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PubMed2002

Food-grade delivery system for controlled gene expression in Lactococcus lactis.

A food-grade system for the delivery of desired genes to Lactococcus lactis, their inducible expression, and their transfer to related strains was established. Based on the thermosensitive pG(+)host replicon, two types of plasmid vectors were constructed which contained sections of either the chromosomal leu operon of L. lactis or the tel operon from the lactococcal sex factor. Genes cloned into the leu or tel sequences of these vectors were delivered to the homologous regions of the chromosome or the sex factor through two single crossovers, leading to integration of the recombinant plasmids and subsequent excision of the vector portions. Inducible transcription of integrated genes was achieved by using the nisin-controlled expression (NICE) system. To establish the signal transduction genes nisRK in L. lactis, the vectors pLNG1363 (targeted to the chromosome) and pUK500 (targeted to the sex factor) were constructed. Fusions of six different peptidase genes (pep) from Lactobacillus delbrueckii with the nisin-inducible promoter P(nisA) were delivered to the sex factor with derivatives of the vector pUK300. Food-grade recombinants of L. lactis were constructed which had the nisRK genes and individual P(nisA)::pep fusions integrated either separately into the chromosome and the sex factor or simultaneously into the sex factor. With both types of recombinants, expression of P(nisA)::pep fusions after induction with nisin was demonstrated. Depending on the loci used for integration of nisRK, variable induction rates were observed. Furthermore, an engineered sex factor carrying a P(nisA)::pepI fusion was transfered by conjugation between two strains of L. lactis at a frequency of 4 x 10(-4).

Bacterial Proteins↗
PubMed2005

Alternative lactose catabolic pathway in Lactococcus lactis IL1403.

In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-beta-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-beta-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-beta-glucosidase, it also exhibits phospho-beta-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.

Bacterial Proteins↗
PubMed1990

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1.

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.

Base Sequence↗
PubMed1992

Transfer of Tn916 between Lactococcus lactis subsp. lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363.

Lactococcus lactis subsp. lactis MG1363 can act as a conjugative donor of chromosomal markers. This requires a chromosomally located fertility function that we designate the lactococcal fertility factor (Laff). Using inter- and intrastrain crosses, we identified other L. lactis strains (LMO230 and MMS373) that appear to lack Laff. The selectable marker in our crosses was Tcr, carried by Tn916, a transposon present on the chromosome. The transfer of Tcr was not due to Tn916-encoded conjugative functions, because (i) L. lactis cannot act as a donor in Tn916-promoted conjugation (F. Bringel, G. L. Van Alstine, and J. R. Scott, Mol. Microbiol. 5:2983-2993, 1992) and (ii) transfer occurred when the Tcr marker was present in a Tn916 derivative containing a mutation, tra-641, that prevents Tn916-directed conjugation in any host. In addition, we isolated a strain in which Tn916 appears to be linked to Laff; this strain should be useful for further analysis of this fertility factor. In this strain, Tn916 is on the same 600-kb SmaI fragment as Clu, a fertility factor previously shown to promote lactose plasmid transfer in L. lactis. Thus, it is possible that Clu and Laff are identical.

Base Sequence↗
PubMed1998

Transcriptional regulation and evolution of lactose genes in the galactose-lactose operon of Lactococcus lactis NCDO2054.

The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes. Initially the beta-galactosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment. Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order is galK-galT-lacA-lacZ-galE; the gal genes encode enzymes of the Leloir pathway for galactose metabolism, and lacA encodes a galactoside acetyltransferase. The galT and galE genes of L. lactis LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting L. lactis C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening lacA-lacZ strain NCDO2054. Analysis of mRNA for the gal and lac genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the lac genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless beta-glucuronidase gene (gusA) from E. coli, which identified three lactose-inducible intergenic promoters in the gal-lac operon. The greater similarity of the lacA and lacZ genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the gal genes, suggests that the genes within the gal operon of L. lactis NCDO2054 have been recently acquired. Thus, the lacA-lacZ genes appear to have engaged the promoters of the gal operon in order to direct and control their expression.

Amino Acid Sequence↗
PubMed2005

Lactococcin MMT24, a novel two-peptide bacteriocin produced by Lactococcus lactis isolated from rigouta cheese.

Lactococcin MMT24 is a novel bacteriocin produced by Lactococcus lactis MMT24, a strain isolated from a Tunisian traditional cheese. The bacteriocin shows a narrow antimicrobial activity against closely related lactic acid bacteria. Lactococcin MMT24 is heat resistant, remains active after incubation at pH 3 to 10, lyophilization, long-term storage at -20 degrees C and is sensitive to treatment with proteolytic enzymes. The mode of action of lactococcin MMT24 was identified as bactericidal. Purification of the active compound showed that lactococcin MMT24 consists of two distinct peptides, named pepalpha and pepbeta, whose complementary action is necessary for full antibacterial activity. Optimal antibacterial activity was obtained when the complementary peptides pepalpha and pepbetawere present in equal amounts. Mass spectrometry analysis showed masses of 3765.33 Da and 3255.26 Da for pepalpha and pepbeta, respectively. These molecular masses do not correspond to those of so far described bacteriocins. Addition of 50 nmol l(-1) of lactococcin MMT24 to cells of L. lactis ssp. cremoris ATCC11603 induced increase in the concentration of K+ in supernatant indicating a massive leakage of this ion from the cells. This release was most likely caused by pores formation by the pepalphaand pepbeta peptides in the target bacterial membrane.

Anti-Bacterial Agents↗
PubMed2002

How to distinguish between the vacuum cleaner and flippase mechanisms of the lmrA multi-drug transporter in Lactococcus lactis.

A numerical model of the LmrA multi-drug transport system of Lactococcus lactis is used to explore the possibility of distinguishing experimentally between two putative transport mechanisms, i.e., the vacuum-cleaner and the flippase mechanisms. This comparative model also serves as an example of numerical simulation with the scripting language Python and its scientific add-on Scipy.

Bacterial Proteins↗
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↗
PubMed1992

Structure of the extracellular polysaccharide from slime-forming Lactococcus lactis subsp. cremoris SBT 0495.

The extracellular polysaccharide obtained from slime-forming Lactococcus lactis subsp. cremoris SBT 0495 is composed of D-glucose, D-galactose, L-rhamnose, and phosphate. Methylation analysis of the native and dephosphorylated polysaccharides provided information on the linkage of the sugar residues and the location of the phosphate group. N.m.r. spectroscopy confirmed the structure of the polysaccharide, which is assigned the following repeating-unit: [formula: see text]

Carbohydrate Sequence↗
PubMed2006

Cloning and production of a novel bacteriocin, lactococcin K, from Lactococcus lactis subsp. lactis MY23.

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml(-1) in 12 h.

Amino Acid Sequence↗
PubMed2001

Tolerance to high osmolality of Lactococcus lactis subsp. lactis and cremoris is related to the activity of a betaine transport system.

Lactococcus lactis strains from the subsp. cremoris are described as more sensitive to osmotic stress than subsp. lactis strains. We examined the relation between osmotic tolerance and the activity of the betaine transporter BusA among 34 strains of L. lactis. The cremoris strains that showed reduced growth at high osmolality failed to accumulate betaine. The nature of the defect was found to vary among cremoris strains: lack of the busA encoding region, absence of synthesis or synthesis of an inactive form of BusA. The results suggest that the selection of strains well fitted to the dairy production lead to the loss of an otherwise efficient adaptation mechanism.

ATP-Binding Cassette Transporters↗
PubMed1991

Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide.

Lactococcin, a bacteriocin produced by Lactococcus lactis subsp. lactis ADRIA 85LO30, was purified as a 2.3-2.4 kDa peptide. Six non-bacteriocin-producing (Bac-) and non-immune (Imm-) strains were isolated after curing experiments. These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb). By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.

Bacteriocins↗
PubMed1990

Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis.

The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.

Chromosomes, Bacterial↗
PubMed2002

Rapid PCR-based method which can determine both phenotype and genotype of Lactococcus lactis subspecies.

A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.

Amino Acid Sequence↗
PubMed1992

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Bacteriophages↗
PubMed2005

The Lactococcus lactis CodY regulon: identification of a conserved cis-regulatory element.

CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. DNA microarray analysis, comparing the expression profiles of L. lactis MG1363 and an isogenic strain in which codY was mutated, was used to determine the CodY regulon. In peptide-rich medium and exponentially growing cells, where CodY exerts strong repressing activity, the expression of over 30 genes was significantly increased upon removal of codY. The differentially expressed genes included those predominantly involved in amino acid transport and metabolism. In addition, several genes belonging to other functional categories were derepressed, stressing the pleiotropic role of CodY. Scrutinizing the transcriptome data with bioinformatics tools revealed the presence of a novel over-represented motif in the upstream regions of several of the genes derepressed in L. lactis MG1363DeltacodY. Evidence is presented that this 15-bp cis-sequence, AATTTTCWGAAAATT, serves as a high affinity binding site for CodY, as shown by electrophoretic mobility shift assays and DNase I footprinting analyses. The presence of this CodY-box is sufficient to evoke CodY-mediated regulation in vivo. A copy of this motif is also present in the upstream region of codY itself. It is shown that CodY regulates its own synthesis and requires the CodY-box and branched-chain amino acids to interact with its promoter.

Bacterial Proteins↗
PubMed2005

In vitro expression of the restriction endonucleases LlaMI and ScrFI isolated from Lactococcus lactis M19 and UC503.

A new restriction endonuclease LlaMI has been characterized in Lactococcus lactis subsp. cremoris M19. LlaMI recognizes the sequence 5'-CCNGG-3' and cuts after the second cytosine. This restriction endonuclease is related to commercially available ScrFI but not identical to it. Comparative analysis of the predicted amino acid sequences of LlaMI and ScrFI indicates five non-conservative amino acid changes between these two restriction enzymes. These two enzymes were expressed in vitro as histidine-tagged fusion proteins. LlaMI was shown to be more sensitive to high salt concentration than ScrFI. Southern blotting and hybridization analysis indicate that the gene for LlaMI R/M system is chromosomally encoded.

Amino Acid Substitution↗
PubMed1997

Heterologous gene expression of bovine plasmin in Lactococcus lactis.

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active.

Animals↗