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PubMed2004

Acid-inducible transcription of the operon encoding the citrate lyase complex of Lactococcus lactis Biovar diacetylactis CRL264.

Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

Adaptation, Physiological↗
PubMed2006

[Progress on lactococcus lactis expressing heterologous antigens as live mucosal vaccines].

Lactic acid bacteria (LAB) are natural commensals of the gastro-intestinal tracts of humans and most animals. They are considered to be safe bacteria with a GRAS (generally regarded as safe) status. The studies on LAB as live vehicles for expression of heterologous proteins or antigens have gained great progress in the past decades. Lactococcus lactis, an important species of LAB, possesses many properties that make it an ideal candidate for expressing and delivering heterologous proteins. With the significant advances in the genetic study of LAB, a series of constitutive or inducible gene expression systems as well as protein targeting systems had developed in Lactococcus lactis. Several antigens of pathogenic microorganisms, such as C subunit of tetanus toxin (TTFC) and the Brucella abortus ribosomal protein L7/L12, were successfully expressed in Lactococcus lactis. And it was showed that the recombinant Lactococcus lactis were able to induce specific mucosal immune response. It will be a great challenge how to make the recombinant Lactococcus lactis present heterologous proteins to the mucosal immune system in an effective way and induce specific immune response. One of the important directions for the further scientific development in this field is exploring protein targeting system to express heterologous antigens in precise locations of Lactococcus lactis. Expressing the antigen and cytokine simultaneously in Lactococcus lactis is an attractive direction as well. Using recombinant Lactococcus lactis that present antigens as live mucosal vaccines provides an effective way to prevent microorganisms invading and shows a promising future.

Animals↗
PubMed2001

Genetic localization and regulation of the maltose phosphorylase gene, malP, in Lactococcus lactis.

Maltose phosphorylase (MP) from Lactococcus lactis was purified and the corresponding gene was cloned and expressed in Escherichia coli. The isoelectric point of the pure enzyme was determined to be 7.0. According to zymogram analysis and SDS-PAGE, the native MP was shown to be a monomeric enzyme with a molecular mass of 75 kDa. A polyclonal antiserum was produced to assess the regulation of the gene encoding MP in LC: lactis. According to immunoblot analysis, synthesis of the enzyme was markedly repressed by both glucose and lactose in the growth medium. When the lactococci were cultivated in the presence of other sugars, including maltose, trehalose or galactose, there was a pronounced expression of the MP gene. In addition, when the cells were grown in media without any added sugar, there was also pronounced expression of the enzyme, according to immunoblot analysis and specific activity data. These results indicated that no particular sugar specifically induces the gene encoding MP. However, an effect of glucose on MP expression was demonstrated by performing fermentations in the presence of both maltose and glucose. When glucose was added to maltose-grown lactococci in the mid-exponential growth phase, both the specific activity and amount of MP per millilitre of cell extract decreased rapidly. The genetic locus for the MP gene was found to be in the vicinity of the region encoding a possible regulator belonging to the LacI-GalR family of transcriptional regulators. Furthermore, this genetic location was separated from the previously characterized maltose-inducible and glucose-repressible beta-phosphoglucomutase (beta-PGM) gene. The different genetic loci for the genes encoding MP and beta-PGM explains the different gene regulation behaviour.

Amino Acid Sequence↗
PubMed1990

Relationship between utilization of proline and proline-containing peptides and growth of Lactococcus lactis.

Proline, which is the most abundant residue in beta-casein, stimulates growth of Lactococcus lactis in a proline-requiring strain (Lactococcus lactis subsp. cremoris Wg2) and in a proline-prototrophic strain (Lactococcus lactis subsp. lactis ML3). Both strains lack a proline-specific uptake system, and free proline can enter the cell only by passive diffusion across the cytoplasmic membrane. On the other hand, lactococci can actively take up proline-containing peptides via the lactococcal di- and tripeptide transport system, and these peptides are the major source of proline. Consequently, lactococcal growth on amino acid-based media is highly stimulated by the addition of proline-containing di- and tripeptides. Growth of L. lactis subsp. lactis ML3 on chemically defined media supplemented with casein does not appear proline limited. Addition of dipeptides (including proline-containing peptides) severely inhibits growth on a casein-containing medium, which indicates that the specific growth rate is determined by the balanced supply of different di- or tripeptides which compete for the same di- and tripeptide transport system.

Amino Acid Sequence↗
PubMed2001

DNA sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms.

The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system. When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons. Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications.

Amino Acid Sequence↗
PubMed2004

Identification of Lactococcus lactis genes required for bacteriophage adsorption.

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.

Adsorption↗
PubMed1997

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase.

The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL. protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.

Acetyltransferases↗
PubMed2006

[Relief effect of beta-galactosidase genetically engineered lactococcus lactis on the cell toxicity caused by lactose].

OBJECTIVE: To assess the relief effect of beta-galactosidase genetically engineered Lactococcus lactis on the cell toxicity caused by lactose in vitro. METHODS: An in vitro toxic Caco-2 cell model caused by lactose was established to evaluate the relief effect of beta-galactosidase genetically engineered Lactococcus lactis. Cell morphological parameters and proliferation activity parameter were used. RESULTS: The in vitro toxic Caco-2 cell model caused by lactose was successfully established; the genetically engineered Lactococcus lactis constructed in the authors' laboratory could enable the Caco-2 cell to have normal appearance with the presence of lactose and could improve the proliferation activity with the presence of high concentration of lactose (P < 0.01). CONCLUSION: The beta-galactosidase genetically engineered Lactococcus lactis has significant relief effect on the cell toxicity caused by lactose in vitro, which lays a foundation for food-grade alternation of this bacterium.

Caco-2 Cells↗
PubMed1996

Transfer of conjugative plasmid pAM beta 1 from Lactococcus lactis to mouse intestinal bacteria.

Conjugal transfer of plasmid pAM beta 1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 x 10-3 and with lower frequencies to other species. In vivo, using gastric intubation with the pAM beta 1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis. However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 x 10-3, in mice whose anus was artificially closed after gastric intubation with pAM beta 1-bearing Lactococcus lactis. These results demonstrate clearly that pAM beta 1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.

Animals↗
PubMed1994

A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis.

A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced. Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L. lactis. Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).

Base Sequence↗
PubMed1991

A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis.

Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression. Mutations were made such that the DNA sequence upstream of the ATG start codon was not changed. Moreover, care was taken that the substitutions, which were all within the first six codons, neither affected the amino acid sequence of the gene product nor introduced codons rarely used in L. lactis. The results suggest that mRNA secondary structure contributes to the efficiency of translation initiation in L. lactis.

Base Sequence↗
PubMed1990

Simultaneous loss of N5-(carboxyethyl)ornithine synthase, nisin production, and sucrose-fermenting ability by Lactococcus lactis K1.

A spontaneous derivative of Lactococcus lactis subsp. lactis K1 (formerly Streptococcus lactis K1) lacking N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) was isolated. This mutant had also lost the abilities to ferment sucrose and to produce the antibiotic nisin. Hybridization studies indicate that these linked traits are encoded on the chromosome of L. lactis K1 and that they may be located on a conjugative transposon.

Amino Acid Oxidoreductases↗
PubMed1995

Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis.

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

Amino Acids↗
PubMed1995

Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80.

Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.

Amino Acid Sequence↗
PubMed1988

Utilization of dipeptides by Lactococcus lactis ssp. cremoris.

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.

Chromatography, High Pressure Liquid↗
PubMed2007

Expression of plant flavor genes in Lactococcus lactis.

Lactic acid bacteria, such as Lactococcus lactis, are attractive hosts for the production of plant-bioactive compounds because of their food grade status, efficient expression, and metabolic engineering tools. Two genes from strawberry (Fragaria x ananassa), encoding an alcohol acyltransferase (SAAT) and a linalool/nerolidol synthase (FaNES), were cloned in L. lactis and actively expressed using the nisin-induced expression system. The specific activity of SAAT could be improved threefold (up to 564 pmol octyl acetate h-1 mg protein-1) by increasing the concentration of tRNA1Arg, which is a rare tRNA molecule in L. lactis. Fermentation tests with GM17 medium and milk with recombinant L. lactis strains expressing SAAT or FaNES resulted in the production of octyl acetate (1.9 microM) and linalool (85 nM) to levels above their odor thresholds in water. The results illustrate the potential of the application of L. lactis as a food grade expression platform for the recombinant production of proteins and bioactive compounds from plants.

Acyltransferases↗
PubMed1994

A variant of the staphylococcal chloramphenicol resistance plasmid pC194 with enhanced ability to transform Lactococcus lactis subsp. lactis.

In our attempts to transform Lactococcus lactis subsp. lactis with pC194, a staphylococcal chloramphenicol resistance plasmid, only a few transformants could be obtained and only when relatively large amounts of plasmid DNA were used. However, when pC194 DNA from lactococcal transformants was introduced back to Staphylococcus aureus and reisolated, it could be retransformed into L. lactis at substantially higher frequencies. It was concluded that pC194 had undergone mutation expanding its host range. By exchanging DNA fragments between the original pC194 and the variant transforming L. lactis (named pVS41), the mutation could tentatively be located within the 1.1-kbp AccI-HaeIII fragment. Comparison of the DNA sequences in the vicinity of the replication plus-origin revealed the formation of an "opal" stop codon (TGA) apparently interrupting the synthesis of the putative protein C for which no function has been described. Cloning this mutation within a 194 bp AccI-MspI fragment on pC194 made this plasmid able to transform L. lactis. Whether the mutation somehow affects functions controlling plasmid host-specificity, or whether the extended host range reflects, for example, mutational inactivation of some lactococcal restriction site, cannot yet be stated on the basis of these data.

Base Sequence↗