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PubMed1995

Cloning and molecular analysis of the dihydrofolate reductase gene from Lactococcus lactis.

The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis. Several lines of evidence indicate that the cloned gene encodes dihydrofolate reductase (DHFR). (i) It fully complements the fol "null" mutation in E. coli. (ii) Nucleotide sequencing of the cloned fragment revealed the presence of one open reading frame encoding a protein that shares homology with the family of bacterial DHFR enzymes. (iii) Overexpression of this open reading frame in E. coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dramatic increase of DHFR activity. In cell extracts, the DHFR activity was not inhibited by low trimethoprim concentration. By Northern (RNA) blotting and primer extension analyses, the size and the start point of the dhfr transcript, respectively, have been determined. Results of these experiments indicate that in L. lactis the dhfr gene represents part of a larger transcription unit.

Amino Acid Sequence↗

PubMed2006

LmrCD is a major multidrug resistance transporter in Lactococcus lactis.

When Lactococcus lactis is challenged with drugs it displays a multidrug resistance (MDR) phenotype. In silico analysis of the genome of L. lactis indicates the presence of at least 40 putative MDR transporters, of which only four, i.e. the ABC transporters LmrA, LmrC and LmrD, and the major facilitator LmrP, have been experimentally associated with the MDR. To understand the molecular basis of the MDR phenotype in L. lactis, we have performed a global transcriptome analysis comparing four independently isolated drug-resistant strains of L. lactis with the wild-type strain. The results show a strong and consistent upregulation of the lmrC and lmrD genes in all four strains, while the mRNA levels of other putative MDR transporters were not significantly altered. Deletion of lmrCD renders L. lactis sensitive to several toxic compounds, and this phenotype is associated with a reduced ability to secrete these compounds. Another gene, which is strongly upregulated in all mutant strains, specifies LmrR (YdaF), a local transcriptional repressor of lmrCD that belongs to the PadR family of transcriptional regulators and that binds to the promoter region of lmrCD. These results demonstrate that the heterodimeric MDR ABC transporter LmrCD is a major determinant of both acquired and intrinsic drug resistance of L. lactis.

ATP-Binding Cassette Transporters↗

PubMed2000

Characterization of OpuA, a glycine-betaine uptake system of Lactococcus lactis.

A Lactococcus lactis glycine-betaine transport system was identified by functional complementation of an Escherichia coli proP proU mutant with a gene library from L. lactis sbsp. cremoris. The cloned locus forms an operon highly homologous to opuA, encoding a glycine-betaine uptake system of Bacillus subtilis. Disruption of opuA in L. lactis abolished protection by glycine-betaine against elevated osmolarity. OpuA belongs to the so-called "ABC transporters" family, which comprise an extracellularly localized substrate-binding protein. In B. subtilis OpuA system, this binding protein is a lipoprotein, attached to the external face of the cytoplasmic membrane by its lipidic moiety. In contrast, in the L. lactis opuA operon, and in other gram-positive homologues as well, a fusion between the gene encoding the integral membrane protein and the substrate-binding protein components gave rise to a hybrid protein presumably attaching the substrate-binding protein to the surface of the cell via its covalent link to the integral membrane component. Mapping of L. lactis opuA transcription start identified one mRNA, more abundant in cells grown at elevated osmolarity. Construction of an opuA-gusA fusion confirmed that opuA transcription is directed by a promoter osmotically inducible in L. lactis. When recombined upstream from a lac transcriptional fusion in the chromosome of E. coli, the opuA promoter appeared as very strong, and only poorly stimulated by elevated osmotic pressure, suggesting the existence of a specific machinery involved in the osmotic signal transduction in L. lactis.

ATP-Binding Cassette Transporters↗

PubMed1989

Structure and expression of the Lactococcus lactis gene for phospho-beta-galactosidase (lacG) in Escherichia coli and L. lactis.

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).

Amino Acid Sequence↗

PubMed2003

Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis.

The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.

ATP-Binding Cassette Transporters↗

PubMed2005

Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis.

In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the las operon. We used metabolic control analysis to study the role of this organization. Earlier studies have shown that, at wild-type levels, LDH has no control over glycolysis and growth rate, but high negative control over formate production (C(Jformate)LDH=-1.3). We found that PFK and PK exert no control over glycolysis and growth rate at wild-type enzyme levels but both enzymes exert strong positive control on the glycolytic flux at reduced activities. PK exerts high positive control over formate (C(Jformate)PK=0.9-1.1) and acetate production (C(Jacetate)PK=0.8-1.0), whereas PFK exerts no control over these fluxes at increased expression. Decreased expression of the entire las operon resulted in a strong decrease in the growth rate and glycolytic flux; at 53% expression of the las operon glycolytic flux was reduced to 44% and the flux control coefficient increased towards 3. Increased las expression resulted in a slight decrease in the glycolytic flux. At wild-type levels, control was close to zero on both glycolysis and the pyruvate branches. The sum of control coefficients for the three enzymes individually was comparable with the control coefficient found for the entire operon; the strong positive control exerted by PK almost cancels out the negative control exerted by LDH on formate production. Our analysis suggests that coregulation of PFK and PK provides a very efficient way to regulate glycolysis, and coregulating PK and LDH allows cells to maintain homolactic fermentation during glycolysis regulation.

Bacterial Proteins↗

PubMed1997

Expression of ropy and mucoid phenotypes in Lactococcus lactis.

Strains of Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris were cultured under aerobic and anaerobic conditions on plates of whey agar, Elliker agar, and M17L agar at 15, 20, and 30 degrees C to determine the environmental conditions required for the expression of the ropy phenotype. Two strains, L. lactis ssp. cremoris Ropy 352 and L. lactis ssp. cremoris Hollandicus, exhibited two distinct polysaccharide phenotypes, ropy and mucoid. Expression of these phenotypes could be induced individually or simultaneously. The inducible nature of this response suggests that genetic regulators were present. Western blots were used to determine whether or not Lon protease and RcsA, two regulators of polysaccharide expression in Escherichia coli, were present in lactococci. Lon, a negative regulator, and RcsA, an unstable positive regulator, have been shown at the structural level to be conserved in a number of Gram-negative and Gram-positive microorganisms. The present study found evidence for structural conservation of Lon protease in lactococci. Less of the Lon-like protein was observed in the ropy strains than in the nonropy strains.

ATP-Dependent Proteases↗

PubMed1994

Mosaic structure of large regions of the Lactococcus lactis subsp. cremoris chromosome.

Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris are closely related phenotypically and genetically. Here we report that certain regions of their chromosomes diverge considerably more than others. Conserved regions differ by less than 20%, whilst variable regions differ by more than 60%. This mosaic structure may have arisen by horizontal gene transfer from distantly related bacteria since in a particular region of the L. lactis subsp. cremoris chromosome the G+C content and the codon bias are not typical for lactococci. Such an exchange, which conserves the function of the gene and cannot be achieved under selective pressure, may be of considerable importance in the evolution of bacteria.

Amino Acid Sequence↗

PubMed1991

Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors. The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits. Our purpose was to identify unique rRNA sequences that could be used to discriminate L. lactis subsp. cremoris from related subspecies. The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L. lactis subsp. cremoris. All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus. In this region, all L. lactis subsp. lactis biovar diacetylactis strains examined had a sequence identical to that of L. lactis subsp. lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J. Brosius, J. L. Palmer, J. P. Kennedy, and H. F. Noller, Proc. Natl. Acad. Sci. USA 75:4801-4805, 1978). Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells. Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested. No hybridization was seen with the control species. All five strains of the subspecies cremoris hybridized to the subspecies-specific probe.

Base Sequence↗

PubMed1992

Is thermotolerance correlated to heat-shock protein synthesis in Lactococcus lactis subsp. lactis?

Exposure of Lactococcus lactis subsp. lactis cells to a heat shock at 40 degrees C for 30 min induces thermotolerance, the increased ability of bacterial cells to survive exposure to lethal temperature (52 degrees C for 25 min). This transient state of thermal resistance is accompanied, as in Escherichia coli, by the synthesis of a new set of specific proteins termed heat-shock proteins (Hsps). Pre-treatment of the bacterial cells by antibiotics (streptomycin, spiramycin, kanamycin and erythromycin) known to act on translation, induces the major Hsps synthesis but no thermal protection; conversely, puromycin and amino acid analogues treatments, known to produce abnormal and incomplete peptides, triggers the thermotolerance state without inducing significant Hsps synthesis. These results demonstrate that heat-shock response and induced thermotolerance are not tightly correlated phenomena in L. lactis subsp. lactis.

Adaptation, Physiological↗

PubMed2000

Inactivation of the glutamate decarboxylase gene in Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. lactis strains show glutamate decarboxylase activity, whereas L. lactis subsp. cremoris strains do not. The gadB gene encoding glutamate decarboxylase was detected in the L. lactis subsp. cremoris genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.

Base Sequence↗

PubMed1997

Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis.

The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L. lactis subsp. cremoris laboratory strain MG1363, which was originally derived from a dairy strain. After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels. Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress. Two classes of heat stress proteins were identified from their temporal induction pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min. GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min. The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress. Unlike other organisms, all salt stress-induced proteins in L. lactis were also subjected to heat stress induction. DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis.

Amino Acid Sequence↗

PubMed1994

Expression of Lactobacillus casei ATCC 393 beta-galactosidase encoded by plasmid pLZ15 in Lactococcus lactis CNRZ 1123.

Lactococcus lactis subsp. lactis CNRZ 1123, a Lac- derivative of CNRZ 1122 was transformed by electroporation with the Lactobacillus casei ATCC 393 plasmid pLZ15, which bears a beta-galactosidase gene. The transformants expressed a constitutive beta-galactosidase activity at a higher level than in Lact. casei, and in the cell-free extract two additional protein bands were detected by SDS-PAGE which could correspond to lactose metabolism enzymes. Both plasmid and beta-gal activity were stable in Lactococcus after 100 generations in glucose-containing medium.

Gene Expression Regulation, Enzymologic↗

PubMed2006

Identification of adsorption inhibition, restriction/modification and abortive infection type phage resistance systems in Lactococcus lactis strains.

98 Lactococcus lactis strains were isolated from traditional fermented milk products in Turkey tested against 60 lactococcal lytic phages to determine their resistance levels. While 82 L. lactis strains were sensitive against lactic phages at different levels, 16 L. lactis strains showed resistance to all phages tested. Types of phage resistance among 16 L. lactis strains were identified as phage adsorption inhibition in eight strains, restriction/modification in six strains and abortive infection (heat sensitive phage resistance) in two strains, using three broad-spectrum phages phi pll 98-32, phi pld 67-42 and phi pld 67-44.

Acriflavine↗

PubMed2002

Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552.

A Lactococcus lactis subsp. lactis strain (DPC5552), which causes the lysis of other lactococcal cultures, was isolated during a screening of raw milk samples for bacteriocin producers. Purification of the bacteriocin produced revealed that production of the lantibiotic, lacticin 481, was associated with the bacteriolytic capability of the strain. However, unlike bacteriocin-induced lysis observed with bacteriocins such as lacticin 3147 and lactococcins A, B, and M (where the target strain is killed), the DPC5552 supernatant gave rise to a situation whereby the target strain continued to grow (albeit at a lower rate) with simultaneous release of the intracellular enzymes lactate dehydrogenase (LDH) and post-proline dipeptidyl aminopeptidase (Pep X). In parallel experiments, 32 AU/ml of the inhibitory activity from L. lactis DPC5552 resulted in a 10- and 6-fold-higher LDH release after 5 h than that with 32 AU/ml of either lacticin 3147 or lactococcin A, B, and M. Laboratory-scale Cheddar cheese-making trials also demonstrated that lacticin 481-producing cultures induced the release of elevated levels of LDH from the starter L. lactis HP, without severely compromising its acid-producing capabilities. These results indicate that lacticin 481-producing strains may provide improved adjuncts for delivering lactococcal intracellular enzymes into the cheese matrix and, thus, improve cheese quality and flavor.

Bacterial Proteins↗

PubMed1991

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

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

Amino Acid Sequence↗

PubMed2000

Towards a proteomic map of Lactococcus lactis NCDO 763.

Lactococcus lactis is a widely used bacteria in dairy industry, specially in cheese ripening. Numerous lactococcal enzymes and proteins are involved in this process. Proteomics makes it possible to deal with a high number of proteins and identify modification of their patterns in two-dimensional (2-D) gels. However, an annotated reference map is necessary prior to analyzing protein variations. We have begun to construct such a map in easily reproducible conditions and identify proteins.

Bacterial Proteins↗

PubMed1991

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

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

Amino Acid Sequence↗