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PubMed2002

Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR.

The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using (13)C and (31)P NMR to monitor in vivo the kinetics of the pools of NAD(+), NADH, ATP, inorganic phosphate (P(i)), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of (13)C-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH(-) deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (approximately 5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH(-) strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH(-) strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P(i) content could play an important role.

Aerobiosis↗
PubMed2001

Nisin production from Lactococcus lactis A.T.C.C. 7962 using supplemented whey permeate.

The influence of pH control and aeration (20% dissolved oxygen) on nisin production in a supplemented cheese whey permeate was examined during batch fermentation with Lactococcus lactis subsp. lactis A.T.C.C. 7962. A maximum nisin activity of 5280 i.u./ml of medium was observed in the raw extract of nisin after 9 h of fermentation with a constant pH at 4.9. However, the fermentation was continued until 24 h, when a decrease in the nisin activity was observed. The pH control did not influence the nisin production and aeration of the culture medium increased cell growth (biomass) but not nisin activity. The yeast Kluyveromyces marxianus, used as an alternative method to control pH, has not been efficient.

Animals↗
PubMed2003

Induction of partial protection in mice after oral administration of Lactococcus lactis producing Brucella abortus L7/L12 antigen.

The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

Administration, Oral↗
PubMed2005

Modelling the production of nisin by Lactococcus lactis in fed-batch culture.

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l(-1) h(-1), respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml(-1), by constant addition of sucrose at a feeding rate of 7 g l(-1) h(-1); an increase in 58% over that of the batch culture (2,658 IU ml(-1)). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.

Culture Media↗
PubMed1996

Cloning, DNA sequence, and regulation of expression of a gene encoding beta-galactosidase from Lactococcus lactis.

The beta-galactosidase from Escherichia coli is one of the most important enzymes in molecular biology. Here we report the cloning and sequencing of a gene encoding beta-galactosidase from Lactococcus lactis and compare the predicted amino acid sequence to that from other organisms. The beta-galactosidase from L. lactis was found to be a protein of 996 residues with 68.7% similarity to the E. coli enzyme and 65.8% similarity to the enzyme from Klebsiella pneumoniae. The lactococcal beta-galactosidase has lower similarity (approx 55%) to the enzymes from other lactic acid bacteria and no significant similarity to the beta-galactosidase enzymes from Agrobacterium radiobacter, Bacillus stearothermophilus, or Clostridium thermosulfurogenes. Expression of the lacZ gene from L. lactis was found to be higher when cells were grown in medium containing lactose than when grown in glucose, and expression was higher when cells were grown at 30 degrees C than at 35 degrees C.

Amino Acid Sequence↗
PubMed1992

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor. II. Plasmid transfer in mixed cultures.

The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10(-7) to 10(-8) after 12 h of fermentation. These frequencies were 60-400 times lower than in unstirred M17 broth and 100,000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10(-5) - 10(-6).

Biotechnology↗
PubMed2006

Intracellular accumulation of trehalose protects Lactococcus lactis from freeze-drying damage and bile toxicity and increases gastric acid resistance.

Interleukin-10 (IL-10) is a promising candidate for the treatment of inflammatory bowel disease. Intragastric administration of Lactococcus lactis genetically modified to secrete IL-10 in situ in the intestine was shown to be effective in healing and preventing chronic colitis in mice. However, its use in humans is hindered by the sensitivity of L. lactis to freeze-drying and its poor survival in the gastrointestinal tract. We expressed the trehalose synthesizing genes from Escherichia coli under control of the nisin-inducible promoter in L. lactis. Induced cells accumulated intracellular trehalose and retained nearly 100% viability after freeze-drying, together with a markedly prolonged shelf life. Remarkably, cells producing trehalose were resistant to bile, and their viability in human gastric juice was enhanced. None of these effects were seen with exogenously added trehalose. Trehalose accumulation did not interfere with IL-10 secretion or with therapeutic efficacy in murine colitis. The newly acquired properties should enable a larger proportion of the administered bacteria to reach the gastrointestinal tract in a bioactive form, providing a means for more effective mucosal delivery of therapeutics.

Animals↗
PubMed1993

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon.

Amino Acid Sequence↗
PubMed1998

Cloning and expression of the Lactococcus lactis purDEK genes, required for growth in milk.

An operon containing the genes purD and purE and part of the purK gene was cloned from the facultative anaerobic gram-positive bacterium Lactococcus lactis by complementation of the purD mutation in Escherichia coli SO609. The genes encode enzymes in the de novo pathway of purine nucleotides. The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of purD indicated that a region of 145 bp is enough to give regulated expression of the reporter lacLM genes, which encode beta-galactosidase. Deletion of a region 79 bp upstream of the transcription start point reduced the promoter activity 33-fold when incubated in a purine-free medium and to values below the detection limit when incubated in a purine-containing medium. No secondary transcription start points were mapped in or close to this region, indicating that a putative activator site and not a promoter was deleted or partly destroyed.

Amino Acid Sequence↗
PubMed1993

Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis.

Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis. In L. lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid. Transconjugants of strain HID113 carrying this plasmid were unstable. Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome.

Conjugation, Genetic↗
PubMed1998

Cell membrane integrity and lysis in Lactococcus lactis: the detection of a population of permeable cells in post-logarithmic phase cultures.

A method was developed that enabled an analysis of the proportion of permeable cells in a culture of Lactococcus lactis. This used the fluorescence of propidium iodide (PI) when in contact with DNA and the impermeability of the intact cell membrane to this compound. A permeability index was suggested that expresses the PI-induced fluorescence of a cell suspension as a percentage of the value obtained from wholly permeabilized cells after treatment with cetyltrimethylammonium bromide. This method was applied to the determination of cell permeability in death phase cultures. A large proportion of unlysed cells was freely permeable to PI, a finding that may have some significance for the investigation of the role of cell lysis in cheese maturation. This method is suggested as a useful addition to the techniques available for the study of cell damage in a variety of fields, and for the screening of cheese starter bacteria.

Bacteriolysis↗
PubMed2006

Spontaneous resistance in Lactococcus lactis IL1403 to the lantibiotic lacticin 3147.

The ability and frequency at which target organisms can develop resistance to bacteriocins is a crucial consideration in designing and implementing bacteriocin-based biocontrol strategies. Lactococcus lactis ssp. lactis IL1403 was used as a target strain in an attempt to determine the frequency at which spontaneously resistant mutants are likely to emerge to the lantibiotic lacticin 3147. Following a single exposure to lacticin 3147, resistant mutants only emerged at a low frequency (10(-8)-10(-9)) and were only able to withstand low levels of the bacteriocin (100 AU mL(-1)). However, exposure to increasing concentrations, in a stepwise manner, resulted in the isolation of eight mutants that were resistant to moderately higher levels of lacticin 3147 (up to 600 AU mL(-1)). Interestingly, in a number of cases cross-resistance to other lantibiotics such as nisin and lacticin 481 was observed, as was cross-resistance to environmental stresses such as salt. Finally, reduced adsorption of the bacteriocin in to the cell was documented for all resistant mutants.

Adsorption↗
PubMedDate not supplied

[Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture].

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.

Acetolactate Synthase↗
PubMed1995

Lactococcus lactis glyceraldehyde-3-phosphate dehydrogenase gene, gap: further evidence for strongly biased codon usage in glycolytic pathway genes.

The gene gap, encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), was isolated from a genomic library of Lactococcus lactis LM0230 DNA. Plasmids containing the L. lactis gene were able to complement a gap mutant of Escherichia coli. The nucleotide sequence of gap predicted a polypeptide chain of 337 amino acids for the enzyme and a subunit molecular mass of 36,043. The codon usage in gap and four other glycolytic genes from L. lactis showed a high degree of bias, when compared with 84 other chromosomal genes. Northern blot analysis of total L. lactis RNA showed that gap hybridized strongly with a 1.3 kb transcript. The 5' end of the transcript was determined by primer extension analysis to be a C located 35 bp upstream from the gap start codon. These transcript analyses, and the orientation of the open reading frames in the DNA flanking gap, indicated that in L. lactis gap is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to gap did not encode other glycolytic pathway enzymes. The DNA sequence flanking gap contained two open reading frames (ORF156 and ORF211) of unknown function. The 3' end of a clpA homologue was identified in the sequence upstream of ORF156. The location of gap on the L. lactis DL11 chromosome map was determined to be between map coordinates 0.530 and 0.660.

Amino Acid Sequence↗
PubMed2006

Permeabilization and lysis induced by bacteriocins and its effect on aldehyde formation by Lactococcus lactis.

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.

Aldehydes↗