Rapid and Efficient Purification Method for Small, Hydrophobic, Cationic Bacteriocins: Purification of Lactococcin B and Pediocin PA-1.

The bacteriocins lactococcin B and pediocin PA-1 were purified by ethanol precipitation, preparative isoelectric focusing, and ultrafiltration. The procedure reproducibly leads to high final yields in comparison to the generally low yields obtained by column chromatography. Specifically, during isoelectric focusing no loss of activity occurs. The method, in general, should be applicable to small, hydrophobic, cationic bacteriocins.

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.

Topology of LcnD, a protein implicated in the transport of bacteriocins from Lactococcus lactis.

Four in-frame translational fusions to both the reporter proteins beta-galactosidase and alkaline phosphatase support a topological model of LcnD, a protein implicated in the transport of several bacteriocins from Lactococcus lactis, in which the N-terminal part is located intracellularly and one transmembrane helix spans the cytoplasmic membrane.

Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.

In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.

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 alpha-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.

Expression of a chitinase gene from Serratia marcescens in Lactococcus lactis and Lactobacillus plantarum.

A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in the same transcriptional orientation as the gene specifying the replication protein of the vector pIL253. Using the expression vectors pMG36e and pGKV259 with lactococcal promoter fragments p32 and p59, the expression in L. lactis was increased nine- and 27-fold, respectively. An additional twofold increase was obtained after cloning the gene under the control of p59 in the high-copy number replicon pIL253. In Lactobacillus plantarum, chitinase activity was expressed from p32, and the activity was at the same level as under p32 control in L. lactis.

The di- and tripeptide transport protein of Lactococcus lactis. A new type of bacterial peptide transporter.

Lactococcus lactis takes up di- and tripeptides via a proton motive force-dependent carrier protein. The gene (dtpT) encoding the di-tripeptide transport protein of L. lactis was cloned by complementation of a dipeptide transport-deficient and proline auxotrophic Escherichia coli strain. Functional expression of the dipeptide transport gene was demonstrated by uptake studies of alanyl-[14C]glutamate and other peptides in E. coli cells. The di-tripeptide transport protein catalyzes proton motive force-driven peptide uptake and dipeptide exchange activity. The nucleotide sequence of dtpT was determined and the translated sequence corresponds with a protein of 463 amino acid residues. Hydropathy profiling indicates that the protein could form 12 membrane-spanning segments with the amino and carboxyl termini at the outer surface of the membrane. A secondary structure model is presented which is substantiated by analysis of DtpT-PhoA fusion constructs. Amino acid sequence comparisons showed no significant homology with other bacterial peptide transport systems nor with any other known protein. Flanking regions of the di-tripeptide transport gene were used to delete dtpT from the chromosome of L. lactis. Genetic and biochemical characterization of this mutant indicates that DtpT is the only transport protein in L. lactis for hydrophilic di- and tripeptides.

Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis.

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac and Prt, the Lac Prt determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.

Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase.

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.

Mode of Action of Lactococcin B, a Thiol-Activated Bacteriocin from Lactococcus lactis.

Lactococcin B (LcnB) is a small, hydrophobic, positively charged bacteriocin produced by Lactococcus lactis subsp. cremoris 9B4. Purified LcnB has a bactericidal effect on sensitive L. lactis cells by dissipating the proton motive force and causing leakage of intracellular substrates. The activity of LcnB depends on the reduced state of the Cys-24 residue. Uptake and efflux studies of different solutes suggest that LcnB forms pores in the cytoplasmic membrane of sensitive L. lactis cells in the absence of a proton motive force. At low concentrations of LcnB, efflux of those ions and amino acids which are taken up by proton motive force-driven systems was observed. However, a 150-fold higher LcnB concentration was required for efflux of glutamate, previously taken up via a unidirectional ATP-driven transport system. Strains carrying the genetic information for the immunity protein against LcnB were not affected by LcnB. The proton motive force of immune cells was not dissipated, and no leakage of intracellular substrates could be detected.

Lactococcal plasmid pWV01 as an integration vector for lactococci.

A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.

The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during growth in milk in the absence of antibiotics. The proteolytic activity of strain MG611 was approximately 11-fold higher than that of strain MG610 and about 1.5 times higher than that of strain MG1363(pGKV552), which carried the proteinase genes on an autonomously replicating plasmid with a copy number of approximately 5. All three strains showed rapid growth in milk with concomitant rapid production of acid. The results suggest that a limited number of copies of the proteinase genes prtP and prtM per genome is sufficient for good growth in milk.

Replacement recombination in Lactococcus lactis.

In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP). The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence. Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis. In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L. lactis chromosome. The other transformants in which the pepXP gene had not been inactivated carried a Campbell-type integrated copy of the plasmid. Loss of part of the Campbell-type integrated plasmid via recombination between 1.6-kb nontandem repeats occurred with low frequencies that varied between less than 2.8 x 10(-6) and 8.5 x 10(-6), producing cells with a chromosomal structure like that of cells in which replacement recombination had taken place.

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis.

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in L. lactis, we suggest that this activity destabilized the amplified structures in L. lactis.

Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis.

Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci.

Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2.

A 6.5-kilobase HindIII fragment that specifies the proteolytic activity of Streptococcus cremoris Wg2 was sequenced entirely. The nucleotide sequence revealed two open reading frames (ORFs), a small ORF1 with 295 codons and a large ORF2 containing 1,772 codons. For both ORFs, there was no stop codon on the HindIII fragment. A partially overlapping PstI fragment was used to locate the translation stop of the large ORF2. The entire ORF2 contained 1,902 coding triplets, followed by an apparently rho-independent terminator sequence. The inferred amino acid sequence would result in a protein of 200 kilodaltons. Both ORFs have their putative transcription and translation signals in a 345-base-pair ClaI fragment. ORF2 is preceded by a promoter region containing a 15-base-pair complementary direct repeat. Both the truncated 33- and the 200-kilodalton proteins have a signal peptide-like N-terminal amino acid sequence. The protein specified by ORF2 contained regions of extensive homology with serine proteases of the subtilisin family. Specifically, amino acid sequences involved in the formation of the active site (viz., Asp-32, His-64, and Ser-221 of the subtilisins) are well conserved in the S. cremoris Wg2 proteinase. The homologous sequences are separated by nonhomologous regions which contain several inserts, most notably a sequence of approximately 200 amino acids between the His and Ser residues of the active site.