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PubMed2001

Molecular physiology of sugar catabolism in Lactococcus lactis IL1403.

The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.

Acetyltransferases↗
PubMed2003

Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403.

The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and two ATP-binding protein genes (dppD and dppF). In this work, the gene specifying the second peptide-binding protein (DppP) was cloned under the control of the nisin promoter. The protein fused to a carboxyl-terminal histidine tag (DppP-His(6)) was purified and its binding properties were determined by monitoring the intrinsic fluorescence changes observed upon ligand binding. The major features of peptide binding to DppP-His(6) include: (i) a requirement for a free N-terminal alpha-amino group in the ligand; (ii) a high affinity for di-, tri- and tetra-peptides; (iii) affinity constants for peptide binding independent of pH; and (iv) a high affinity for D-isomer-containing peptides. Remarkably, the features (ii), (iii) and (iv) differ from those previously reported for DppA-His(6), suggesting that DppP-His(6) is a more versatile peptide-binding protein that could have additional functions.

ATP-Binding Cassette Transporters↗
PubMed1998

RNA processing is involved in the post-transcriptional control of the citQRP operon from Lactococcus lactis biovar diacetylactis.

The importance of Lactococcus lactis biovar diacetylactis (L. diacetylactis) in the dairy industry is due to its ability to produce aroma compounds, such as acetoin and diacetyl, from citrate. The first step in citrate utilization is its uptake by the cells. In L. diacetylactis, the citrate transport system is encoded by the citQRP operon. We have previously proposed that expression of citQRP operon is regulated at the post-transcriptional level. In this paper, we show that the cit mRNA is processed at a complex secondary structure in L. diacetylactis and Escherichia coli. This secondary structure includes the 5'-terminal two-thirds of citQ and the overlap between citQ and citR. Primer-extension analysis revealed that the major cleavage sites are located upstream of citR and within citQ. In an attempt to identify the enzyme(s) responsible for this cleavage, we have analyzed this processing in E. coli mutants deficient in endoribonucleases. A comparative analysis of cit mRNA degradation was performed in RNase E and RNase III mutants and in wild-type strains using Northern blot hybridization. This analysis revealed that the cit transcript is degraded into several breakdown products, which are significantly stabilized in the mutant lacking RNase III. Our results indicate that the complex secondary structure has a critical role in the control of the expression of cit mRNA. A model for processing is discussed.

Bacterial Proteins↗
PubMed2000

Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCDO 763.

In Lactococcus lactis, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases. We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT. In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT. We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription. BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5'-phosphate-dependent aminotransferases. In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric. It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively. The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine. However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors. Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.

Amino Acids, Branched-Chain↗
PubMed1995

A Starter Culture Rotation Strategy Incorporating Paired Restriction/ Modification and Abortive Infection Bacteriophage Defenses in a Single Lactococcus lactis Strain.

Three derivatives of Lactococcus lactis subsp. lactis NCK203, each with a different pair of restriction/ modification (R/M) and abortive infection (Abi) phage defense systems, were constructed and then rotated in repeated cycles of a milk starter culture activity test (SAT). The rotation proceeded successfully through nine successive SATs in the presence of phage and whey containing phage from previous cycles. Lactococcus cultures were challenged with 2 small isometric-headed phages, (phi)31 and ul36, in one rotation series and with a composite of 10 industrial phages in another series. Two native lactococcal R(sup+)/M(sup+) plasmids, pTRK68 and pTRK11, and one recombinant plasmid, pTRK308, harboring a third distinct R/M system were incorporated into three NCK203 derivatives constructed separately for the rotation. The R(sup+)/M(sup+) NCK203 derivatives were transformed with high-copy-number plasmids encoding four Abi genes, abiA, abiC, per31, and per50. Various Abi and R/M combinations constructed in NCK203 were evaluated for their effects on cell growth, level of phage resistance, and retardation of phage development during repeated cycles of the SAT. The three NCK203 derivatives chosen for use in the SAT exhibited additive effects of the R/M and Abi phenotypes against sensitive phages. In such combinations, phage escaping restriction are prevented from completing their infective cycle by an abortive response that kills the host cell. The rotation series successfully controlled modified, recombinant, and mutant phages which were resistant to any one of the individual defense systems by presenting a different set of R/M and Abi defenses in the next test of the rotation.

Journal Article↗
PubMed2002

An X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis: cloning, expression in Escherichia coli, and application for removal of N-terminal Pro-Pro from recombinant proteins.

A novel pepX gene was cloned from isolated DNA of Lactococcus lactis by PCR. The deduced amino acid sequence of the 89-kDa protein showed 94, 93, 65, and 44% identity with the pepX protein from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactobacillus delbruecki subsp. bulgaricus, and Lactobacillus helveticus, respectively, and contained a serine protease G-K-S-Y-L-G consensus motif. The pepX gene has been cloned into pET17b and was expressed at a high level in Escherichia coli BL21 (DE3) LysS. PepX was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE Sephadex A-50 chromatography. Optimal pepX activity was observed at pH 8.0 and 37 degrees C. According to SDS-PAGE analysis, pepX has a molecular mass of approximately 89 kDa. The peptidase can remove completely the unwanted X-Pro from the N-terminal of the target protein, releasing the naturally active protein and peptide, revealing a prospective application of pepX in large-scale production of pharmaceutical protein and peptide products.

Amino Acid Sequence↗
PubMed2002

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures.

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.

DNA, Bacterial↗
PubMed1999

Effect of alpha-acetolactate decarboxylase inactivation on alpha-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis.

Strains of Lactococcus lactis subsp. lactis biovar diacetylactis deficient in alpha-acetolactate decarboxylase produce alpha-acetolactate. This unstable compound is a precursor of acetoin and an aromatic compound, diacetyl. Following random mutagenesis of strain CNRZ 483, alpha-acetolactate decarboxylase-negative mutant 483 M1 was selected. When grown in milk, its growth and acidification characteristics were similar to those of the parental strain. In anaerobic conditions, the parental strain produced 2.10 mM acetoin and less than 0.05 mM diacetyl. The mutant accumulated up to 2.11 mM alpha-acetolactate, which spontaneously degraded to acetoin and diacetyl. After 24 h of culture, the alpha-acetolactate concentration was only 0.49 mM and the acetoin and diacetyl concentrations reached 1.50 mM and 0.26 mM, respectively. Diacetyl production by both strains increased in aerobic conditions, as well as when citrate was added. In contrast to cultures of the parental strain, however, diacetyl and acetoin concentrations in mutant cultures continued to increase without reaching a plateau. The results also showed that diacetyl production by wild type L. lactis subsp. lactis biovar diacetylactis strains cannot be explained uniquely by the spontaneous decarboxylation of the alpha-acetolactate produced in the culture medium.

Journal Article↗
PubMed2002

Effect of exopolysaccharides on phage-host interactions in Lactococcus lactis.

In this study, we report that Lactococcus lactis strains producing exopolysaccharides (EPS) are sensitive to virulent phages. Eight distinct lytic phages (Q61 to Q68) specifically infecting Eps(+) strains were isolated in 47 buttermilk samples obtained from 13 North American factories. The eight phages were classified within the 936 species by the multiplex PCR method, indicating that these phages are not fundamentally distinct from those infecting Eps(-) L. lactis strains. The host range of these phages was determined with 19 Lactococcus strains, including 7 Eps(+) and 12 Eps(-) cultures. Three phages (Q62, Q63, and Q64) attacked only the Eps(+) strain SMQ-419, whereas the five other phages (Q61, Q65, Q66, Q67, and Q68) infected only the Eps(+) strain SMQ-420. The five other Eps(+) strains (H414, MLT2, MLT3, SMQ-461, and SMQ-575) as well as the 12 Eps(-) strains were insensitive to these phages. The monosaccharide composition of the polymer produced by the seven Eps(+) strains was determined. The EPS produced by strains MLT3, SMQ-419, and SMQ-575 contained glucose, galactose, and rhamnose. The EPS fabricated by H414 contained only galactose. The EPS made by MLT2, SMQ-420, and SMQ-461 contained glucose and galactose. These findings indicate that the sugar composition of the EPS has no effect on phage sensitivity. The plasmid encoding the eps operon was cured from the two phage-sensitive strains. The cured derivatives were still phage sensitive, which indicates that EPS are not necessary for phage infection. Phage adsorption assays showed that the production of EPS does not confer a significant phage resistance phenotype.

Bacteriophages↗
PubMed2004

Synonymous codon usage in Lactococcus lactis: mutational bias versus translational selection.

In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed. Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism. However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis. It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets. Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms. The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values. This suggests that amino acid composition exerts constraints on codon usage in this organism. On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values. Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.

Codon↗
PubMed2006

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995.

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strains.

Bacterial Proteins↗
PubMed2002

Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese.

AIMS: This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. METHODS AND RESULTS: The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P < 0.001) different. CONCLUSIONS: Significant (P < 0.001) differences between titrable acidity of F and S strains were observed after the second hour of incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.

Animals↗
PubMed2006

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin.

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.

Adenosine Triphosphate↗
PubMed2002

Increasing acidification of nonreplicating Lactococcus lactis deltathyA mutants by incorporating ATPase activity.

Lactococcus lactis MBP71 deltathyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.

Adenosine Triphosphatases↗
PubMed2004

Recent genetic transfer between Lactococcus lactis and enterobacteria.

The genome sequence of Lactococcus lactis revealed that the ycdB gene was recently exchanged between lactococci and enterobacteria. The present study of ycdB orthologs suggests that L. lactis was probably the gene donor and reveals three instances of gene transfer to enterobacteria. Analysis of ycdB gene transfer between two L. lactis subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris, indicates that the gene can be mobilized, possibly by conjugation.

Base Sequence↗
PubMed2005

Development of an enteric-coated, layered multi-particulate formulation for ileal delivery of viable recombinant Lactococcus lactis.

Layering of recombinant hIL-10 producing Lactococcus lactis (L. lactis Thy12) on inert carriers is a promising technique for the preparation of a multi-particulate formulation of viable, hIL-10 producing L. lactis. To improve viability after layering and storage, L. lactis Thy12 was layered in different matrices (10% skim milk and/or 2.5, 5, 10% inulin). After layering, the highest viability was obtained in the 10% skim milk supplemented with 5% inulin matrix (8.7%). However, upon storage, 10% skim milk alone yielded the highest viability. Thereby, layered L. lactis Thy12 showed superior long term stability in comparison with freeze-dried L. lactis Thy12. The layering process was performed during 3h without encountering technical problems, with good layer consistence and constant viability. Enteric properties were obtained with a 30% Eudragit L30D-55 or 15% Eudragit FS30D coating and maintained during an initial six months storage period (-20 degrees C/20% RH). After in vitro simulation of the gastric stage, only 5% of the bacteria remained viable in Eudragit L30D-55 coated pellets, contrary to 85% in Eudragit FS30D coated pellets, indicating its superior protective capacity against gastric fluid. After eight months storage (-20 degrees C), 80% of the initial L. lactis Thy12 remained viable in the Eudragit FS30D coated pellets.

Chemistry, Pharmaceutical↗