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PubMed1993

Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis.

The nucleotide sequence of a chromosomal DNA fragment of Lactococcus lactis subsp. lactis SSL135, previously implicated in peptide utilization, has been determined. The genes oppDFBCA, encoding the oligopeptide transport system (Opp), and that encoding the endopeptidase PepO were located on this 8.9-kb DNA fragment. The oppDFBCA and pepO genes are probably organized in an operon. Analysis of the deduced amino acid sequences of the genes indicated that the oligopeptide transport system consists of two ATP-binding proteins OppD and OppF, two integral membrane proteins OppB and OppC, and a substrate-binding protein OppA. On the basis of the homology of OppF and OppD of L. lactis with other ABC (ATP-binding cassette) transporter proteins, the L. lactis Opp system can be classified as a member of this group. Two integration mutants, one defective in OppA and the other defective in PepO, were constructed. Growth of these mutants in a chemically defined medium with oligopeptides showed that the transport system, but not the endopeptidase, is essential for the utilization of peptides longer than three residues. Uptake of the pentapeptide Leu-enkephalin in glycolyzing lactococcal cells was followed by rapid hydrolysis of the peptide intracellularly. Importantly, extracellular hydrolysis of Leu-enkephalin is not observed. The OppA-deficient mutant was unable to transport Leu-enkephalin. Growth experiments with pasteurized milk revealed that transport of oligopeptides forms an essential part of the proteolytic system in lactococci.

Amino Acid Sequence↗
PubMed1998

Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA.

The Lactococcus lactis ccpA gene, encoding the global regulatory protein CcpA, was identified and characterized. Northern blot and primer extension analyses showed that the L. lactis ccpA gene is constitutively transcribed from a promoter that does not contain a cre sequence. Inactivation of the ccpA gene resulted in a twofold reduction in the growth rate compared with the wild type on glucose, sucrose and fructose, while growth on galactose was almost completely abolished. The observed growth defects could be complemented by the expression of either the L. lactis or the Bacillus subtilis ccpA gene. The disruption of the ccpA gene reduced the catabolite repression of the gal operon, which contains a cre site at the transcription start site and encodes enzymes involved in galactose catabolism. In contrast, CcpA activates the transcription of the cre-containing promoter of the las operon, encoding the glycolytic enzymes phosphofructokinase, pyruvate kinase and L-lactate dehydrogenase, because its transcription level was fourfold reduced in the ccpA mutant strain compared with the wild-type strain. The lower activities of pyruvate kinase and L-lactate dehydrogenase in the ccpA mutant strain resulted in the production of metabolites characteristic of a mixed-acid fermentation, whereas the fermentation pattern of the wild-type strain was essentially homolactic.

Bacterial Proteins↗
PubMed2006

Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2.

The native lactococcal plasmid, pKR223, from Lactococcus lactis subsp. lactis biovar diacetylactis KR2 encodes two distinct bacteriophage-resistant mechanisms, the LlaKR2I restriction and modification (R/M) system and the abortive infection (Abi) mechanism, AbiR, that impedes bacteriophage DNA replication. This study completed the characterization of AbiR, revealing that it is the first Abi system to be encoded by three genes, abiRa, abiRb, and abiRc, arranged in an operon and that it requires the methylase gene from the LlaKR2I R/M system. An analysis of deletion and insertion clones demonstrated that the AbiR operon was toxic in L. lactis without the presence of the LlaKR2I methylase, which is required to protect L. lactis from AbiR toxicity. The novelty of the AbiR system resides in its original gene organization and the unusual protective role of the LlaKR2I methylase. Interestingly, the AbiR genetic determinants are flanked by two IS982 elements generating a likely transposable AbiR composite. This observation not only substantiated the novel function of the LlaKR2I methylase in the AbiR system but also illustrated the evolution of the LlaKR2I methylase toward a new and separate cellular function. This unique structure of both the LlaKR2I R/M system and the AbiR system may have contributed to the evolution of the LlaKR2I methylase toward a novel role comparable to that of the cell cycle-regulated methylases that include Dam and CcrM methylases. This new role for the LlaKR2I methylase offers a unique snapshot into the evolution of the cell cycle-regulated methylases from an existing R/M system.

Bacteriophages↗
PubMed1990

Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis.

The thymidylate synthase (thyA) gene has been isolated from Lactococcus lactis subsp. lactis. The cloned gene was strongly expressed in Escherichia coli both in vivo and in vitro (maxicells and cell-free transcription and translation systems) and complemented E. coli thyA mutants. DNA-DNA hybridizations demonstrated that the thyA gene is encoded by the chromosome of L. lactis subsp. lactis. By sequential deletion of DNA outside the complementing region, the thyA gene was localized to a 1.1-kilobase DNA fragment. The nucleotide sequence of the lactococcal thyA gene was determined by the dideoxy-chain termination technique. The derived amino acid sequence indicated a protein size of 32,580 daltons, which is in good agreement with results obtained from maxicell and in vitro transcription and translation experiments. The primary sequence is homologous to 12 other thyA proteins from a variety of other organisms. Upstream from the structural gene, -10 and -35 promoter sequences which were almost canonical sigma-70 promoter sequences were identified, which may explain the strong expression of the thyA gene observed in E. coli. An A-T-rich sequence characteristic of gram-positive promoters was also noted adjacent to the -35 region. The thyA gene has potential as a marker for plasmid maintenance and selection in food systems.

Amino Acid Sequence↗
PubMed1991

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator.

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.

Calibration↗
PubMed2001

Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

Amino Acid Sequence↗
PubMed1996

Molecular analysis of the replication origin of the Lactococcus lactis plasmid pCJ305.

The replication origin region, ori, of the Lactococcus lactis subsp. lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (RepB) essential for plasmid replication. Gel retardation assays, using lactococcal crude cell extracts in which RepB was overproduced, were used to demonstrate that the replication protein interacts with DNA sequences within the origin region. IR1 was identified as a RepB binding site. The -35 region of the repB promoter is contained within the loop of the potential stem-loop structure of IR1, suggesting autoregulation of repB. The pCI305 RepB failed to interact with DNA sequences within the minimal replicons of nine other members of the pCI305 family of plasmids and it was concluded that this DNA-protein interaction was replicon specific. In vivo studies were performed to determine the role of the three-and-one-half copies of the 22-bp sequences. When this sequence was provided in trans on a compatible vector, it resulted in the loss of pCI305 from the cell population (incompatibility).

Bacterial Proteins↗
PubMed1989

Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis.

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.

Amino Acids↗
PubMed1994

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11.

An endopeptidase has been purified from Lactococcus lactis subsp. cremoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor p-chloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl fluoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. Two of the oligopeptides produced during the early stages of beta-casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic system.

Amino Acid Sequence↗
PubMed1997

Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis.

This study demonstrates that exposure of log-phase Lactococcus lactis subsp. cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol. The intracellular pH (pHi) played a major role in the induction of this multistress resistance response. The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo. When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death. Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo. Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells. Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred. Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.

Acetic Acid↗
PubMed1992

Sequence of a gene (lap) encoding a 95.3-kDa aminopeptidase from Lactococcus lactis ssp. cremoris Wg2.

A gene (lap) coding for a Lactococcus lactis ssp. cremoris Wg2 aminopeptidase was cloned from genomic libraries of size-fractionated lactococcal DNA. The 5' end of the lap gene was isolated by using a polymerase chain reaction hybridization probe of 77 nucleotides (nt) synthesized from two degenerate primers derived from the N-terminal amino acid (aa) sequence of the lactococcal lysine-aminopeptidase (LAP). The remaining part(s) of the gene were recovered by a search for overlapping sequences in Southern blots of variably restricted genomic DNA. The complete nt sequence of the lap gene has been determined. A large open reading frame of 2538 nt is predicted to encode a polypeptide of 846 aa (approx. 95.3 kDa; pI, 5.93). A recombinant plasmid containing the lap gene with its flanking sequences was shown to direct in vivo synthesis of LAP activity in Escherichia coli, indicating that the cloned DNA fragment is the lap gene. Primer extension analysis of lap mRNA and Northern blot hybridization indicated the gene transcript to be approx. 3.0 kb in size with a 5'-untranslated region of 19-22 nt. Comparison of the deduced aa sequence indicates that the LAP has extensive homology with the super family of Zn(2+)-metallohydrolases and shows identity in the core deca-peptide consensus sequence for the Zn(2+)-binding motif of these enzymes.

Amino Acid Sequence↗
PubMed2002

Characterization of the promoter regions involved in galactose- and nisin-mediated induction of the nisA gene in Lactococcus lactis ATCC 11454.

The nisA promoter is positively regulated in Lactococcus lactis ATCC 11454 by autoinduction via a two-component NisRK-mediated system. However, induction of this promoter can also occur when introduced into the plasmid-free L. lactis LM0230 during growth in galactose or lactose, independent of the NisRK system. In this study, we also characterized this galactose-mediated induction by determining the nisA start site during growth in galactose, which was identical to the nisA start site upon nisin induction. The region involved in the galactose-mediated induction of the nisA promoter was investigated by directed deletion analysis of a 200 bp region upstream of the nisA promoter in the transcription fusion pDOC99. The induction of the deletion derivatives by galactose and nisin was compared phenotypically using beta-galactosidase measurements, and the regions necessary for the induction were determined by sequence analysis. Analysis of these regions revealed two sets of a TCT direct repeat [TCT-N8-TCT] present at positions (-107 to -94) and (-39 to -26) relative to the transcription initiation site. Disruption of the upstream repeat abolished galactose induction and significantly reduced the nisin induction capacity, suggesting a potential pivotal role for these repeats in transcription induction of the nisA promoter. It was also observed that the galactose-mediated induction was abolished when a plasmid containing the phosphotransferase system (PTS), phospho-beta-galactosidase and tagatose pathway genes was introduced into this strain. As this effectively made the Leloir pathway redundant, it points to some component of this pathway as the specific inducer of the nisA promoter.

Bacterial Proteins↗
PubMed1999

Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis.

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6. 6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.

Adenosine Triphosphate↗
PubMed2000

Gene cloning, sequencing, and inactivation of the branched-chain aminotransferase of Lactococcus lactis LM0230.

A branched-chain aminotransferase gene (ilvE) from Lactococcus lactis LM0230 was identified on a 9-kb chromosomal insert by complementation in Escherichia coli DL39. Sequencing of a 2.0-kbp fragment resulted in the identification of a 1,023-bp open reading frame that could encode a 340-amino-acid protein. Sequence analysis of the deduced amino acid sequence revealed 62% identity to IlvE of Haemophilus influenzae and high similarity to IlvEs from a variety of organisms found in GenBank classified as class IV aminotransferases. Under logarithmic growth in complex medium, ilvE is transcribed monocistronically as a 1.1-kb transcript. Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein. A derivative of LM0230 lacking IlvE activity was constructed by gene replacement. Comparison of the IlvE-deficient strain's ability to grow in defined media lacking an amino acid but containing its alpha-keto acid biosynthetic precursor to that of the wild-type strain indicated that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors. Comparison of the aminotransferase activity of the IlvE mutant to LM0230 revealed that the mutant retained <2, 4.5, 43, 40, and 76% of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively. No difference in growth or acidification rate between LM0230 and the IlvE-deficient strain was observed in milk.

Amino Acids, Branched-Chain↗
PubMed1991

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.

Bacterial Proteins↗
PubMed2001

Survival response and rearrangement of plasmid DNA of Lactococcus lactis during long-term starvation.

The survival response of Lactococcus lactis during long-term starvation was investigated. The cells were cultured with different levels of glucose (the sole energy source) and either were kept in the resultant spent medium or transferred to fresh medium (without glucose) for up to 2 years. The survival of the cells during starvation was not dependent on the nature of transition phase, as expected, but on the nature of medium in which the cells were kept. The proliferation of cells, despite the apparent lack of glucose, could have been due to some cells being able to utilize the small amounts of peptides still present in the spent medium or to use energy sources provided by the breakup of dead cells. The 1- and 2-year-old cultures contained cells with vastly changed morphotypes. When these isolates were examined, it was revealed that the original plasmids present in the parent were rearranged in a certain way, and an entirely new plasmid was generated. Changes were also evident in the chromosomal DNA and in gene expression. Furthermore, all of the isolates exhibited a growth advantage relative to the parent cells when grown in energy-limiting media. When they were tested against different types of stresses, they exhibited a higher resistance against the bile salt and hydrogen peroxide stresses compared to the parent. Because of the similar changes observed in the 2-year-old isolates, a similar survival strategy may be operational in those cells that survive for that length of time.

Bacterial Proteins↗
PubMed1998

tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

Artificial Gene Fusion↗
PubMed1993

Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes.

Lactococcus lactis subsp. cremoris is widely used in the manufacture of fermented milk products. Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful. Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L. lactis subsp. cremoris from related strains. These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures. A total of 170 strains of L. lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L. lactis. Fifty-nine of these isolates also hybridized to L. lactis subsp. cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L. lactis subsp. cremoris-specific probe had the L. lactis subsp. cremoris phenotype.

Animals↗