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PubMed2000

Lactococcus lactis, a bacterial model for stress responses and survival.

The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes. To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis. Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation). Our results revealed that metabolic flux plays an important role in L. lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors. As previously shown, we also observed an increase of stress resistance during the stationary phase. We have evidence that stationary phase actually initiates very early during growth. Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether. These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L. lactis.

Lactococcus lactis↗
PubMed2002

Contribution of Lactococcus lactis cell envelope proteinase specificity to peptide accumulation and bitterness in reduced-fat Cheddar cheese.

Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of alpha(S1)-casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.

Cheese↗
PubMed1990

Molecular cloning, transcriptional analysis, and nucleotide sequence of lacR, a gene encoding the repressor of the lactose phosphotransferase system of Lactococcus lactis.

The repressor gene (lacR) of the lactose phosphotransferase system of Lactococcus lactis subsp. lactis strain MG1820 has been cloned and characterized. Transcription of lacR, into a 1.2-kilobase monocistronic messenger, is repressed approximately 5-fold during growth on lactose. Nucleotide sequence analysis of the lacR gene showed the presence of an open reading frame of 861 base pairs. The deduced amino acid sequence of LacR is homologous to three Escherichia coli regulatory proteins (DeoR, FucR, and GutR) and includes a N-terminal domain (helix-turn-helix) involved in DNA binding and a C-terminal domain that may be responsible for inducer binding. The in vivo function of LacR has been determined by introducing multiple copies of lacR into L. lactis, under control of its own or the unrelated prtP promoter. Growth rates and lactose phosphotransferase system enzyme activities were measured during growth on lactose and glucose. The presence of lacR on a multicopy plasmid resulted in the decrease of lactose phosphotransferase system activity, whereas only on lactose a decrease (25%) of growth rate was observed. No significant difference in growth rate was observed on glucose, indicating that LacR specifically represses the lactose genes of L. lactis.

Amino Acid Sequence↗
PubMed1991

Functional alteration of macrophages by a slime-forming Lactococcus lactis ssp. cremoris.

The effect of a slime-forming, encapsulated Lactococcus lactis ssp. cremoris KVS20 on macrophage function has been examined in vivo and in vitro in short-term studies. Peritoneal macrophages in which 21 to 34% of macrophage was presenting Fc gamma-receptor positive macrophages were elicited by intraperitoneal injection of 10 to 50 mg/kg of L. lactis ssp. cremoris KVS20. The peritoneal macrophage exhibited cytotoxic activity against Sarcoma-180 cells in which the maximum activity was obtained in macrophage from mice injected with 10 mg/kg on d 5. However, L. lactis ssp. cremoris KVS20 rendered the elicited macrophage cytotoxic in vitro. The cytotoxicity was significantly augmented by 6- and 24-h treatment at the concentration of 50 to 500 micrograms/ml. These results obtained in the short-term studies demonstrated that the antitumor activity of L. lactis ssp. cremoris KVS20 may be mediated through the enhanced cytotoxic activity of macrophage.

Animals↗
PubMed2005

Complete sequences of four plasmids of Lactococcus lactis subsp. cremoris SK11 reveal extensive adaptation to the dairy environment.

Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted alpha-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these "adaptation" genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer.

Base Sequence↗
PubMed1997

Duplication of the pepF gene and shuffling of DNA fragments on the lactose plasmid of Lactococcus lactis.

The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF [V. Monnet, M. Nardi, A. Chopin, M.-C. Chopin, and J.-C. Gripon, J. Biol. Chem. 269:32070-32076, 1994]) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp. cremoris NCDO763. Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp. lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763. From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters. The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases. Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403. Traces of several recombination events are visible on the lactose-proteinase plasmid. This suggests that the duplication of pepF occurred by recombination from the chromosome of an L. lactis subsp. lactis strain followed by gene transfer. We discuss the possible functions of PepF and the role of its amplification.

Amino Acid Sequence↗
PubMed2000

Molecular pharmacological characterization of two multidrug transporters in Lactococcus lactis.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in Lactococcus lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).

Animals↗
PubMed1998

The citrate transport system of Lactococcus lactis subsp. lactis biovar diacetylactis is induced by acid stress.

Citrate transport in Lactococcus lactis subsp. lactis biovar diacetylactis is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. We have shown previously that citP is included in the citQRP operon, which is mainly transcribed from the P1 promoter in L. lactis subsp. lactis biovar diacetylactis. furthermore, transcription of citQRP and citrate transport are not induced by the presence of citrate in the growth medium. In this work, we analyzed the influence of the extracellular pH on the expression of citP. The citrate transport system is induced by natural acidification of the medium during cell growth and by a shift to media buffered at acidic pHs. This inducible response to acid stress takes place at the transcriptional level and seems to be due to increased utilization of the P1 promoter. Increased transcription correlates with increased synthesis of CitP and results in higher citrate transport activity catalyzed by the cells. Finally, this acid stress response seems to provide L. lactis subsp. lactis biovar diacetylactis with a selective advantage resulting from cometabolism of glucose and citrate at low pHs.

Bacterial Proteins↗
PubMed2000

The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis. Catalytic properties and allosteric regulation of the pure enzyme system.

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Allosteric Regulation↗
PubMed2004

A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression.

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P(nisA) as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.

Bacterial Proteins↗
PubMed2006

Proteolysis of Hispanico cheese manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639.

Hispánico cheese was manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639, bacteriocin-nonproducing L. lactis ssp. lactis INIA 437, or a combination of both strains, as starter cultures. Lactobacillus helveticus LH 92, a culture of high amino-peptidase activity sensitive to lacticin 481, was added to all vats. Milk inoculation with the bacteriocin producer promoted early lysis of Lb. helveticus cells in cheese. Cell-free aminopeptidase activity in cheese made with the 3 lactic cultures was 1.8 times the level reached in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 15 d of ripening. Proteolysis (as estimated by the o-phthaldialdehyde method) in cheese made with the 3 lactic cultures was twice as high, and the level of total free amino acids 2.4 times the level found in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 25 d of ripening. Hydrophobic and hydrophilic peptides and their ratio were at the lowest levels in cheese made with the 3 lactic cultures, which received the lowest scores for bitterness and the highest scores for taste quality.

Amino Acids↗
PubMed2005

Lactococcus lactis is capable of improving the riboflavin status in deficient rats.

Lactococcus lactis is a commonly used starter strain that can be converted from a vitamin B2 consumer into a vitamin B2 'factory' by over-expressing its riboflavin biosynthesis genes. The present study was conducted to assess in a rat bioassay the response of riboflavin produced by GM or native lactic acid bacteria (LAB). The riboflavin-producing strains were able to eliminate most physiological manifestations of ariboflavinosis such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegalia that were observed using a riboflavin depletion-repletion model. Riboflavin status and growth rates were greatly improved when the depleted rats were fed with cultures of L. lactis that overproduced this vitamin whereas the native strain did not show the same effect. The present study is the first animal trial with food containing living bacteria that were engineered to overproduce riboflavin. These results pave the way for analysing the effect of similar riboflavin-overproducing LAB in human trials.

Animals↗
PubMed1989

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase.

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.

Cell Membrane↗
PubMed1997

A novel plasmid-encoded phage abortive infection system from Lactococcus lactis biovar. diacetylactis.

A 16-kb plasmid (pND859) was identified from Lactococcus lactis biovar. diacetylactis UK12922 which encodes phage resistance to the small isometric phage 712 when tested in L. lactis LM0230. The gene encoding phage abortive infection, designated abi-859, was localized on a 1.2-kb region which consists of an open reading frame (ORF) of 846 bp preceded by a potential ribosome-binding site and a putative promoter region. A helix-turn-helix region typical of DNA-binding motifs was identified near the N-terminal of the abi-859 product, suggesting a possible interaction with the phage DNA.

Amino Acid Sequence↗
PubMed2002

Respiration capacity and consequences in Lactococcus lactis.

We recently reported that the well-studied fermenting bacterium Lactococcus lactis could grow via a respirative metabolism in the presence of oxygen when a heme source is present. Respiration induces profound changes in L. lactis metabolism, and improvement of oxygen tolerance and long-term survival. Compared to usual fermentation conditions, biomass is approximately doubled by the end of growth, acid production is reduced, and large amounts of normally minor end products accumulate. Lactococci grown via respiration survive markedly better after long-term storage than fermenting cells. We suggest that growth and survival of lactococci are optimal under respiration-permissive conditions, and not under fermentation conditions as previously supposed. Our results reveal the uniqueness of the L. lactis respiration model. The well-studied 'aerobic' bacteria express multiple terminal cytochrome oxidases, which assure respiration all throughout growth; they also synthesize their own heme. In contrast, the L. lactis cydAB genes encode a single cytochrome oxidase (bd), and heme must be provided. Furthermore, cydAB genes mediate respiration only late in growth. Thus, lactococci exit the lag phase via fermentation even if heme is present, and start respiration in late exponential phase. Our results suggest that the spectacularly improved survival is in part due to reduced intracellular oxidation during respiration. We predict that lactococcal relatives like the Enterococci, and some Lactobacilli, which have reported respiration potential, will display improved survival under respiration-permissive conditions.

Electron Transport Complex IV↗
PubMed1997

[Cloning and expression of promoter and signal peptide function fragments from Lactococcus lactis in Escherichia coli].

Promoter and signal peptide function fragments from Lactococcus lactis were cloned in E. coli using a promoter-signal sequence probe vector pGPB14. Forty-two clones were obtained, whose level of resistance to ampicillin ranged from 100 to 8000 micrograms/ml. Eight clones were selected for beta-lactamase distribution assay. The beta-lactamase activity was found mainly in the periplasm, which indicated successful secretion of the enzyme. Southern hybridization test demonstrated that the inserted fragments were indeed from L. lactis. Restriction enzyme analysis revealed that the size of inserted fragments ranged from 80bp to 400 bp, four of which were sequenced on the vector pGEM-3Zf. It was found that the inserted fragments of pSEQ8 and pSEQ12 turned out to be part of the inserted fragments of pSEQ4 and pSEQ17 respectively. Promoter and translation initiation codon were found among all four fragments signal sequenced. One typical S. D. sequence and one atypical signal sequence were found in pHSB4 and pHSB8, while the other two contained no typical S. D. sequence and signal peptide sequence. In addition, it was found that the upstream regions of the promoter contributed to the efficiency of transcription initiation.

Base Sequence↗
PubMed2005

Molecular characterization of the CmbR activator-binding site in the metC-cysK promoter region in Lactococcus lactis.

The metC-cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the metC promoter. This binding was stimulated by O-acetyl-L-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter.

Bacterial Proteins↗