences encoding the lactococcin A leader and the structural part of mature pediocin PA-1. The hybrid genes were introduced into L. lactis IL1403, and the ability of these strains to produce and secrete pediocin PA-1 was investigated.     

Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.     

Combinational variation of restriction modification specificities in Lactococcus lactis

Three genes coding for a type I R-M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R-M specificity. The presence of these plasmids in IL1403 conferred a new R-M phenotype on the host, indicating that the plasmid-encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R-M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.     

Presence of STA gene sequences in brewer's yeast genome

I. BALOGH AND A. MARÁZ. 1996. STA genes are responsible for producing extracellular glucoamylase enzymes in Saccharomyces cerevisiae var. diastaticus . These genes exist in three forms, which are located on three different chromosomes. The nucleotide sequences of the STA genes are highly homologous. A sporulation-specific glucoamylase gene called SGA1 exists in every Saccharomyces cerevisiae strain, this also having a partly homologous DNA sequence with the STA genes. In this study S. cerevisiae var. diastaticus and brewer's yeast strains were characterized by pulsed-field gel electrophoresis. In many cases chromosome length polymorphism (CLP) was found. The chromosomes were hybridized with a DNA probe which was homologous with STA genes and the SGA1 gene. Presence of the SGA1 gene was detected in each strain used. Four brewing yeasts were found to have homologous sequences with the STA3 gene on chromosome XIV despite the fact that these strains were not able to produce extracellular glucoamylase enzyme.     

The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner.

Lactococcin A is a bacteriocin produced by Lactococcus lactis. Its structural gene has recently been cloned and sequenced (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). Purified lactococcin A increased the permeability of the cytoplasmic membrane of L. lactis and dissipated the membrane potential. A significantly higher concentration of lactococcin A was needed to dissipate the membrane potential in an immune strain of L. lactis. Lactococcin A at low concentrations (0.029 microgram/mg of protein) inhibited secondary and phosphate-bond driven transport of amino acids in sensitive cells and caused efflux of preaccumulated amino acids. Accumulation of amino acids by immune cells was not affected by this concentration of lactococcin A. Lactococcin A also inhibited proton motive force-driven leucine uptake and leucine counterflow in membrane vesicles of the sensitive strain but not in membrane vesicles of the immune strain. These observations indicate that lactococcin A makes the membrane permeable for leucine in the presence or absence of a proton motive force and that the immunity factor(s) is membrane linked. Membrane vesicles of Clostridium acetobutylicum, Bacillus subtilis, and Escherichia coli were not affected by lactococcin A, nor were liposomes derived from phospholipids of L. lactis. These results indicate that lactococcin A acts on the cytoplasmic membrane and is very specific towards lactococci. The combined results obtained with cells, vesicles, and liposomes suggest that the specificity of lactococcin A may be mediated by a receptor protein associated with the cytoplasmic membrane.     

Mode of action of LciA, the lactococcin A immunity protein

Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A. The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein. It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA. The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right-side-out vesicles by proteinase K, it is located at the outside of the cell. The immunity protein contains a putative a-amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell. Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

Alternative lactose catabolic pathway in Lactococcus lactis IL1403

In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-beta-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-beta-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-beta-glucosidase, it also exhibits phospho-beta-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.