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PubMed1996

Concentration and recovery of the bacteriocin nisin from Lactococcus lactis subsp. lactis.

This investigation compared various techniques to concentrate and recover nisin from Lactococcus lactis cells. Centrifugation, combined with pH manipulation, was initially investigated as it is known that nisin will adsorb to producer cells at pH 6.5 and desorb at pH < or = 3.0. Unfortunately, centrifugation stripped producer cells of nisin even at pH 6.5; therefore a milder separation process (microfiltration) was evaluated. Results using a medium (LTB) containing peptone, tryptone, yeast extract, NaCl, Na2HPO4 and glucose demonstrated that nisin could be at least partially concentrated with cells via microfiltration. However, when a filtered stillage-based medium was used, nisin production was boosted to levels which exceeded the holding capacity of producer cells, resulting in release of nisin from cells at pH 6.5. Since it appears unfeasible to use producer cells for nisin recovery, an alternative may be to separately immobilize cells/ fragments in a re-usable column to act as a "resin' to adsorb nisin. Microfiltration could then be used to release nisin from cells, with nisin recovered by passing the permeated material through the immobilized-cell columns at pH 6.5.

Bacteriocins↗
PubMed2002

Catabolism of mannitol in Lactococcus lactis MG1363 and a mutant defective in lactate dehydrogenase.

Mannitol metabolism in Lactococcus lactis MG1363 and in a derivative strain deficient in lactate dehydrogenase (LDH(d)) was characterized. Both strains had the ability to grow on mannitol as an energy source, although this polyol was a poorer substrate for growth than glucose. When compared to glucose, the metabolism of mannitol caused an NADH burden due to formation of an additional NADH molecule at the reaction catalysed by mannitol-1-phosphate dehydrogenase (Mtl1PDH). This resulted in a prominent accumulation of mannitol 1-phosphate (Mtl1P) both in growing and resting cells, suggesting the existence of a severe bottleneck at Mtl1PDH. Growth on mannitol induced the activity of Mtl1PDH in both the LDH(d) and MG1363 strains. The lower accumulation of Mtl1P in mannitol-grown cells when compared to glucose-grown LDH(d) cells, as monitored by in vivo (13)C-NMR, reflects this induction. A clear shift towards the production of ethanol was observed on mannitol, indicating pressure to regenerate NAD(+) when this substrate was used. A strategy to obtain a mannitol-overproducing strain is proposed.

Biomass↗
PubMed1990

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.

Amino Acid Sequence↗
PubMed1998

Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147.

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.

Bacteriocins↗
PubMed1997

Crystal structures and mechanism of 6-phospho-beta-galactosidase from Lactococcus lactis.

The initial structural model of 6-phospho-beta-galactosidase from Lactococcus lactis was refined to an R-factor of 16.4% (R[free] = 23.6%) to 2.3 A resolution (1 A = 0.1 nm), and the structures of three other crystal forms were solved by molecular replacement. The four structural models are essentially identical. The catalytic center of the enzyme is approximately at the mass center of the molecule and can only be reached through a 20 A long channel, which is observed with an "open" or "closed" entrance. The closed entrance is probably too small for the educt lactose-6-phosphate to enter, but large enough for the first product glucose to leave. Among the presented structures is a complex between an almost inactive mutant and the second product galactose-6-phosphate, which is exclusively bound at side-chains. A superposition (onto the native enzyme) of galactose-6-phosphate as bound to the mutant suggests the geometry of a postulated covalent intermediate. The binding mode of the educt was modeled, starting from the bound galactose-6-phosphate. A tightly fixed tryptophan is used as a chopping-board for splitting the disaccharide, and several other aromatic residues in the active center cavity are likely to participate in substrate transport/binding.

Bacterial Proteins↗
PubMed1992

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.

Alginates↗
PubMed1996

Purification and characterization of dihydroorotate dehydrogenase A from Lactococcus lactis, crystallization and preliminary X-ray diffraction studies of the enzyme.

Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A- and B-forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5-9.0. It is specific for L-dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate (III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P21 with unit cell dimensions a = 54.19 A, b = 109.23 A, c = 67.17 A, and beta = 104.5 degrees. A native data set has been collected with a completeness of 99.3% to 2.0 A and an Rsym value of 5.2%. Analysis of the solvent content and the self-rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.

Bacterial Proteins↗
PubMed2001

Relationship between glycolysis and exopolysaccharide biosynthesis in Lactococcus lactis.

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps(+)) and in nonproducer strain MG5267 (Eps(-)) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by (31)P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps(+) strain than in the Eps(-) strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps(+) strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, alpha-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. (13)C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.

Carbon Isotopes↗
PubMed2002

The dimeric dihydroorotate dehydrogenase A from Lactococcus lactis dissociates reversibly into inactive monomers.

The flavoenzyme dihydroorotate dehydrogenase A from Lactococcus lactis is a homodimeric protein of 311 residues/subunit, and the two active sites are positioned at a distance from the dimer interface. To promote formation of the monomeric form of the enzyme, we changed the residues involved in formation of two salt bridges formed between the residues Glu206 of the one polypeptide and Lys296 of the other polypeptide. The mutant enzymes formed inactive precipitates when cells were grown at 37 degrees C, but remained soluble and active when cells were grown at 25 degrees C. The salt bridges were not needed for activity, because the mutant enzymes in which one of the residues was converted to an alanine (E206A or K296A) retained almost full activity. The mutant enzymes in which the charge of one of the residues of the salt bridge was inverted (i.e., E206K or K296E) were severely impaired. The double mutant E206K-K296E, which has the possibility of forming salt bridges in the opposite orientation of the wild type, was fully active in concentrated solutions, but dissociated into inactive monomers upon dilution. The K(D) for the dimer to monomer dissociation reaction was 12 microM, and dimer formation was favored by the product, orotate, or by high ionic strength, indicating that the hydrophobic interactions are important for the subunit contacts. Wild-type dihydroorotate dehydrogenase A was similarly found to dissociate into inactive monomers, but with a K(D) for dissociation equal to 0.12 microM. These results imply that the dimeric state is necessary for activity of the enzyme.

Dihydroorotate Dehydrogenase↗
PubMed2006

Subcellular location of phage infection protein (Pip) in Lactococcus lactis.

The amino acid sequence of the phage infection protein (Pip) of Lactococcus lactis predicts a multiple-membrane-spanning region, suggesting that Pip may be anchored to the plasma membrane. However, a near-consensus sortase recognition site and a cell wall anchoring motif may also be present near the carboxy terminus. If functional, this recognition site could lead to covalent linkage of Pip to the cell wall. Pip was detected in both plasma membranes and envelopes (plasma membrane plus peptidoglycan) isolated from the wild-type Pip strain LM2301. Pip was firmly attached to membrane and envelope preparations and was solubilized only by treatment with detergent. Three mutant Pip proteins were separately made in which the multiple-membrane-spanning region was deleted (Pip-Deltammsr), the sortase recognition site was converted to the consensus (Pip-H841G), or the sortase recognition site was deleted (Pip-Delta6). All three mutant Pip proteins co-purified with membranes and could not be solubilized except with detergent. When membranes containing Pip-Deltammsr were sonicated and re-isolated by sucrose density gradient centrifugation, Pip-Deltammsr remained associated with the membranes. Strains that expressed Pip-H841G or Pip-Delta6 formed plaques with near unit efficiency, whereas the strain that expressed Pip-Deltammsr did not form plaques of phage c2. Both membranes and cell-free culture supernatant from the strain expressing Pip-Deltammsr inactivated phage c2. These results suggest that Pip is an integral membrane protein that is not anchored to the cell wall and that the multiple-membrane-spanning region is required for productive phage infection but not phage inactivation.

Animals↗
PubMed1990

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.

Amino Acid Sequence↗
PubMed2004

Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02.

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.

Bacterial Proteins↗
PubMed1991

Distance-dependent translational coupling and interference in Lactococcus lactis.

The possibility of raising the expression level of a heterologous gene in Lactococcus lactis by exploiting the principle of translational coupling was investigated. For this purpose, the Escherichia coli lacZ gene was transcriptionally fused to a short open reading frame (ORF) of lactococcal origin. A Shine-Dalgarno (SD) sequence was introduced at the boundary of the two ORFs. In a series of otherwise identical plasmids, the relative positions of the translational stop codon of the upstream ORF and the translational start codon of the downstream ORF (lacZ) were varied. The expression of lacZ gradually increased as the stop and start codons were placed in closer proximity. A concomitant switch from translational interference to translational coupling was observed. Best results were obtained with partially overlapping stop and start codons. It is concluded that the principle of translational coupling offers good possibilities to increase the level of heterologous gene expression in L. lactis.

Amino Acid Sequence↗
PubMed2005

Specificity and selectivity determinants of peptide transport in Lactococcus lactis and other microorganisms.

Peptide transport in microorganisms is important for nutrition of the cell and various signalling processes including regulation of gene expression, sporulation, chemotaxis, competence and virulence development. Peptide transport is mediated via different combinations of ion-linked and ATP-binding cassette (ABC) transporters, the latter utilizing single or multiple peptide-binding proteins with overlapping specificities. The paradigm for research on peptide transport is Lactococcus lactis, in which the uptake of peptides containing essential amino acids is vital for growth on milk proteins. Differential expression and characteristics of peptide-binding proteins in several Lactococcus lactis strains resulted in apparent conflicts with older literature. Recent developments and new data now make the pieces of the puzzle fall back into place again and confirm the view that the oligopeptide-binding proteins determine the uptake selectivity of their cognate ABC transporters. Besides reviewing the current data on binding specificity and transport selectivity of peptide transporters in L. lactis, the possible implications for peptide utilization by other bacterial species are discussed.

ATP-Binding Cassette Transporters↗
PubMed1999

Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis.

A plasmid-encoded phage abortive infection mechanism (AbiL) was identified from Lactococcus lactis biovar. diacetylactis LD10-1. AbiL conferred complete resistance to the small isometric-headed phage phi 712 (936 species) and partial resistance to the prolate-headed phage phi c2 (c2 species) when introduced into L. lactis LM0230. However, AbiL was not effective against the small isometric-headed phage ul36 (P335 species). The AbiL determinant was sequenced and it consists of two open reading frames, abiLi and abiLii. Their encoded proteins did not share significant homology with any known proteins in the protein databases. Transcriptional analysis indicated that abiLi and abiLii are organized as a single operon. Deletion within abiLii abolished the phage resistance. The levels of four phi c2-specific transcripts, three within the early transcribed region and one within the late transcribed region, were examined by RT-PCR, no effect of AbiL on synthesis of these transcripts was detected, suggesting that AbiL may act at a point after the transcription of phi c2 in L. lactis.

Amino Acid Sequence↗
PubMed2004

ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis.

The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia coli and Bacillus subtilis, respectively. A unique property of these regulators is that they form hexameric protein complexes, mediating repression of arginine biosynthetic pathways as well as activation of arginine catabolic pathways. The gltS-argE operon of Lactococcus lactis encodes a putative glutamate or arginine transport protein and acetylornithine deacetylase, which catalyzes an important step in the arginine biosynthesis pathway. By random integration knockout screening we found that derepression mutants had ISS1 integrations in, among others, argR and ahrC. Single as well as double regulator deletion mutants were constructed from Lactococcus lactis subsp. cremoris MG1363. The three arginine biosynthetic operons argCJDBF, argGH, and gltS-argE were shown to be repressed by the products of argR and ahrC. Furthermore, the arginine catabolic arcABD1C1C2TD2 operon was activated by the product of ahrC but not by that of argR. Expression from the promoter of the argCJDBF operon reached similar levels in the single mutants and in the double mutant, suggesting that the regulators are interdependent and not able to complement each other. At the same time they also appear to have different functions, as only AhrC is involved in activation of arginine catabolism. This is the first study where two homologous arginine regulators are shown to be involved in arginine regulation in a prokaryote, representing an unusual mechanism of regulation.

Amino Acid Sequence↗
PubMed2004

Methylthioacetaldehyde, a possible intermediate metabolite for the production of volatile sulphur compounds from L-methionine by Lactococcus lactis.

Volatile sulphur compounds (VSCs) production from L-methionine was studied in Lactococcus lactis. In vitro studies with radiolabelled L-methionine and resting cells of L. lactis revealed that L-methionine was initially converted to alpha-keto-gamma-methylthiobutyrate (KMBA) by a transamination reaction. A part of KMBA was subsequently chemically converted to methylthioacetaldehyde, methanethiol and dimethylsulphides. Chemical conversion of KMBA to methylthioacetaldehyde was dependent on pH, Mn(II) and oxygen. Since methanethiol and dimethylsulphide production was highly related to that of methylthioacetaldehyde, the latter compound was proposed as being an intermediate in VSCs production by L. lactis.

Acetaldehyde↗
PubMed2006

Stimulation of nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae.

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.

Animals↗