The supernatant obtained was loaded onto an Ni-nitrilotriacetic acid (NTA) column (QIAGEN) and washed with 4 column volumes of 50 mM Na-HEPES, 50 mM K2SO4, 20 mM imidazole, 1 mM β mercaptoethanol (pH 7.4). These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work. Thus, further metabolism of lactate to acetate or formate takes place at a low rate, if at all. Mithilfe dieser sogenannten Homofermentation säuert L. lactis die Umgebung an und schützt sich hierdurch vor Nahrungskonkurrenten, die bei den niedrigen pH-Werten nicht überleben können.

6). The equilibrated strips were loaded on a standard 12% sodium dodecyl sulfate-polyacrylamide gel (7) and run for 4 h at 180 V. The gels were washed three times for 5 min each with distilled water, followed by staining with homemade colloidal Coomassie blue and destaining with distilled water. In any case, the interaction of CopR with the cop box provides further support for the start of the lctO gene at Met-18. Lanes 2 to 4, 16 pmol of cop box DNA, 160 pmol of CopR, and 0, 0.5, and 50 μM copper, respectively. Moreover, the highly conserved sequence VxGSGTSLDTARFR in the substrate binding site of NAD-linked lactic oxidases of lactic bacteria was not present in L. lactis LctO. Methionine oxidation was included as a possible modification in all searches, as was alkylation of cysteines, where appropriate. The protein sequences were aligned with ClustalW, and conserved amino acids are displayed in inverse type. bulgaricus was concluded to probably serve in eliminating oxygen (12). When cytoplasmic copper becomes excessive, the zinc ion of CopY is replaced by two Cu+ ions to form Cu+ The Genus Lactococcus has the characteristics of using fermentation as their metabolic pathway (Holt 528).

At the end of the growth phase, the pHs of the wild-type and the lctO knockout cultures were again the same; this effect was not influenced by 1 mM copper in the medium (not shown). lactis IL1403 is a gram-positive bacterium which belongs to the group of lactic acid bacteria, among which several genera play an essential role in the manufacture of food products. Proof of this concept will require further investigations. Meist liegen in den probiotischen Lebensmitteln und Medikamenten L. lactis-Stämme in Kombination mit anderen Bakterienarten vor. By using proteomics, we identified LctO as a protein which was markedly upregulated by copper in L. lactis IL1403. Analysis of purified LctO showed this protein to be a lactate oxidase. This protein would have a calculated pI of 6.0, compared to 6.87 for the annotated protein. The oxidation of lactate to pyruvate by LctO would provide an ideal mechanism to use molecular oxygen, as lactate, the end product of fermentation by lactic bacteria, is plentiful in the cell.

We determined cellular pyruvate levels in control cells and cells exposed to copper. The final pellets were resuspended in 5 ml of 20 mM Tris-Cl, 1 mM EDTA (pH 8) and broken by three passages through a French press at 40 MPa. Other details are as described in Materials and Methods. In Rohmilch kommt L. lactis natürlicherweise vor. Transformants were selected at 30°C on plates with brain heart infusion medium (Difco Laboratories, Detroit, MI) containing 5 μg/ml of erythromycin. One protein which was highly upregulated by copper was LctO. The gel pieces were dried in a SpeedVac vacuum drier and rehydrated at 4°C for 60 min in 15 μl of digestion solution (20 mM NH4HCO3 [pH 7.8], 2 ng/μl of sequencing-grade trypsin [Promega]). Biochimica et Biophysica Acta (BBA) - Biomembranes, https://doi.org/10.1016/S0005-2736(02)00712-5. It uses molecular oxygen to catalyze the oxidation of l-lactate to pyruvate under generation of hydrogen peroxide.

It exhibits 42% and 46% sequence identity with the lactate oxidases of Streptococcus iniae and Aerococcus viridans, respectively. Lane 1, 16 pmol of cop box DNA. 2-CopY, which is released from the DNA and allows transcription of the downstream genes to proceed (1, 2). Induction of the lctO gene could be verified by measuring lctO mRNA levels by real-time quantitative PCR (Fig. L. lactis gilt gemeinhin als sehr sicher und wirkt sich selbst bei Säuglingen und Kleinkindern positiv auf die Gesundheit aus. Da das Bakterium außerdem sehr leicht zu kultivieren ist, wird es in der modernen Lebensmittelindustrie gezielt als Starterkultur während der frühen Produktion von unterschiedlichsten Käsesorten eingesetzt (beispielsweise Roquefort, Brie, Camembert, Cheddar und Parmesan). LctO was identified with a MASCOT score of 139 and a sequence coverage of 46% (18 peptides).

External calibration was performed for each run using peptide calibrants (Bruker Daltonics, part no.

For gene knockout, L. lactis IL1403 was transformed with pTELO1 by electroporation as described previously (4). The cloned protein was composed of an N-terminal His6-thioredoxin tag and a C-terminal V5 epitope. Real-time quantitative PCR.RNA was isolated from L. lactis with the RNeasy kit from QIAGEN, according to the manufacturer's instructions. Bacterial strains and culture conditions. Übrigens: Wenn Sie solche Informationen interessieren, dann fordern Sie unbedingt meinen Praxis-Newsletter mit den “5 Wundermitteln” an: Kleine Anmerkung: Die Sache mit den “5 Wundermitteln” ist mit Abstand der beliebteste Newsletter, den meine Patienten gerne lesen…. The presence of a cop box in the promoter region of the lctO gene raised the intriguing possibility that LctO is under direct control of a CopY-type repressor. To obtain more information about the function of LctO, an lctO knockout strain was constructed by homologous, Campbell-like recombination (4). Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro.

Lanes 6 to 8, 16 pmol of control DNA, 160 pmol of CopR, and 0, 0.5, and 50 μM copper, respectively.

The pellet was air dried and dissolved in 300 μl of isoelectric focusing buffer (6). Meinen Namen, E-Mail und Website in diesem Browser speichern, bis ich wieder kommentiere. Erste Ergebnisse zeigen gute Erfolge (www.ncbi.nlm.nih.gov/pubmed/20532816).

Although copper is an essential cofactor of many enzymes, toxicity arises by excess copper accumulation in the cell (9). Escherichia coli Top10 cells (Invitrogen) used for cloning were grown in Luria-Bertani (LB) medium, with 100 μg/ml of ampicillin when required. Two additional mutations were localized in the sequence upstream of the proposed ATG start codon, affecting nucleotides −51 and −20. For the synthesis of cDNA, the instructions of the iScript cDNA synthesis kit (Bio-Rad) were followed. Hydrogen peroxide production has also been studied for L. lactis ATCC 19435 with NADH oxidase (29) and for Streptococcus pyogenes with lactate oxidase (22), but the physiological roles of these mechanisms remained enigmatic. With the mass spectrometry data, searches were performed against the NCBInr and Swiss-Prot databases using the MASCOT peptide mass fingerprint database search software (Matrix Science). Construction of the L. lactis lctO knockout strain.A vector unable to replicate in L. lactis and containing an erythromycin resistance marker, pTE1, was constructed by excising the erythromycin resistance gene with its native promoter from plasmid pVA838 (10) with AvaI and HindIII. Mass accuracy was estimated at ±0.2 Da. The CopR repressor-DNA interaction was abolished by copper, providing a molecular basis for the induction of LctO by copper. Diese Website benutzt Cookies. Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. We use cookies to help provide and enhance our service and tailor content and ads. We had observed similar divergence between the published L. lactis genome and genes sequenced from our strain (Magnani and Solioz, unpublished observation). The reaction was started by the addition of 100 μg of LctO, and O2 consumption was recorded. The protein concentration in the supernatant (cytosolic fraction) was determined with the Bio-Rad protein assay, using bovine serum albumin as a standard.

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