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PubMed1996

Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr.

By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars. Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase. Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L. lactis or B. subtilis or in intact cells of B. subtilis. Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion.

Adenosine Triphosphate↗
PubMed1993

Improved cloning vectors and transformation procedure for Lactococcus lactis.

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L. lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v. Although growth of L. lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.

Base Sequence↗
PubMed1998

Crystallization and preliminary X-ray diffraction studies of 6-phosphogluconate dehydrogenase from Lactococcus lactis.

6-Phosphogluconate dehydrogenase is one of the seven enzymes involved in the pentose phosphate pathway. Crystals of a mammalian and a protozoan enzyme have been obtained previously and structures determined. It is reported here that a bacterial 6-phosphogluconate dehydrogenase, from Lactococcus lactis, has been purified and used in crystallization trials. Large prisms suitable for a detailed structural analysis have been obtained and characterized as orthorhombic, space group F222, with a = 70.4, b = 105.7, c = 474.6 A. Diffraction has been observed to 2.2 A resolution using synchrotron radiation. Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationships of this enzyme.

Bacterial Proteins↗
PubMed1995

recA gene involvement in oxidative and thermal stress in Lactococcus lactis.

The recA gene is best known for its effects on homologous recombination and DNA repair via SOS induction. There is gathering evidence that recA also affects expression of genes associated with different types of stress. We studied recA properties in Lactococcus lactis by generating a recA-disrupted mutant of MG1363 and comparing it with the wild type strain. recA appears to have an important role in cell survival upon oxygen or thermal stress, in addition to its conserved role in DNA repair. Oxygen toxicity appears to be due to the production of hydroxyl radicals via the Fenton reaction; recA would be involved in the repair of DNA damage generated by these radicals. Surprisingly, the recA strain stops growing at elevated temperature (37 degrees C). Immunological tests indicate that amounts of three heat shock proteins are reduced in the recA strain compared to the wild type strain. In contrast, the amount of heat shock regulator HflB is markedly increased, even at low temperature. HflB is known to degrade heat shock transcription factor sigma 32 in Escherichia coli. We propose that heat shock response is reduced in the recA mutant due to overproduction of HflB.

ATP-Dependent Proteases↗
PubMed1993

15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis.

A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [13C]bicarbonate and/or sodium [15N]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [13C,15N]nisin A using H[13C]MQC, H[13C]MBC, 2D INADEQUATE, and H[15N]MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H[13C]MQC to unlabeled and [13C]leucocin A afforded the complete 1H and 13C assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.

Amino Acid Sequence↗
PubMed2001

Reassessing the role of Staphylococcus aureus clumping factor and fibronectin-binding protein by expression in Lactococcus lactis.

Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, both clfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as the S. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in > or =80% of the rats (80% infective dose [ID80]) with the parent lactococcus was > or =10(7) CFU. In contrast, clfA-expressing and fnbA-expressing lactococci required only 10(5) CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID80 = 10(4) to 10(5) CFU) in this model. The results confirmed the role of clfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of the clfA and fnbA products should be blocked for the therapy to be effective.

Adhesins, Bacterial↗
PubMed2005

Application of PCR, rep-PCR and RAPD techniques for typing of Lactococcus lactis strains.

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.

Bacterial Typing Techniques↗
PubMed1998

The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically.

The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis, L. lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified.

Amino Acid Sequence↗
PubMed2005

Lactococcus lactis SpOx spontaneous mutants: a family of oxidative-stress-resistant dairy strains.

Numerous industrial bacteria generate hydrogen peroxide (H(2)O(2)), which may inhibit the growth of other bacteria in mixed ecosystems. We isolated spontaneous oxidative-stress-resistant (SpOx) Lactococcus lactis mutants by using a natural selection method with milk-adapted strains on dairy culture medium containing H(2)O(2). Three SpOx mutants displayed greater H(2)O(2) resistance. One of them, SpOx3, demonstrated better behavior in different oxidative-stress situations: (i) higher long-term survival upon aeration in LM17 and milk and (ii) the ability to grow with H(2)O(2)-producing Lactobacillus delbrueckii subsp. delbrueckii strains. Furthermore, the transit kinetics of the SpOx3 mutant in the digestive tract of a human flora-associated mouse model was not affected.

Animals↗
PubMedDate not supplied

[Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis].

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.

Acetolactate Synthase↗
PubMed2001

Cold shock proteins of Lactococcus lactis MG1363 are involved in cryoprotection and in the production of cold-induced proteins.

Members of the group of 7-kDa cold-shock proteins (CSPs) are the proteins with the highest level of induction upon cold shock in the lactic acid bacterium Lactococcus lactis MG1363. By using double-crossover recombination, two L. lactis strains were generated in which genes encoding CSPs are disrupted: L. lactis NZ9000 Delta AB lacks the tandemly orientated cspA and cspB genes, and NZ9000 Delta ABE lacks cspA, cspB, and cspE. Both strains showed no differences in growth at normal and at low temperatures compared to that of the wild-type strain, L. lactis NZ9000. Two-dimensional gel electrophoresis showed that upon disruption of the cspAB genes, the production of remaining CspE at low temperature increased, and upon disruption of cspA, cspB, and cspE, the production of CspD at normal growth temperatures increased. Northern blot analysis showed that control is most likely at the transcriptional level. Furthermore, it was established by a proteomics approach that some (non-7-kDa) cold-induced proteins (CIPs) are not cold induced in the csp-lacking strains, among others the histon-like protein HslA and the signal transduction protein LlrC. This supports earlier observations (J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. De Vos, O. P. Kuipers, and T. Abee, Appl. Environ. Microbiol. 66:3756-3763, 2000). that the CSPs of L. lactis might be directly involved in the production of some CIPs upon low-temperature exposure. Remarkably, the adaptive response to freezing by prior exposure to 10 degrees C was significantly reduced in strain NZ9000 Delta ABE but not in strain NZ9000 Delta AB compared to results with wild-type strain NZ9000, indicating a notable involvement of CspE in cryoprotection.

Adaptation, Physiological↗
PubMed1999

Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis.

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cremoris (B. Christiansen, L. Brondsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164-5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoris when the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. lactis subsp. cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.

Attachment Sites, Microbiological↗
PubMed1993

Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.

Biological Transport↗
PubMed2004

ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassette-type multidrug transporter.

Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs. The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L. lactis or the mammalian P-glycoprotein. The ydaG/ydbA genes were cloned and expressed separately and jointly in L. lactis using the nisin-inducible system. When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell. YdaG and YdbA could be co-purified as a stable heterodimer. ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits. Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L. lactis strains selected for growth in the presence of a variety of toxic compounds. This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.

ATP-Binding Cassette Transporters↗
PubMed2006

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice.

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST-Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli-L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracellular secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.

Administration, Oral↗
PubMed1989

Product of the Lactococcus lactis gene required for malolactic fermentation is homologous to a family of positive regulators.

Malolactic fermentation is a secondary fermentation that many lactic acid bacteria can carry out when L-malate is present in the medium. The activation of the malolactic system in Lactococcus lactis is mediated by a locus we call mleR. Induction of the genes necessary to perform malolactic fermentation occurs only in bacteria with a functional copy of mleR. The mleR gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 33,813 daltons. The amino acid sequence of the predicted MleR gene product is homologous to that of positive activators in gram-negative bacteria: LysR, IlvY gene products of Escherichia coli, MetR, CysB of Salmonella typhimurium, AmpR of Enterobacter cloacae, NodD of Rhizobium sp., and TrpI of Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed1993

Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis.

The DNA sequence encoding the leader peptide of the lantibiotic subtilin from Bacillus subtilis was fused to the sequence encoding pronisin Z, and this hybrid gene was expressed in a Lactococcus lactis strain that produces nisin A. This strain simultaneously secreted nisin A and a protein of approximately 6 kDa. Amino acid sequencing of the purified 6 kDa protein and structural analysis of its main tryptic fragment by two-dimensional 1H-NMR showed that it consists of the unmodified leader peptide of subtilin, without the N-terminal methionine residue, linked to a fully matured nisin Z part. The hybrid protein and its main tryptic fragment [ITPQ]-nisin Z, showed at least 200-fold lower antimicrobial activities than nisin Z against three different indicator strains.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary structural studies of lactose-specific enzyme IIA from Lactococcus lactis.

Lactose-specific enzyme IIA of the phosphoenol:pyruvate-dependent sugar phosphotransferase system from Lactococcus lactis has been crystallized in phosphate buffer. The crystals belong to space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with unit-cell axes a = b = 90.9 and c = 82.4 A. The packing parameter (Matthews parameter) V(m) of 2.48 A(3) Da(-1) is consistent with one trimer per asymmetric unit and non-crystallographic threefold symmetry has been confirmed by calculating a self-rotation function. The crystals diffract X-rays to at least 2.3 A resolution, are stable in an X-ray beam and are therefore appropriate for structure determination. Native data to 2.3 A resolution have been collected using a MAR image-plate system at a synchrotron source. One isomorphous heavy-atom derivative has been identified and the presence of an isomorphous signal in the data has been confirmed by Patterson methods.

Journal Article↗