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PubMed1990

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.

Bacillus subtilis↗
PubMed1991

Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown.

Amino Acid Sequence↗
PubMed1991

Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763.

Lactococcus lactis subsp. lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate. A DNA bank from L. lactis subsp. lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay. The X-PDAP gene, designated pepXP, was further subcloned and sequenced. It codes for a protein containing 763 residues. Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide. The pepXP gene from L. lactis subsp. lactis NCDO 763 in more than 99% homologous to the pepXP gene from L. lactis subsp. cremoris P8-2-47 described elsewhere (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and is also conserved in other lactococcal strains.

Amino Acid Sequence↗
PubMed1991

Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.

Bacteriophages↗
PubMed1991

Phage abortive infection mechanism from Lactococcus lactis subsp. lactis, expression of which is mediated by an Iso-ISS1 element.

A 5-kb DNA fragment conferring a phage abortive infection phenotype (Abi+) has been cloned from Lactococcus lactis subsp. lactis IL416. The Abi+ determinant was subcloned on a 2-kb fragment which carried an Iso-ISS1 element and an open reading frame of 753 bp designated ORFX. Deletion within ORFX entailed the loss of the Abi+ phenotype, establishing that ORFX is the structural abi-416 gene. The expression of abi-416 was shown to be mediated by the Iso-ISS1 element, which contains a sequence fitting the consensus sequence for gram-positive promoters.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.

Base Sequence↗
PubMed1991

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis.

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.

Amino Acid Sequence↗
PubMed1991

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.

Conjugation, Genetic↗
PubMed1991

Molecular characterization of the nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3.

The nisin resistance determinant of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3 was localized onto a 1.3-kb EcoRI-NdeI fragment by subcloning and interrupting the NdeI site by cloning random NdeI fragments into it; the nisin resistance determinant was then sequenced. The nucleotide sequence revealed a large open reading frame containing 318 codons. Putative transcription and translation signal sequences were located directly upstream from the initiation codon. Immediately downstream of the termination codon was a palindromic region resembling a rho-independent termination sequence. This 957-nucleotide open reading frame and its associated transcription and translation signal sequences were cloned into plasmid-free L. lactis subsp. lactis LM0230 and conferred an MIC of 160 IU of nisin per ml. This level of nisin resistance is equivalent to that of the initial nisin-resistant subclone, pFM011, used for further subcloning in this study. The inferred amino acid sequence would result in a protein with a molecular mass of 35,035 Da. This value was in agreement with the molecular mass of a protein detected after in vitro transcription and translation of DNA encoding the nisin resistance gene, nsr. This protein contained a hydrophobic region at the N terminus that was predicted to be membrane associated but did not contain a typical signal sequence cleavage site. No significant homology was detected when the DNA sequence of the nsr gene and the amino acid sequence of its putative product were compared with other available sequences. When subjected to Southern hybridization, a 1.2-kb DraI fragment encoding the nsr gene did not hybridize with the genomic DNA of the nisin-producing strain L. lactis subsp. lactis 11454.

Amino Acid Sequence↗
PubMed1991

Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis ATCC 11454.

The location and organization of the nisin locus in Lactococcus lactis ATCC 11454 were studied. Primer extension of in vivo mRNA transcripts of the gene that encodes the nisin prepropeptide sequence indicated the presence of a promoter at least 4 kb upstream from the nisin gene and that the mRNA has several processing sites. Restriction fragment patterns using rare-cutting enzymes, orthogonal pulsed-field clamped homogeneous electric field (CHEF) agarose gel electrophoresis, and hybridization with nisin gene probes showed that the nisin prepropeptide gene was located on a megabase-size restriction fragment, which was taken as proof of a chromosomal location. This is contrary to earlier reports, which had indicated that genes for nisin production were located on plasmids. There was no evidence of more than one chromosomal location or more than one copy of the nisin gene. The restriction patterns indicated that the size of the L. lactis genome is about 2,500 kb. The previously observed (G. W. Buchman, S. Banerjee, and J. N. Hansen, J. Biol. Chem. 263: 16260-16266, 1988) downstream open reading frame (ORF) was fully sequenced to reveal an 851-amino-acid coding region, an upstream putative mRNA processing site, and a putative rho-independent terminator. The ORF was analyzed for secondary structural features, and the sequence data bases were searched for homologies. The ORF contained many amphipathic helices, a C-terminal transmembrane helix, and homologies to some membrane-associated proteins. It lacked an N-terminal membrane insertion sequence and accordingly appears to be associated with, and anchored to, the cytoplasmic side of the membrane. An additional ORF that possessed a ribosome-binding sequence and tandem promoters, indicating the beginning of a new operon, was identified still farther downstream. The results were consistent with the nisin gene being part of a polycistronic operon with a size greater than 8.5 kb.

Amino Acid Sequence↗
PubMed1991

Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Konings, Appl. Environ. Microbiol. 56:526-532, 1990). The L. lactis pepN gene appeared to complement an E. coli strain carrying a mutation in its pepN gene. High-level expression of the pepN gene in E. coli was obtained by using the T7 system. The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots. Cloning of the pepN gene on a multicopy plasmid in L. lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein. Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N. The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein. The intracellular location of aminopeptidase N in L. lactis was confirmed by immunogold labeling of lactococcal cells.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acids↗
PubMed1991

Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919.

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.

Chromosomes, Bacterial↗
PubMed1992

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.

Animals↗
PubMed1992

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.

Cloning, Molecular↗
PubMed1992

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.

The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a probe the plasmid-linked prtP gene from L. lactis subsp. cremoris Wg2. The prtM gene necessary for the maturation of the proteinase was also chromosomally located adjacent to prtP in BC101. By using as a hybridization probe the ISS1-like element ISS1W, which is found adjacent to the proteinase genes in both pWV05 and pSK111, specific homology to the chromosomal fragment containing the proteinase gene was found. DNA sequencing of a polymerase chain reaction product of chromosomal DNA upstream from prtM revealed a 123-nucleotide sequence which was 100% identical to the equivalent sequence in the ISS1W-containing plasmid. The terminal inverted repeat (18 nucleotides) of the ISS1W element was found in this sequenced DNA. These findings suggest that the chromosomal proteinase gene is organized in a fashion similar to that of the plasmid-linked proteinase gene.

Base Sequence↗
PubMed1992

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.

Alginates↗
PubMed1992

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.

Animals↗