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PubMed2005

Proteomic characterization of the acid tolerance response in Lactococcus lactis MG1363.

Exponentially growing cells of Lactococcus lactis MG1363 are able to develop an Acid Tolerance Response (ATR) when incubated at pH 5, in both rich (M17)--and chemically defined (SA)--culture media. Physiological and proteomic characterization of this adaptive response indicated that L. lactis reorganizes its metabolism in response to acid stress to a great extent and quite differently in the two media. The development of ATR was fully dependent on protein de novo synthesis in SA and only partly dependent in M17. 2D gel electrophoresis revealed a total of 90 spots induced by acidity, 80 of which were identified by mass spectrometry. Only 10 proteins (BglA, PycA, GlmS, HasC, ArgS, GatA, AtpA, ArcB, Cfa, and SodA) were overproduced in the two media. A transcriptional analysis of the corresponding genes suggested that for half of them the mode of regulation may differ in the two media. Among the protein spots upregulated during the ATR in SA but not in M17, 13 already displayed an elevated rate of synthesis in M17 at neutral pH. These proteins could play an important role in the development of the protein de novo synthesis-independent ATR observed in M17.

Adaptation, Physiological↗
PubMed1994

Expression, purification, and characterization of thymidylate synthase from Lactococcus lactis.

The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q-Sepharose and phenyl-Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3-dimensional homology model, we have predicted covariant changes that might compensate for these differences. With the large amounts of L. lactis TS now available, studies can be pursued to understand the structure-function relationships of this enzyme compared to other TSs and to confirm the presumed roles of the compensatory changes predicted in the homology model.

Amino Acid Sequence↗
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↗
PubMed1998

The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function.

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.

Binding Sites↗
PubMed1995

Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

Amino Acid Sequence↗
PubMed1996

Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin).

A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus. The recombinant enzyme was expressed to high levels in Escherichia coli (approximately 10 mg/liter of culture) and purified to homogeneity in a two-step procedure. A number of peptides were studied as substrates for the enzyme. The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A-17, and alpha-neo-endorphin. In addition the enzyme hydrolyzes bradykinin, substance P, beta-endorphin, ACTH, and VIP. Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates. As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher kcat/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine. Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine. This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme. Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme. These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme.

Amino Acid Sequence↗
PubMed1997

The N-terminal sequence of Lactococcus lactis phosphoglucose isomerase purified by affinity chromatography differs from the other species.

A specific monoclonal antibody, M3A, was produced to rapidly purify Lactococcus lactis phosphoglucose isomerase (PGI) for amino acid sequence analysis. M3A recognized the Lac. lactis PGI specifically and sensitively with both enzyme-linked immunosorbent assay and Western blot analysis. The enzyme was rapidly purified to a specific activity of 21.8 U/mg with a yield of 20% by a three-step procedure, including M3A-bound Sepharose chromatography. The specific activity of PGI was increased about 64.1-fold from the cell lysate. The molecular mass of Lac. lactis PGI was estimated to be about 50 kDa by SDS-PAGE. The N-terminal amino acid sequence of Lac. lactis PGI exhibited no significant similarity to other PGIs, except for a 52.6% identity to Bacillus stearothermophilus PGI A and PGI B. These results suggest that there might be some molecular types of PGI.

Amino Acid Sequence↗
PubMed1993

The primary structure of phosphofructokinase from Lactococcus lactis.

The primary amino acid sequence of phosphofructokinase (EC2.7.1.11) from Lactococcus lactis, obtained by Edman analysis of peptides obtained from proteolytic digestions, is MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGIEVYGINHGYAGMVAGDIF PLTSASVGDKIGRGGTFLYSARYPEFAQVEGQLAGIEQLKKFGIEGVVVI GGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDFTIGFDTAVSTVVDAL DKIRDTSSSHNRTFVVEVMGRNAGDIALNAGIAAGADDISIPELEFKFEN VVNNINKGYEKGKNHHIIIVAEGVMTGEEFATKLKEAGYKGDLRVSVLGH IQRGGSPTARDRVLASRMGARAVELLRDGIGGVAVGIRNEELVESPILGT AEEGALFSLTTEGGIKVNNPHKAGLELYRLNSALNNLNL.

Amino Acid Sequence↗
PubMed1998

Effect of cold shock on protein synthesis and on cryotolerance of cells frozen for long periods in Lactococcus lactis.

Aspects of the cold-shock response in Lactococcus lactis subsp. lactis LL41-1 were investigated. First, it was determined whether new proteins were synthesized in response to cold shock. Cell-free extracts were prepared from a cold-shocked (exposed to 10 degreesC for 5 h) culture (cfe-cs) and from a non-cold-shocked (held at 30 degreesC continuously) culture (cfe-non), and were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A protein of approximately 6.3 kDa was present in the cfe-cs and appeared not to be present in the cfe-non. No other changes were evident. Second, the effect of cold shock on cryotolerance of cells that have been frozen at -20 degreesC for up to 1 year was examined. Without the cold-shock treatment prior to freezing the cell viability following freezing for 1 day was 34%, 14 days 32%, 182 days 7%, and 364 days 0.2%. However, with the cold shock treatment it was 83%, 82%, 12%, and 0.8%, respectively. It appears that cold shock significantly improves cryotolerance of the cells for short periods of freezing, but the protective effect was less marked following longer storage periods.

Bacterial Proteins↗
PubMed2002

Resistance to serum killing may contribute to differences in the abilities of capsulate and non-capsulated isolates of lactococcus garvieae to cause disease in rainbow trout (Oncorhynchus mykiss L.).

Three capsulated and two non-capsulated isolates of Lactococcus garvieae were investigated in terms of their wall proteins, virulence and interactions with rainbow trout immunoglobulin (Ig). All isolates were similar in integral membrane protein profile, and all were able to bind non-immune rainbow trout Ig, although different proteins appeared to be involved in Ig binding. However, whilst capsulated isolates were highly virulent, non-capsulated isolates were avirulent. This appeared to correlate with susceptibility of the non-capsulated isolates to rainbow trout normal serum. In contrast, the capsulated isolates were resistant to both normal and immune serum killing. In spite of this, passive immunisation of rainbow trout with specific anti-serum to L. garvieae was able to protect against challenge by capsulated isolates of L. garvieae. This suggests the antibody may have some other role in protection against disease caused by this important Gram-positive bacterial fish pathogen.

Animals↗
PubMed2002

Antibody increases phagocytosis and killing of Lactococcus garvieae by rainbow trout (Oncorhynchus mykiss, L.) macrophages.

The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.

Animals↗
PubMed1993

Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis.

Southern blot analysis of genomic DNA of the mesophilic lactic bacterium Lactococcus lactis subsp. lactis strain IL1403, illuminated six rRNA gene clusters. Each cluster contains one copy each of three rRNA genes, displaying the typical eubacterial organization of physically linked 16 S, 23 S and 5 S rRNA genes. Five of the six rRNA clusters were cloned into plasmid pBR322. One recombinant plasmid, pSLCM6, containing a 6500 base-pair genomic DNA fragment, was characterized by physical mapping and the sequences encoding rRNAs and tRNAs were localized by Southern hybridization. This fragment contains a single operon composed of one promoter, a leader sequence, a 16 S rRNA gene, a tRNA(Ala) gene, a 23 S rRNA gene, a 5 S rRNA gene and a tRNA(Asn) gene. S1 nuclease mapping and primer extension analysis of in vivo transcripts localized one transcriptional initiation site 150 base-pairs upstream from the start of the 16 S rRNA gene. These procedures also suggest that this transcript is processed by an RNAse III-like activity similar to Bacillus subtilis; i.e. the L. lactis nuclease might be sequence-specific. The chronology of specific cleavages occurring during the maturation process of the precursor transcript is described. One interesting observation is that the regions flanking the 16 S and 23 S rRNAs containing the primary processing sites are identical and contain sequences that could be involved in transcriptional antitermination. S1 mapping of the 3' ends of in vivo transcripts indicate that a terminator-like sequence a few base-pairs downstream from the distal tRNA(Asn) gene is inefficient in arresting transcription.

Base Sequence↗
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↗
PubMed1999

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity.

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.

Bioreactors↗
PubMed1999

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase.

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.

Biotechnology↗
PubMed2001

Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.

Base Sequence↗
PubMed1999

Identification of genes in a KG- phenotype of Lactococcus garvieae, a fish pathogenic bacterium, whose proteins react with antiKG- rabbit serum.

Five different clones (SA1B05, SA1B10, SA2F01, SA8A11 and SA9H10) were isolated from the gene library of the Lactococcus garvieae SA8201 (KG-) strain by immunological screening using rabbit serum against L. garvieae (KG-) phenotype cells. A Western blot analysis indicated that the molecular sizes of immunologically detected proteins of SA1B05, SA1B10, SA2F01, SA8A11 and SA9H10, which were fused with LacZ protein, were 25, 30, 28, 26 and 13 kDa, respectively. The amino acid sequences of the immunologically detected proteins of SA1B05, SA1B10, SA2F01 and SA8A11 were homologous to a processing protease of Bacillus subtilis (36.6%), dihydropteroate synthase of Escherichia coli (34.6%), trigger factor of B. subtilis (45.8%) and N-acetylglucosamine-6-phosphate deacetylase of Vibrio furnissii (37.1%), respectively. There was no significant homologous sequence of SA9H10 in DDBJ/EMBL/GenBank and SwissProt. We cloned and sequenced a longer DNA fragment (SA9H10L) of SA9H10 from the gene library. The predicted amino acid sequence of this clone was weak homology to M protein of Streptococcus pyogenes (22.7%). Five genes were specifically expressed in the KG- phenotype strains. However, SA8A11 and SA9H10 was expressed in the mutated strain SA8201-TTC, whose serological phenotype was changed from KG- to KG+ by 2,3,5-triphenyltetrazolium chloride.

Amino Acid Sequence↗
PubMed1993

Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis.

Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis. In L. lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid. Transconjugants of strain HID113 carrying this plasmid were unstable. Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome.

Conjugation, Genetic↗