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PubMed2005

[Influence of expression of transglutaminase on the growth of Lactococcus lactis].

To improve the aerobic growth performance of Lactococcus lactis subsp. cremoris NZ9000, the gene mtg encoding the mature microbial transglutaminase was amplified from the chromosomal DNA of Streptoverticillium mobaraense and then cloned into the nisin-inducible expression vector pNZ8148. The resulting plasmid pFL001 was transformed into strain NZ9000 by electroporation. Compared with strain NZ9000 harboring pNZ8148 (the control strain), strain NZ9000 harboring pFL001 (the recombinant strain) had a remarkably improved aerobic growth performance. When grown aerobically under non-pH-controlled conditions, the maximal biomass of the recombinant strain reached 4.13 g/L, which was 11-fold higher than the growth of the control strain (0.34 g/L). When grown aerobically with the pH controlled at 6.5 +/- 0.1, the maximal biomass of the recombinant strain reached 4.73 g/L, which was an 80% increase compared with the growth of the control strain. In addition, the efficiency of biomass synthesis relative to glucose consumption (Y(x/s)) of the recombinant strain, 71.7 g of biomass per mol of glucose, was 1.6-fold higher than that of the control strain. The significantly improved growth performance of the recombinant strain might be attributed to the expression of mtg in the recombinant strain, which might increase intracellular pH and save part of the energy(ATP) that was originally used for pumping the cytoplasmic H+, and as a consequence, the energy used for growth increased accordingly.

Hydrogen-Ion Concentration↗
PubMed1997

Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis.

Transport of hydrophilic di- and tripeptides into Lactococcus lactis is mediated by a proton motive force-driven peptide transport protein (DtpT) that shares similarity with eukaryotic peptide transporters, e.g., from kidney and small intestine of rabbit, man, and rat. Hydropathy profiling in combination with the "positive inside rule" predicts for most of the homologous proteins an alpha-helical bundle of 12 transmembrane segments, but the positions of these transmembrane segments and the location of the amino and carboxyl termini are by no means conclusive. The secondary structure of DtpT was investigated by analyzing 42 DtpT-alkaline phosphatase fusion proteins, generated by random or directed fusions of the corresponding genes. These studies confirm the presence of 12 transmembrane segments but refute several other predictions made of the secondary structure. Data obtained from the fusion proteins were substantiated by studying the accessibility of single cysteine mutants in putative cytoplasmic or extracellular loops by membrane (im)permeant sulfhydryl reagents. The deduced topology model of DtpT consists of a bundle of 12 alpha-helixes with a short amino and a large carboxyl terminus, both located at the cytoplasmic site of the membrane. On the basis of sequence comparisons with DtpT, it seems likely that the structure model of the amino-terminal half of DtpT also holds for the eukaryotic peptide transporters, whereas the carboxyl-terminal half is largely different.

Alkaline Phosphatase↗
PubMed2002

In situ determination of the intracellular pH of Lactococcus lactis and Lactobacillus plantarum during pressure treatment.

Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs of Lactococcus lactis and Lactobacillus plantarum during and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts of Lactococcus lactis and Lactobacillus plantarum by 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH of L. lactis was decreased to the value of the extracellular pH during compression. The same result was observed during treatment of Lactobacillus plantarum at 300 MPa. Lactobacillus plantarum was unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5. Lactococcus lactis lost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions.

Animals↗
PubMed1994

Expression of a chitinase gene from Serratia marcescens in Lactococcus lactis and Lactobacillus plantarum.

A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in the same transcriptional orientation as the gene specifying the replication protein of the vector pIL253. Using the expression vectors pMG36e and pGKV259 with lactococcal promoter fragments p32 and p59, the expression in L. lactis was increased nine- and 27-fold, respectively. An additional twofold increase was obtained after cloning the gene under the control of p59 in the high-copy number replicon pIL253. In Lactobacillus plantarum, chitinase activity was expressed from p32, and the activity was at the same level as under p32 control in L. lactis.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1.

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with lambda NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was expressed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded beta-casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plasmid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.

Cloning, Molecular↗
PubMed1991

Molecular characterization of the nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3.

The nisin resistance determinant of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3 was localized onto a 1.3-kb EcoRI-NdeI fragment by subcloning and interrupting the NdeI site by cloning random NdeI fragments into it; the nisin resistance determinant was then sequenced. The nucleotide sequence revealed a large open reading frame containing 318 codons. Putative transcription and translation signal sequences were located directly upstream from the initiation codon. Immediately downstream of the termination codon was a palindromic region resembling a rho-independent termination sequence. This 957-nucleotide open reading frame and its associated transcription and translation signal sequences were cloned into plasmid-free L. lactis subsp. lactis LM0230 and conferred an MIC of 160 IU of nisin per ml. This level of nisin resistance is equivalent to that of the initial nisin-resistant subclone, pFM011, used for further subcloning in this study. The inferred amino acid sequence would result in a protein with a molecular mass of 35,035 Da. This value was in agreement with the molecular mass of a protein detected after in vitro transcription and translation of DNA encoding the nisin resistance gene, nsr. This protein contained a hydrophobic region at the N terminus that was predicted to be membrane associated but did not contain a typical signal sequence cleavage site. No significant homology was detected when the DNA sequence of the nsr gene and the amino acid sequence of its putative product were compared with other available sequences. When subjected to Southern hybridization, a 1.2-kb DraI fragment encoding the nsr gene did not hybridize with the genomic DNA of the nisin-producing strain L. lactis subsp. lactis 11454.

Amino Acid Sequence↗
PubMed2004

Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis.

Despite the fact that almost 39% of the theoretical expressed proteins of Lactococcus lactis have a predicted isoelectric point above 7, these proteins have not been studied in previous proteome analyses. In the present study, we set up a reference map of alkaline lactococcal proteins by using immobilized pH gradients (IPG) spanning pH 6 to 12 and 9 to 12, and protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Different electrophoresis systems for isoelectric focusing were evaluated to optimize the first dimension. Best results were obtained by sample application using cup-loading at the anodic side and increasing the final voltage up to 8000 V for IPGs, using N,N-dimethylacrylamide as monomer. After two-dimensional gel electrophoresis of extracts obtained from exponentially growing cells, about 200 protein spots were selected for identification by peptide mass fingerprinting. With MALDI-TOF MS, 153 proteins were identified that were the products of 85 different genes. Their predicted isoelectric points range from as high as 11.31 to as low as 6.34. Ribosomal proteins, hypothetical proteins and proteins with unknown function represent the largest groups of identified proteins. For further classification, the codon adaptation index (CAI) and grand average of hydropathicity (GRAVY) for each lactococcal protein were calculated. The protein with the lowest CAI identified in this study is the manganese ABC transporter ATP-binding protein. Less than 10% of the alkaline lactococcal proteins have a smaller CAI. The highest GRAVY for an identified protein is 0.26. The complete in silico data of Lactococcus lactis as well as clickable reference maps are available at www.wzw.tum.de/proteomik/lactis.

Bacterial Proteins↗
PubMed2006

Exopolysaccharide expression in Lactococcus lactis subsp. cremoris Ropy352: evidence for novel gene organization.

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.

Animals↗
PubMed1998

Molecular diversity and relationship within Lactococcus lactis, as revealed by randomly amplified polymorphic DNA (RAPD).

Lactococcus lactis strains are widely used in industrial dairy fermentations. Conventional phenotypic tests have been used for years to classify members of this species into two subspecies, lactis and cremoris, and play a key role in the choice of strains to be used in particular cheese fermentations. DNA hybridisation techniques have also been used for strain classification, giving rise to two genome homology groups. However, results showed discrepancies between the two methods of classification. We applied the randomly amplified polymorphic DNA fingerprinting (RAPD) technique to resolve previous contradictions in lactococcal classifications. Unlike usual RAPD methods, we use three primers to classify 113 strains and integrate the resulting information by a digitised programme used for this purpose. Our analysis revealed three major RAPD groups, designated G1, G2 and G3. G1 and G3 contain strains of the lactis subspecies, and G2 contains strains of the cremoris subspecies, as previously defined by phenotypic characteristics. Moreover, group G1 corresponds to one genome homology group, and groups G2 and G3 correspond to the second one. The taxonomic structure within L. lactis is therefore unusual: two distinct genetic groups of strains show indistinguishable phenotypes, while conversely, two phenotypically distinct groups are genetically homologous. We hypothesize that a subfamily of the subsp. lactis group gave rise to the cremoris subspecies.

DNA Fingerprinting↗
PubMed1992

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.

Animals↗
PubMed2001

Hemin reconstitutes proton extrusion in an H(+)-ATPase-negative mutant of Lactococcus lactis.

H(+)-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H(+)-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells are capable of coupling NADH oxidation to proton translocation.

Cell Membrane↗
PubMed1999

The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids.

Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein. For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L. lactis phospholipids. The detergent was removed by adsorption onto polystyrene beads. The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L. lactis.

ATP-Binding Cassette Transporters↗
PubMed2000

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis.

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.

Acetyltransferases↗
PubMed1998

Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture.

Lactococcus lactis DPC4268 is widely used for Cheddar cheese manufacture in Ireland where it is recognised for its reliable fast acid producing ability in dairy environments. A transconjugant of this strain, L. lactis DPC4275, was generated which produces the broad spectrum, two-component bacteriocin lacticin 3147, which is inhibitory to a variety of undesirable gram-positive bacteria including Clostridium, Bacillus, Enterococcus, Listeria, Staphylococcus and Streptococcus. Both DPC4268 and DPC4275 were used as single strain starters for manufacture of salamis. These products were compared with a salami manufactured with a conventional starter (Lactobacillus sake and Staphylococcus carnosus) in terms of pH development, a(w)-value, weight loss, colour development and sensory characteristics. Salamis produced with either lactococcal culture exhibited pH values below 5.1 and a(w)-values below 0.90 which is favourable for preservation and hygienic stability. In addition, these salamis had good sensory and colorimetric qualities. A minimum lacticin 3147 concentration of 640 AU/g was detected in the salamis which were produced with L. lactis DPC4275.

Animals↗
PubMed2004

Natural-abundance isotope ratio mass spectrometry as a means of evaluating carbon redistribution during glucose-citrate cofermentation by Lactococcus lactis.

The cometabolism of citrate and glucose by growing Lactococcus lactis ssp. lactis bv. diacetylactis was studied using a natural-abundance stable isotope technique. By a judicious choice of substrates differing slightly in their 13C/12C ratios, the simultaneous metabolism of citrate and glucose to a range of compounds was analysed. These end-products include lactate, acetate, formate, diacetyl and acetoin. All these products have pyruvate as a common intermediate. With the objective of estimating the degree to which glucose and citrate metabolism through pyruvate may be differentially regulated, the delta13C values of the products accumulated over a wide range of concentrations of citrate and glucose were compared. It was found that, whereas the relative accumulation of different products responds to both the substrate concentration and the ratio between the substrates, the delta13C values of the products primarily reflect the availability of the two substrates over the entire range examined. It can be concluded that in actively growing L. lactis the maintenance of pyruvate homeostasis takes precedence over the redox status of the cells as a regulatory factor.

Carbon↗
PubMed2000

Dissolution of xylose metabolism in Lactococcus lactis.

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-). Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.

Aldose-Ketose Isomerases↗
PubMed2006

Glyceraldehyde-3-phosphate dehydrogenase regulation in Lactococcus lactis ssp. cremoris MG1363 or relA mutant at low pH.

AIMS: To analyse the phenotype of a relA acid-resistant mutant of Lactococcus lactis ssp. cremoris MG1363, and to compare the glyceraldehyde-3-phosphate dehydrogenase regulation in both strains. METHODS AND RESULTS: Lactococcus lactis ssp. cremoris MG1363 and the relA mutant affected in the (p)ppGpp synthetase were grown in a series of batch-mode fermentation at different pH-regulated conditions with glucose as carbon substrate. All the determinants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regulation were quantified. In L. lactis MG1363, the GAPDH was strongly inhibited in vitro by decreased pH values, but this inhibition was totally compensated in vivo by the lower NADH/NAD+ ratio and more efficiently by the important increase in the intracellular amount of GAPDH. In contrast to the wild type, GAPDH activity of the relA strain was not increased when grown at low pH but the level of GAPDH remained constitutively high. However, pH homeostasis was not improved in the relA mutant and it grew slower and exhibited a lower glycolytic flux than the wild-type strain at low pH. CONCLUSIONS: Despite a better resistance to acid stress, the increased survival in L. lactis relA mutant at low pH was not related with an improved pH homeostasis but was associated with a diminished capacity to maintain a high flux through glycolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The phenotype of a strong acid-resistant L. lactis strain was established in acid conditions and some key metabolic parameters compared with the wild type. This analysis led to the conclusion that growth and survival seem to be antinomic parameters, since improving one of them leads to a decrease in the other one.

Bacteriology↗
PubMed1998

Autolysis of Lactococcus lactis is influenced by proteolysis.

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.

Bacteriolysis↗