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PubMed1996

Characterization of a mosaic ISS1 element and evidence for the recent horizontal transfer of two different types of ISS1 between Streptococcus thermophilus and Lactococcus lactis.

A 12-kb region of the Streptococcus thermophilus CNRZ368 chromosome was found to contain two copies of IS981 (one complete and one truncated) and three copies of ISS1 (two complete, ISS1SA and ISS1SC, and one truncated, delta ISS1SB). Comparison of the nucleotide sequences of these ISS1 elements with those of previously identified iso-ISS1 elements from Lactococcus lactis and the Enterococcus genus indicated that the ISS1 group is divided into three distinct subgroups which we have named alpha, beta and gamma. Nucleotide sequences of elements belonging to the same subgroup share more than 97% identity whereas sequences of elements from different groups share only 75-85% identity. Sequence analysis of ISS1SA and delta ISS1SB showed that they are members of the alpha group. We found that ISS1SC from S. themophilus CNRZ368, an ISS1 from L. lactis IL964 and IS946 from L. lactis TEK1 resulted from recombinations between alpha and beta elements. In addition, ISS1W from L. lactis Wg2 resulted from a recombination event between a gamma element and an ISS1 belonging to an unidentified subgroup. ISS1 sequences belonging to the alpha and beta subgroups were found in both S. thermophilus and L. lactis and gamma sequences were found in both the Enterococcus genus and L. lactis. The quasi-identity of some ISS1 elements in S. thermophilus and L. lactis and the distribution of alpha and beta elements suggest that horizontal transfer of ISS1 elements recently took place from L. lactis to S. thermophilus, two lactic acid bacteria used in the manufacture of cheeses. Since the presence of IS981 in S. thermophilus CNRZ368 also probably resulted from a horizontal transfer from L. lactis [Guédon et al. (1995) Mol. Microbiol. 16, 69-78], the 12-kb region bearing IS981 and ISS1 elements could be due to the integration of a lactococcal DNA fragment into the chromosome.

Base Sequence↗
PubMed2005

A food-grade expression/secretion vector for Lactococcus lactis that uses an alpha-galactosidase gene as a selection marker.

A new food-grade expression/secretion vector for lactococci, pFMN30, was developed using an alpha-galactosidase gene (melA) of Lactobacillus plantarum as a selection marker. The 4.9-kb pFMN30 is a derivative of the lactococcal vector pMG36e containing a broad-host-range replicon of pWV01. In Lactococcus lactis, transformants carrying the vector were easily detectable by the appearance of a blue colony on a X-alpha-gal-containing medium and also by the growth on a medium containing melibiose as a sole carbon source. The expression/secretion vector was equipped with the controllable and strong nisA promoter. In addition, usp45 signal peptide was inserted for the efficient secretion of a foreign protein outside cells. The vector pFMN30 was used for the expression and secretion of alpha-amylase as a reporter gene, lacking a signal sequence derived from Bacillus licheniformis in L. lactis. These results show that the food-grade expression/secretion vector constructed in the present study could be used for the production of foreign proteins in L. lactis for the production food materials and also for the medicinal purposes.

Bacterial Proteins↗
PubMed2004

Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator.

CodY is a pleiotropic transcriptional regulator conserved in low-G+C Gram-positive bacteria. Two distinct signals have been shown independently to influence the activity of this regulator: the level of intracellular GTP in Bacillus subtilis and the level of intracellular branched-chain amino acids (BCAA) isoleucine, leucine and valine in Lactococcus lactis. Measurement of BCAA and GTP levels in several environmental conditions showed that L. lactis CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. In addition, we demonstrated that CodY from L. lactis did not respond to intracellular GTP even when complementing CodY activity in B. subtilis. However, L. lactis CodY activity could still be modulated in B. subtilis by adding a rich nitrogen source to the growth media. This finding suggests that only BCAA are sensed by L. lactis CodY, whereas both GTP and BCAA signals may be integrated by B. subtilis CodY. The difference in the function of CodY from B. subtilis and L. lactis seems to reflect the difference in the physiology of these two bacteria.

Adaptation, Physiological↗
PubMed2000

Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.

The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine. Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. This approach may lead to better methods for cost-effective and long-term management of IBD in humans.

Animals↗
PubMed2003

Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis.

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the anti-estrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [3H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2004

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729.

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.

Anti-Infective Agents↗
PubMed2003

Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection.

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.

Bacterial Proteins↗
PubMed1993

Gene transfer from engineered Lactococcus lactis strains to Enterococcus faecalis in the digestive tract of gnotobiotic mice.

The introduction of genetically modified organisms into food products requires an evaluation of the behaviour and the dissemination of foreign genes of such organisms among the human intestinal microflora. The conjugal transfer, both in vitro and in vivo (in mice digestive tract) of DNA from Lactococcus lactis donor strains to an Enterococcus faecalis strain isolated from human faecal flora was studied. We followed the transfer of (1) the self-transmissible plasmid pIL205; (2) two non-self-transmissible but mobilizable plasmids, pIL252 and pIL253; (3) one plasmid, pMS1.5B, integrated into the chromosome of L. lactis. In vitro, the transfer frequency of pIL205 (expressed as the number of transconjugants per donor cell) was 9.6 x 10(-4); mobilization of one of the non-self-transmissible plasmids, pIL253, was observed (4.9 x 10(-7)). In vivo, only transfer of pIL205 and pIL253 occurred, but the frequency was not determined. The transfer of pMS1.5B was not detected in vitro or in vivo.

Animals↗
PubMed2004

Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis.

Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000DeltahtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.

Adhesins, Bacterial↗
PubMed2000

Investigation of the relationship between lysogeny and lysis of Lactococcus lactis in cheese using prophage-targeted PCR.

The ability of lactococcal strains to lyse (and release intracellular enzymes) during cheese manufacture can be a very desirable trait and has been associated with improvement in flavor and acceleration of cheese ripening. Using a laboratory-scale cheese manufacturing assay, the autolytic behavior of 31 strains of Lactococcus lactis was assessed. In general, marked variation was observed between strains with a 20-fold difference between the best and worst lysing strains based on the release of the intracellular enzyme lactate dehydrogenase. In a parallel experiment, the genomes of these strains were examined for the presence of prophage integrase (int) sequences by using conserved primer sequences from known lysogenic phage. Results demonstrated that the lytic behavior of lactococcal starter strains significantly correlates with the presence of prophage sequences. These results highlight not only the contribution of prophage to starter cell lysis but also the potential of PCR as a useful initial screen to assess strains for this important industrial trait.

Cheese↗
PubMed1994

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: relationships with malic enzymes.

Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.

Amino Acid Sequence↗
PubMed1990

Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis.

A number of host-encoded phage resistance mechanisms have been described in lactococci. However, the phage genome has not been exploited as a source of additional resistance determinants. A 4.5-kb BamHI-HindIII fragment of phage nck202.50 (phi 50) was subcloned in streptococcus-Escherichia coli shuttle plasmid pSA3 and introduced into Lactococcus lactis NCK203 and MG1363 by protoplast transformation. This cloned phage fragment directed a bacteriophage resistance phenotype designated Per (phage-encoded resistance). Both phi 50 and a distantly related phage, nck202.48 (phi 48), formed small plaques on strain NCK213 at a slightly reduced efficiency of plaquing on the Per+ host. The per locus was further reduced to a 1.4-kb fragment through in vitro deletion analysis. The 1.4-kb fragment was sequenced, and the Per phenotype was found to be associated with a ca. 500-bp region rich in direct and inverted repeats. We present evidence that the Per region contains a phage origin of replication which, in trans, may interfere with phage replication by titration of DNA polymerase or other essential replication factors. It was demonstrated that the Per+ phenotype is not a result of reduced adsorption or action of a restriction and modification system. Per+ activity was not detected against six independent phages which were previously shown to be sensitive to the Hsp+ mechanism. The mutually exclusive resistance mechanisms could be combined to confer resistance to both types of phages (Hsp resistant and Per resistant) in a single host. This is the first description in lactococci of a phage resistance phenotype, other than superinfection immunity, originating from a lactococcal phage genome.

Amino Acid Sequence↗
PubMed2005

Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains.

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.

Lactococcus lactis↗
PubMedDate not supplied

Worst-case scenarios for horizontal gene transfer from Lactococcus lactis carrying heterologous genes to Enterococcus faecalis in the digestive tract of gnotobiotic mice.

Since genetically modified (GM) lactic acid bacteria (LAB) might be released in open environments for future nutritional and medical applications, the purpose of this study was to determine an upper limit for the horizontal gene transfer (HGT) in the digestive tract (DT) from Lactococcus lactis carrying heterologous genes (lux genes encoding a bacterial luciferase) to Enterococcus faecalis. Two enterococcal wide host-range conjugative model systems were used: (i) a system composed of a mobilizable plasmid containing the heterologous lux genes and a native conjugative helper plasmid; and (ii) a Tn916-lux transposon. Both systems were tested under the most transfer-prone conditions, i.e. germfree mice mono-associated with the recipient E. faecalis. No transfer was observed with the transposon system. Transfers of the mobilizable plasmid carrying heterologous genes were below 10(2) transconjugants per g of faeces for a single donor dose and reached between 10(3) and 10(4) transconjugants per g of faeces when continuous inoculation of the donor strain was used. Once established in mice, transconjugants persisted at low levels in the mouse DT.

Animals↗
PubMed1994

Comparative analysis of gene expression in Streptococcus pneumoniae and Lactococcus lactis.

The pFL10 plasmid vector for translational fusions was constructed. pFL10 is based in the promiscuous pLS1 replicon and contains the pC194 cat gene deprived of its transcriptional promoter and Shine-Dalgarno (SD) sequence. Three promoters (Pcit, PpolA and PtetL) from Gram-positive bacteria, inserted in pFL10, were tested for their ability to drive transcription in Lactococcus lactis and Streptococcus pneumoniae. These promoters were coupled to the SD sequence of the lactococcal citP gene fused to the cat gene. Determination of the 5' ends of the three mRNAs by primer extension revealed the same start sites in both bacterial systems. However, it was observed a differential efficiency of promoter utilization by the RNA polymerases from the two hosts. The transcriptional behavior correlates with expression of the cat gene measured by determinations of chloramphenicol acetyltransferase (CAT) activity. Substitution of the SD of citP by that of the T7 phi 10 gene rendered a similar decrease of the CAT production in both bacterial systems.

Base Sequence↗
PubMed2000

ctsR of Lactococcus lactis encodes a negative regulator of clp gene expression.

Bacteria undergo a complex programme of differential gene expression in response to stress. In Bacillus subtilis, it was recently shown that CtsR, a negative transcriptional regulator, mediates stress-induced expression of components of the Clp protease complex. In this study, a gene was identified in the Gram-positive bacterium Lactococcus lactis that encodes a 17 kDa product with 38% identity to the CtsR protein of B. subtilis. By Northern analyses it was found that in a L. lactis strain carrying a large internal deletion of ctsR, including the region encoding a putative helix-turn-helix motif, the amounts of clpC, clpP, clpB and clpE mRNAs were increased 3-8-fold compared to those present in wild-type L. lactis MG1363. In another ctsR mutant strain in which only one-third of CtsR was deleted, leaving the putative DNA-binding domain and the C-terminal 29 amino acids intact, only minor derepression of clp gene expression was observed and, furthermore, all the clp genes were still induced by heat. These results indicate that the amino acids of CtsR involved in temperature sensing are located either close to the DNA-binding domain or in the C-terminal part of the protein. Thus, in L. lactis in addition to B. subtilis, CtsR is a key regulator of heat-shock-induced gene expression, suggesting that the presence of CtsR-homologous DNA-binding sites observed in many Gram-positive bacteria reflects functional heat-shock regulatory systems.

Adenosine Triphosphatases↗
PubMed2004

A Plasmodium falciparum GLURP-MSP3 chimeric protein; expression in Lactococcus lactis, immunogenicity and induction of biologically active antibodies.

Plasmodium falciparum malaria is a major cause of morbidity and mortality worldwide. To evaluate the efficacy of a possible vaccine antigen against P. falciparum infection, a fusion protein, derived from P. falciparum Glutamate-rich protein (GLURP) genetically coupled to P. falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 fusion protein. The hybrid protein was purified to homogeneity by ion exchange and hydrophobic-interaction chromatography and its composition was verified by MALDI MS, SDS/PAGE and Western blotting with antibodies against antigenic components of GLURP and MSP3. Mice immunized with the hybrid protein produced higher levels of both GLURP- and MSP3-specific antibodies than mice immunized with either GLURP, MSP3 or a mix of both. The protective potential of the hybrid protein was also demonstrated by in vitro parasite-growth inhibition of mouse anti-GLURP-MSP3 IgG antibodies in a monocyte-dependent manner. These results indicate that the GLURP-MSP3 hybrid could be a valuable strategy for future P. falciparum vaccine development.

Animals↗
PubMed2000

Viscoelastic properties of an exocellular polysaccharide produced by a Lactococcus lactis.

The viscoelastic properties of a well-characterized exocellular polysaccharide (EPS) produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain B40 were investigated. Dynamic rheological measurements were made as a function of frequency and EPS concentration. The bead-spring model of Rouse could reasonably describe the dynamic properties. Concentrated EPS solutions have a significant elasticity (G' > G") at high frequencies. The relatively high G' values at high concentrations and high frequencies are indicative of significant normal stress differences, and we put forward a hypothesis that suggests that these normal stresses may explain the contribution of EPSs to the ropy behavior of yogurts.

Algorithms↗