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PubMed1991

Nucleotide sequence of a Lactococcus lactis gene cluster encoding adenylate kinase, initiation factor 1 and ribosomal proteins.

We have previously isolated a putative promoter from the Lactococcus lactis subsp. lactis chromosome. We now report the sequence of the promoter fragment and its extension in the 5'-direction. The region contains several open-reading frames which correspond to ribosomal protein L15, SecY, adenylate kinase, initiation factor 1 and ribosomal proteins B and S13. The order of the genes, rplO (L15), secY, adk, infA, rpmJ (B) and rpsM (S13), is similar to that in the spc and alpha operon region of Bacillus subtilis, with the exception of the map gene, coding for methionine amino peptidase, which is located between adk and infA in B. subtilis. The putative promoter is located between adk and infA.

Adenylate Kinase↗
PubMed1999

Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis.

In a previous study, we described the use of transposon Tn917-LTV1 for identification of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus -35 promoter region, but it contained an 'extended' -10 promoter region. When a 28 bp segment, containing the consensus -35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH confirmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 defined a specific region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150- to 200-fold increase in the level of gene expression, without affecting the regulation. The functionality was confirmed by introducing this modulating element downstream of other lactococcal promoters.

Base Sequence↗
PubMed1999

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH.

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.

Acetates↗
PubMed2005

In situ activity of a bacteriocin-producing Lactococcus lactis strain. Influence on the interactions between lactic acid bacteria during sourdough fermentation.

AIMS: To biochemically characterize the bacteriocin produced by Lactococcus lactis ssp. lactis M30 and demonstrate its effect on lactic acid bacteria (LAB) during sourdough propagation. METHODS AND RESULTS: A two-peptide bacteriocin produced by L. lactis ssp. lactis M30 was purified by ion exchange, hydrophobic interaction and reversed phase chromatography. Mass spectrometry of the two peptides and sequence analysis of the ltnA2 gene showed that the bacteriocin was almost identical to lacticin 3147. During a 20-day period of sourdough propagation the stability of L. lactis M30 was demonstrated, with concomitant inhibition of the indicator strain Lactobacillus plantarum 20, as well as the non-interference with the growth of the starter strain Lact. sanfranciscensis CB1. CONCLUSIONS: In situ active bacteriocins influence the microbial consortium of sourdough LAB and can "support" the dominance of insensitive strains during sourdough fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The in situ bacteriocinogenic activity of selected lactococci enables the persistence of insensitive Lact. sanfranciscensis strains, useful to confer good characteristics to the dough, at a higher cell concentration with respect to other LAB of the same ecosystem.

Anti-Bacterial Agents↗
PubMed1998

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗
PubMed2006

Therapeutic drug delivery by genetically modified Lactococcus lactis.

Food-grade bacteria have been consumed throughout history without associated pathologies and are, therefore, absolutely safe to ingest. Unexpectedly, Lactococcus lactis (L. lactis), known from cheese production, can be genetically engineered to constantly secrete satisfactory amounts of bioactive cytokines. Both of these features enabled the development of a new kind of topical delivery system: topical and active delivery of therapeutic proteins by genetically modified micro-organisms. The host organism's record inspired the development of applications that target intestinal diseases. In a variety of mouse models, chronic colon inflammation can be successfully treated with (interleukin) IL-10-secreting L. lactis. Trefoil factor (TFF) producer strains have also been shown to be very effective in the treatment of acute colitis. Such novel therapeutic strains are textbook examples of genetically modified (GM) organisms. There are legitimate concerns with regard to the deliberate release of GM micro-organisms. On development of these applications, therefore, we have engineered these bacteria in such a way that biological containment is guaranteed. The essential gene thyA, encoding thymidylate synthase, has been exchanged for IL-10. This makes the GM strain critically dependent on thymidine. Lack of thymidine, for example, resulting from thymidine consumption by thyA-deficient strains-will irreversibly lead to induced "thymidine-less death." This accomplishment has created the possibility of using this strategy for application in human medicine.

Animals↗
PubMed1997

Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1.

Bacteriophage sk1 is a small isometric-headed lytic phage belonging to the 936 species. It infects Lactococcus lactis, a commonly used dairy starter organism. Nucleotide sequence data analysis indicated that the sk1 genome is 28,451 nucleotides long and contains 54 open reading frames (ORFs) of 30 or more codons, interspersed with three large intergenic regions. The nucleotide sequence of several of the sk1 ORFs demonstrated significant levels of identity to genes (many encoding proteins of unknown function) in other lactococcal phages of both small isometric-headed and prolate-headed morphotype. Based on this identity and predicted peptide structures, sk1 genes for the terminase, major structural protein and DNA polymerase have been putatively identified. Genes encoding holin and lysin were also identified, subcloned into an Escherichia coli expression vector, and their function demonstrated in vivo. The sk1 origin of replication was located by identifying sk1 DNA fragments able to support the maintenance in L. lactis of a plasmid lacking a functional Gram-positive ori. The minimal fragment conferring replication origin function contained a number of direct repeats and 179 codons of ORF47. Although no similarity between phage sk1 and coliphage lambda at the nucleotide or amino acid sequence level was observed, an alignment of the sk1 late region ORFs with the lambda structural and packaging genes revealed a striking correspondence in both ORF length and isoelectric point of the ORF product. It is proposed that this correspondence is indicative of a strong conservation in gene order within these otherwise unrelated isometric-headed phages that can be used to predict the functions of the sk1 gene products.

Base Sequence↗
PubMed1998

A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences.

Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Lactococcus lactis subsp. cremoris strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized. Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS905 element in each junction. Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase. Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS905 element. The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the oriC-terC symmetry of the chromosome and the local genetic environment.

Base Sequence↗
PubMed1990

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes.

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.

Cloning, Molecular↗
PubMed2004

Two homologous oligopeptide binding protein genes (oppA) in Lactococcus lactis opp2 [corrected].

In previous studies, it has been shown that inactivation of opp or even oppA abolishes the capacity of Lactococcus lactis to utilize oligopeptides. We now show that the opp operon has been duplicated in L. lactis MG1363. The nucleotide sequence of the oppA and oppC homologues (appA and appC) and most of the oppB homologue (appB) indicate that the corresponding protein sequences are 83%, 92% and 91% identical, respectively. Inactivation of appA, via homologous recombination, as well as complementation studies were carried out to determine the possible function of appA in peptide utilization. As anticipated from studies with an oppA knock-out, peptide utilization was not impaired in an appA disruption mutant. Importantly, AppA expressed from a plasmid could restore the ability of oppA deletion mutants to utilize Leu-enkephalin, albeit with a lower efficiency than OppA. The differences in the ability to utilize this pentapeptide were not due to differences in expression levels but most likely reflect a different catalytic efficiency in oligopeptide utilization when AppA is used as ligand receptor.

Amino Acid Sequence↗
PubMed1998

Characterization of multiple regions involved in replication and mobilization of plasmid pNZ4000 coding for exopolysaccharide production in Lactococcus lactis.

We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in Lactococcus lactis NIZO B40. The plasmid contains four highly conserved replication regions with homologous rep genes (repB1, repB2, repB3, and repB4) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in L. lactis. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different L. lactis strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an oriT sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these oriT sequences, demonstrating their functionality. One oriT sequence was followed by a mobA gene, coding for a trans-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and the oriT sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat and nic site of the oriT from the Escherichia coli plasmid R64. The presence on pNZ4000 of four functional replicons, two oriT sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant.

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.

Base Sequence↗
PubMed1995

Cloning and sequencing of LlaDCHI [corrected] restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaDCHI [corrected]. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaDCHI [corrected] endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI [corrected] was localized. Cloning and sequencing of the entire LlaDCHI [corrected] system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaDCHI [corrected] system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlalII shared only 31% identity with R.DpnII.

Amino Acid Sequence↗
PubMed1990

The bacteriophage kh receptor of Lactococcus lactis subsp. cremoris KH is the rhamnose of the extracellular wall polysaccharide.

A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and ml3. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.

Bacteriophages↗
PubMed2005

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning.

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.

Acetylglucosaminidase↗
PubMed1992

Antisense RNA directed against the major capsid protein of Lactococcus lactis subsp. cremoris bacteriophage 4-1 confers partial resistance to the host.

Antisense RNA targeted against the major capsid protein (MCP) of Lactococcus lactis subsp. cremoris bacteriophage F4-1 reduced bacteriophage replication by up to 50%. The region containing the mcp gene was oriented to transcribe the antisense strand using a L. lactis subsp. cremoris Wg2 promoter. The size of the mcp insert transcribed affected the level of bacteriophage inhibition and the greatest level of inhibition was achieved using a 301-bp fragment from the 5' end of the mcp. Antisense mcp RNA constructs were stable and did not alter the endogenous plasmid profile in the host, L. lactis subsp. cremoris F4-1. There were, however, some adverse effects on the host during the stationary phase as exhibited by a decline in cell density.

Bacteriophages↗
PubMed2003

Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.

Adenosine Triphosphatases↗
PubMed1996

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.

Amino Acid Sequence↗