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PubMed1992

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).

Bacteriocins↗
PubMed1992

Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.

Base Sequence↗
PubMed1992

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.

Adenosine Triphosphate↗
PubMed1992

Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.

Amino Acid Sequence↗
PubMed1992

Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L. lactis.

The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp. cremoris Wg2 were mapped by primer extension. The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2. In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259. The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L. lactis. Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription. The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L. lactis Wg2.

Bacterial Proteins↗
PubMed1993

Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis.

Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known. They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M. Mulders, I.J. Boerrigter, H.S. Rollema, R.J. Siezen, and W.M. de Vos, Eur. J. Biochem, 201:581-584, 1991). The nisin variants were purified to apparent homogeneity, and their biological activities were compared. Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria. However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A. This was observed with all tested indicator strains. These results suggest that nisin Z has better diffusion properties than nisin A in agar. The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product. In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L. lactis strains analyzed.

Base Sequence↗
PubMed1993

Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lactococcus lactis subsp. cremoris AM2.

A gene coding for an aminopeptidase (PepC) from Lactococcus lactis subsp. cremoris AM2 was cloned by complementation of an Escherichia coli mutant lacking aminopeptidase activity. The nucleotide sequence was determined. A portion of the predicted amino acid sequence of PepC (436 amino acids) showed strong homology to the active site of cysteine proteases. No signal sequence was found, indicating an intracellular location of the enzyme.

Amino Acid Sequence↗
PubMed1993

Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region.

The biochemical and genetical diversity of the subtilisin-like cell envelope proteinase (CEP) among Lactococcus lactis strains was investigated. The specificities of the proteinases of 16 strains toward the important cheese peptide alpha s1-casein fragment 1 to 23 and toward two differently charged chromophoric peptides have been determined. On the basis of the results, these strains could be classified into seven groups. The contribution to the specificity of specific residues in the large C-terminal segment, which differentiates this proteinase from most other members of the subtilisin family, was established with hybrid proteinases, even in the case of the small substrates. These remote residues and the subtilisin-like substrate-binding region are therefore assumed to be spatially close to each other and together constitute most of the binding region of CEP. DNA sequence analysis of fragments of the gene (prtP) encoding segments of the proteinase which contain the relevant residues of the substrate-binding region shows that among the strains studied, this binding region is the most negatively charged in the CEP group represented by strain HP and the positively charged in the CEP group represented by strains AM1 and SK11. Consequently, these two proteinase groups show the most divergent specificities. Each of the proteinases of the other groups shows a different intermediate specificity which in part is the reflection of an intermediate charge in the binding region. However, the results suggest that amino acid residues outside the segments known to be part of the CEP-binding region also contribute to specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes.

Lactococcus lactis subsp. cremoris is widely used in the manufacture of fermented milk products. Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful. Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L. lactis subsp. cremoris from related strains. These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures. A total of 170 strains of L. lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L. lactis. Fifty-nine of these isolates also hybridized to L. lactis subsp. cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L. lactis subsp. cremoris-specific probe had the L. lactis subsp. cremoris phenotype.

Animals↗
PubMed1993

A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis.

The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase. The vectors incorporated different lactococcal secretion leaders and translation initiation sequences. When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium. However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane. The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain. This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions. Our results indicate that L. lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.

Amino Acid Sequence↗
PubMed1993

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.

Amino Acid Sequence↗
PubMed1994

Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.

The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.

Chromosome Mapping↗
PubMed1994

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3.

The biosynthetic genes of the nisin-producing strain Lactococcus lactis 6F3 are organized in an operon-like structure starting with the structural gene nisA followed by the genes nisB, nisT, and nisC, which are probably involved in chemical modification and secretion of the prepeptide (G. Engelke, Z. Gutowski-Eckel, M. Hammelmann, and K.-D. Entian, Appl. Environ. Microbiol. 58:3730-3743, 1992). Subcloning of an adjacent 5-kb downstream region revealed additional genes involved in nisin biosynthesis. The gene nisI, which encodes a lipoprotein, causes increased immunity after its transformation into nisin-sensitive L. lactis MG1614. It is followed by the gene nisP, coding for a subtilisin-like serine protease possibly involved in processing of the secreted leader peptide. Adjacent to the 3' end of nisP the genes nisR and nisK were identified, coding for a regulatory protein and a histidine kinase, showing marked similarities to members of the OmpR/EnvZ-like subgroup of two-component regulatory systems. The deduced amino acid sequences of nisR and nisK exhibit marked similarities to SpaR and SpaK, which were recently identified as the response regulator and the corresponding histidine kinase of subtilin biosynthesis. By using antibodies directed against the nisin prepeptide and the NisB protein, respectively, we could show that nisin biosynthesis is regulated by the expression of its structural and biosynthetic genes. Prenisin expression starts in the exponential growth phase and precedes that of the NisB protein by approximately 30 min. Both proteins are expressed to a maximum in the stationary growth phase.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis.

The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.

Acetolactate Synthase↗
PubMed1994

Distribution and evolution of nisin-sucrose elements in Lactococcus lactis.

The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed.

Amino Acid Sequence↗