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PubMed1997

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis.

The lytic activity induced by the lactococcal bacteriophage P001 was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage P001-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage P001 genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.

Bacteriophages↗
PubMed1994

The immune response to Lactococcus lactis: implications for its use as a vaccine delivery vehicle.

The development of natural antibodies to Lactococcus lactis in three inbred strains of mice and the effect of inoculating L. lactis into these mice has been investigated. All animals developed detectable levels of natural (systemic and secretory) anti-lactococcal antibodies. Systemic anti-lactococcal antibodies were principally IgG in young mice. An increase in anti-lactococcal IgM occurred in all older animals. Inoculation of L. lactis strains MG1820 or MG1363 by the oral and parenteral routes induced systemic anti-lactococcal antibody responses in a dose-dependent fashion. The relative immunogenicity of individual bacterial proteins varied according to mouse strain and route of inoculation.

Animals↗
PubMed1992

Isolation of purine auxotrophic mutants of Lactococcus lactis and characterization of the gene hpt encoding hypoxanthine guanine phosphoribosyltransferase.

Five purine auxotrophic mutants of Lactococcus lactis were isolated. L. lactis was capable of converting adenine, guanine and hypoxanthine to AMP, GMP and IMP, respectively, indicating the existence of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) activities. A 1.3 kb DNA fragment from L. lactis was cloned by complementation of the hpt mutation in Escherichia coli. Introduction of this fragment into L. lactis resulted in an increase in HGPRT activity. In vitro transcription and translation analysis showed that the fragment coded for a polypeptide with M(r) of 22,000. The nucleotide sequence of this hpt gene was determined.

Adenine Phosphoribosyltransferase↗
PubMed1991

Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons.

A physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 was constructed by using the contour-clamped homogeneous electric field mode of pulsed-field gel electrophoresis in one- and two-dimensional separations to analyze restriction digests of high-molecular-weight genomic DNA. The map, which shows all the observed NotI and SmaI sites (six and 21, respectively) and 8 of approximately 30 SalI sites, is circular and yields a total size of 2.58 megabase pairs for the L. lactis subsp. lactis DL11 chromosome. By using rDNA from Mycoplasma capricolum to probe Southern blots of pulsed-and fixed-field digestion patterns, six putative rRNA operons were identified in L. lactis subsp. lactis DL11 and placed on the map of the chromosome. Five of these loci are clustered in a region representing only 20% of the chromosome. The presence of a SmaI site in each of the putative operons allowed the direction of transcription of each operon to be deduced.

Blotting, Southern↗
PubMed2003

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system.

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of alpha-keto-beta-methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 degrees C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.

Amino Acids↗
PubMed2006

Addition of glucose enables determination of luciferase activity in carbon-starved, stationary phase Lactococcus lactis cells.

We describe a simple method for measuring luciferase activity in the stationary phase of Lactococcus lactis. Due to large fluctuations in the energy and redox pools of stationary phase bacterial cells, measurement of luciferase activities does not yield reliable results. Upon addition of relatively small amounts of glucose, the pools are restored and measurement of luciferase becomes possible. Since luciferase activities are easily measured, our method allows to apply this simple analytical tool in stationary phase cells.

Glucose↗
PubMed2006

Secretion of biologically active porcine interleukin-2 by Lactococcus lactis.

In this study, secretion of two functional recombinant porcine interleukin-2 (rIL-2) proteins by Lactococcus lactis was studied. Two secretion cassettes were constructed in which the secretion was achieved by gene fusion between the lactococcal usp45 secretion signal, a synthetic propeptide and the sequence encoding the mature IL-2. In addition, one of the two secretion cassettes contained the H-domains of L. lactis PrtP. Both of the constructed recombinant IL-2 proteins were found to be secreted in the same quantities, approximately 0.5mg/l. According to a cell proliferative assay using CTLL-2 cell line the specific biological activities of both purified rIL-2 proteins were found to be of similar levels.

Amino Acid Sequence↗
PubMed1998

A PCR fingerprinting technique to distinguish isolates of Lactococcus lactis.

A fingerprinting technique similar to repetitive extragenic palindromic PCR was developed to identify strains of Lactococcus lactis. The method distinguishes closely related strains and discriminates among some with identical ldh sequences. The fingerprinting primer LL-Rep1 complements a moderately repeated sequence found in low G + C Gram-positive bacteria and may therefore prove useful for discriminating among strains of other low G + C Gram-positive species.

Base Composition↗
PubMed2000

Lactococcus lactis as a cell factory for high-level diacetyl production.

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and alpha-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via alpha-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.

Carboxy-Lyases↗
PubMed1995

Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract.

A human feeding study was performed with Lactococcus lactis TC165.5, which is genetically marked by insertion of the sucrose-nisin conjugative transposon Tn5276 and chromosomal resistance to rifampin and streptomycin. The fate of strain TC165.5 and its nucleic acids was monitored by conventional plating methods and by molecular detection techniques based on specific PCR amplification of the nisin (nisA) gene from DNA extracted from human feces. A method was developed for the efficient extraction of microbial DNA from human feces. The results show that a fraction of viable cells of L. lactis TC165.5 survived passage through the human gastrointestinal tract. Only cells that passed within 3 days of ingestion could be recovered from the feces of the volunteers, and they accounted for approximately 1% of the total number of cells consumed. The presence of nisA in DNA extracted from feces could be detected up to 4 days, when viable cells were no longer present.

Base Sequence↗
PubMed1995

Enhancement of antigen-specific antibody production by extracellular slime products from slime-forming Lactococcus lactis subspecies cremoris SBT 0495 in mice.

The effect of extracellular slime products (ESP) produced by Lactococcus lactis subspecies cremoris SBT 0495 on antigen specific antibody production was studied in mice. ESP contained 48.5% protein, 15.4% neutral sugar, and 1.1% of phosphorus. The optimum dose of ESP was between 100 to 500 micrograms per mouse. ESP administered intraperitoneally (200 micrograms per mouse) enhanced the production of specific antibody in mice. These results indicate that ESP may act as an adjuvant.

Animals↗
PubMed1990

In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp. cremoris plasmid pDI-21.

A 4.4-kb EcoR1-EcoR1 DNA fragment from the Lactococcus lactis subsp. cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes. In vitro transcription-translation using Escherichia coli S30 extract showed the synthesis of 41,000-, 23,000- and 12,000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.

Aldehyde-Lyases↗
PubMed2000

Physiological and regulatory effects of controlled overproduction of five cold shock proteins of Lactococcus lactis MG1363.

The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total protein) was obtained. Northern blot analysis revealed low abundance of the cspC transcript, indicating that the stability of cspC mRNA is low. The limited overproduction of CspA is likely to be caused by low stability of CspA since when there was an Arg-Pro mutation at position 58, the level of CspA production increased. Using two-dimensional gel electrophoresis, it was found that upon overproduction of the CSPs several proteins, including a number of cold-induced proteins of L. lactis, were induced. Strikingly, upon overproduction of CspC induction of CspB, putative CspF, and putative CspG was also observed. Overproduction of CspB and overproduction of CspE result in increased survival when L. lactis is frozen (maximum increases, 10- and 5-fold, respectively, after 4 freeze-thaw cycles). It is concluded that in L. lactis CSPs play a regulatory role in the cascade of events that are initiated by cold shock treatment and that they either have a direct protective effect during freezing (e.g., RNA stabilization) or induce other factors involved in the freeze-adaptive response or both.

Adaptation, Physiological↗
PubMedDate not supplied

Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C.

Mice inoculated intranasally (i.n.) with a recombinant strain of live Lactococcus lactis expressing tetanus toxin fragment C (TTFC), produced both serum and secretory antibodies to TTFC. Killed bacteria which had accumulated TTFC intracellularly in vitro also elicited protective serum antibody responses. There was no requirement for either colonization or invasion of the mucosa. In addition secretory antibody responses in the lung and nasal tissues were elicited after i.n. inoculation in the presence of an adjuvant.

Administration, Intranasal↗
PubMed1994

Distribution and evolution of nisin-sucrose elements in Lactococcus lactis.

The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed.

Amino Acid Sequence↗
PubMed2000

A food-grade cloning system for industrial strains of Lactococcus lactis.

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds.

Amino Acid Sequence↗
PubMed1994

Influence of dilution rate and cell immobilization on plasmid stability during continuous cultures of recombinant strains of Lactococcus lactis subsp. lactis.

The influence of dilution rate and cell immobilization on plasmid stability in recombinant strains of Lactococcus lactis subsp. lactis was investigated during continuous cultures. The studied strains, L. lactis IL2682 and IL2683, contained plasmids pIL9 (Lac+), pIL205 (CmR) and plasmids pIL252 (low copy number) and pIL253 (high copy number), respectively, that conferred resistance to erythromycin. Plasmid pIL205 was remarkably stable. Dilution rate did not affect the rate of loss of plasmids pIL252 and pIL253 significantly. Nevertheless, the loss of plasmid pIL253 was apparent after a further 21 generations when the dilution rate was decreased from 0.70 h-1 to 0.55 h-1. Cell immobilization in beads of kappa-carrageenan/locust bean gum improved plasmid stability by factors of 4.5 for pIL253 and 6.5 for pIL252. Thus, 10% of cells containing plasmids pIL252 or pIL253 were still present after 370 or 540 generations, respectively, compared with 50 or 210 generations in free cell cultures.

Culture Media↗
PubMed1995

A study of the substrate specificity of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

A systematic study was made of the ability of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 to hydrolyse different peptide substrates. The enzyme showed a marked preference for substrates containing arginine as the N-terminal residue but, to a lesser extent, was also capable of cleaving other residues such as lysine and leucine. There was a tendency for the activity to increase with the hydrophobicity index of the C-terminal residue of dipeptide substrates. It was also observed that the enzyme tended to have higher affinities but lower Vmax values for tripeptides with hydrophobic C-terminal residues. The values determined for Km and Vmax increased with chain length for oligopeptides of the general formula Lys-Phe-(Gly)n, the optimum, as determined from Vmax/Km, being when n = 4. Typical Km values for the most effective substrates were in the range 0.2-0.6 mM.

Amino Acid Sequence↗