![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland
Received 14 April 2007/ Accepted 4 June 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although copper is an essential cofactor of many enzymes, toxicity arises by excess copper accumulation in the cell (9). The two oxidation states of copper, Cu+ and Cu2+, not only allow this metal to participate in essential redox reactions but can also lead to the formation of reactive oxygen species that cause cellular damage. Thus, copper homeostasis by controlling uptake, accumulation, detoxification, and removal of copper is critical to living organisms. Many proteins involved in copper homeostasis have already been identified in prokaryotes and eukaryotes (18, 20, 23).
The investigation of copper homeostasis in L. lactis is still in its infancy, but it appears to resemble the well-studied copper homeostatic system of Enterococcus hirae (23). Similar to E. hirae, L. lactis possesses a cop operon which encodes a putative CPx-type (24) copper export ATPase, CopA, which is under the control of the copper-responsive repressor CopR. The operon also encodes a copper chaperone, CopZ. A gene similar to copB, which is part of the E. hirae cop operon and also encodes a copper ATPase, is located in a different chromosomal region in L. lactis (D. Magnani and M. Solioz, unpublished data).
To find novel proteins involved in copper homeostasis and resistance in L. lactis, we employed a proteomics approach. By two-dimensional (2D) gel electrophoresis, proteins which responded in their expression level to copper were identified (to be published elsewhere). One protein which was highly upregulated by copper was LctO. Analysis of purified LctO showed this protein to be a lactate oxidase. It was induced by copper, cadmium, and, to a lesser extent, silver ions, as shown by real-time quantitative PCR. Induction of the enzyme in turn raised the intracellular pyruvate concentration via the oxidation of lactic acid under use of molecular oxygen. A general copper-regulatory element with the consensus sequence TACAnnTGTA, also called the cop box (16), was identified in the 5' region of the lctO gene and was shown to interact with the CopR regulator. We suggest that the upregulation of LctO by copper serves primarily to defend cells against oxidative stress by decreasing molecular oxygen.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Isolation of cytosolic fractions. From an overnight culture of L. lactis IL1403 in M17 medium, an inoculum of 1% (vol/vol) was added to 500 ml of fresh M17 medium. At an optical density at 548 nm (OD548) of 0.4 to 0.5, the culture was evenly split, and 200 µM of CuSO4 was added to one of the cultures. Growth was continued for 45 min before the cells were harvested by centrifugation at 5,000 x g for 10 min at 4°C. All further steps were conducted at 4°C. The cells were washed twice with 20 mM Tris-Cl (pH 8), 1 mM EDTA. The final pellet was resuspended in 10 ml of the same buffer. Cells were broken by three passages through a French press at 40 MPa. Cell debris was removed by centrifugation at 2,000 x g for 20 min. The supernatant was subjected to ultracentrifugation at 100,000 x g for 45 min to remove membrane components. The protein concentration in the supernatant (cytosolic fraction) was determined with the Bio-Rad protein assay, using bovine serum albumin as a standard.
2D gel electrophoresis. The cytosolic fraction containing 300 µg of protein was precipitated with methanol/chloroform as described previously (30). The pellet was air dried and dissolved in 300 µl of isoelectric focusing buffer (6). Isoelectric focusing strips (Ready Strips IPG [pH 4 to 7, 17 cm]; Bio-Rad) were passively rehydrated overnight with the sample to be analyzed. Isoelectric focusing was performed with a Bio-Rad Protean IEF cell. Following rapid voltage ramping from 0 to 250 V in 20 min, the tension was raised linearly to 4,000 V in 2 h, followed by ramping to 10,000 V in 10 h. Following electrofocusing, the strips were equilibrated for 20 min with 10 ml of NuPAGE solution (Invitrogen) containing 155 mg of dithiothreitol, followed by a 20-min incubation with 10 ml of the same solution containing 250 mg of iodoacetamide. 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. The protein content of spots was quantified by densitometry.
Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis.
Coomassie blue-stained gel spots were excised from the gels and washed with 50 mM NH4HCO3 in 50% (vol/vol) acetonitrile for 30 min. 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]). Digestion was carried out overnight at 37°C with shaking. The gel slices were then extracted once each with 30 µl of 1% formic acid and with acetonitrile for 30 min at 37°C with shaking. The combined supernatants were dried in a SpeedVac and the pellets dissolved in 10 µl of 0.1% formic acid. These samples were analyzed on an Ultraflex matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany). Samples were spotted on an AnchorChip target (Bruker Daltonics) with 1 µl of freshly prepared 
-cyano-4-hydroxycinnamic acid matrix solution (1 mg/ml). External calibration was performed for each run using peptide calibrants (Bruker Daltonics, part no. 202570). Signals from 100 to 200 laser shots were summed per analysis. Mass accuracy was estimated at ±0.2 Da. 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). The L. lactis IL1403 database (MOLOKO) was also searched using the PeptOko software (http://spock.jouy.inra.fr/). Methionine oxidation was included as a possible modification in all searches, as was alkylation of cysteines, where appropriate. One missed tryptic cleavage was considered.
Real-time quantitative PCR. RNA was isolated from L. lactis with the RNeasy kit from QIAGEN, according to the manufacturer's instructions. RNA concentrations were measured with the Gene Quant Pro instrument. RNA quality was assessed on formaldehyde-agarose gels stained with ethidium bromide. For the synthesis of cDNA, the instructions of the iScript cDNA synthesis kit (Bio-Rad) were followed. Real-time quantitative PCR was performed on a LightCycler (Roche), using SYBR Premix Ex Taq (TaKaRa BIO Inc., Otsu, Japan) according to the manufacturer's instructions. For the lctO gene, primers ob1 (5'-GAGGCTGCGGGAAATAAAGG) and ob2 (5'-TACCGCAACAACATCTGCTC) were used. 16S RNA was determined as an internal reference, using primers dm7 (5'-GTGGCTCAACCATTGTATGC) and dm8 (5'-AGCCTCAGTGTCAGTTAAG). All real-time quantitative PCR results were expressed relative to 16S RNA.
Construction of the LctO expression vector. The lctO gene of L. lactis IL1403 was cloned by PCR amplification of genomic DNA with the primers ob5 (5'-GTACATCTATCATCTACAGATGTAAAC) and ob6 (5'-GATGACGATGACAAGTTAGTCAATCAATGAGGTATGTTTGATTTC), followed by ligation of the crude PCR product into the His tag expression vector pBAD TOPO TA (Invitrogen) and transformation into E. coli Top10 cells (Invitrogen). This and all the following vectors were purified from E. coli using the large-scale Nucleobond AX plasmid isolation kit (Macherey-Nagel, Oensingen, Switzerland). The sequence of the resultant plasmid, pBO3, encoding lactate oxidase with an N-terminal His6 tag linked to a thioredoxin fragment to increase solubility and a C-terminal V5 epitope, was verified by DNA sequencing. All PCR amplifications were conducted with the proofreading DNA polymerase mixture LA Taq (TaKaRa BIO Inc., Otsu, Japan).
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. The ends of the resulting 1.8-kb DNA fragment were polished (3) with Pfu polymerase (Stratagene, La Jolla, CA) and the fragment ligated with the pCR-Blunt II Topo vector (Invitrogen), resulting in plasmid pTE1. The plasmid for lctO knockout, pTELO1, was obtained by PCR amplification of an internal lctO fragment from pBO3, using primers fm16 (5'-GGCGGAAGCTTTGGAGGCTGCGGGAAATAAAGG) and fm 17 (5'-GGCGCGACTAGTAGCAGGTCCTCCATCTAATTG). The resultant 763-bp PCR fragment was digested with SpeI and HindIII and ligated into pTE1, which was digested with the same enzymes. pTELO1 was propagated in E. coli Top10 cells in LB medium with 40 µg/ml of kanamycin or 500 µg/ml of erythromycin. Plasmid integrity was checked by restriction analysis. For gene knockout, L. lactis IL1403 was transformed with pTELO1 by electroporation as described previously (4). Transformants were selected at 30°C on plates with brain heart infusion medium (Difco Laboratories, Detroit, MI) containing 5 µg/ml of erythromycin. Clones were screened for lctO knockout mutants generated by single-crossover recombination by PCR amplification of genomic DNA, using primer fm22 (5'-ACGGTCGTCAAGACAAACCAGAAG), located upstream of the lctO gene, and primer fm23 (5'-AAGCTAAGGGCGAATTCCAGCAC), located on the integrated pTELO1 plasmid. The correctness of the junction between plasmid and genomic DNAs was verified by restriction analysis of the resultant PCR fragment.
Construction of lctO complementation vector. The lctO gene with its native promoter was amplified by PCR from genomic DNA using the primers fm31 (5'-CGGGATCCGAAGGGATGAGTGAGAAAC) and fm32 (5'-CGCGGAATTCTTCGGTTTGGGTAGATAAGG). The resulting 1,519-bp fragment was cut with EcoRI and BamHI and ligated into the E. coli/E. hirae shuttle vector pC3 (25), which had been digested with the same restriction enzymes. E. coli ED8739 was transformed by the CaCl2 method and clones selected at 37°C on LB plates containing 500 µg/ml erythromycin. The resultant construct, pCPLO, was electroporated into the L. lactis IL1403 wild-type and lctO knockout strains, and transformants were selected at 37°C on M17 plates containing 15 µg/ml chloramphenicol and 5 µg/ml of erythromycin.
EMSA. Primers ob11 (5'-TTATAAAGTTTACATCTGTAGATGATAGAT) and ob12 (5'-ATCTATCATCTACAGATGTAAACTTTATAA), corresponding to the lctO cop box, and the negative control primers ob51 (5'-ATCCTGCATTTACAATTGTAAAATATTTTA) and ob 52 (5'-TAAAATATTTTACAATTGTAAATGCAGGAT) were heated to 95°C for 10 min, slowly cooled to room temperature, and used for electrophoretic mobility shift assays (EMSA) as described previously (14a). The incubation buffer was prepared as a 2x stock solution [40 mM Tris-acetate, 5 mM Mg-acetate, 50 mM Na-acetate, 12% glycerol, 1 mM Ca(OH)2, 5 mM Na-ascorbate, 200 µg/ml bovine serum albumin (pH 8)], and aliquots were stored at –20°C. Reaction mixtures were incubated at room temperature for 30 min, followed by separation on 15% TAE (40 mM Tris-acetate, 1 mM EDTA [pH 8]) polyacrylamide gels, which were prerun at a 165 V for 90 min. Gels were stained with 10 µg/ml of ethidium bromide.
Purification of LctO. For LctO expression, 2 liters of LB medium with 100 µg/ml of ampicillin was inoculated with 20 ml of an overnight culture of E. coli Top10 cells containing the LctO expression vector pBO3. Cells were grown under aeration at 37°C to an OD600 of 0.5, cooled to room temperature, and induced by the addition of 0.1% L-arabinose. Growth was continued overnight at room temperature. Cells were then harvested by centrifugation at 5,000 x g for 10 min at 4°C. The cells were washed twice with 50 mM Na-HEPES, 50 mM K2SO4 (pH 7.4), resuspended in the same buffer, and broken by four passages through a French press at 40 MPa. Debris was removed by centrifugation for 15 min at 2,000 x g in the cold. The supernatant was centrifuged at 100,000 x g for 45 min at 4°C. 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 ß mercaptoethanol (pH 7.4). Purified LctO was eluted with 50 mM Na-HEPES, 50 mM K2SO4, 200 mM imidazole, 1 mM ß mercaptoethanol (pH 7.4). The eluted protein was dialyzed against 50 mM Na-HEPES, 50 mM K2SO4, 1 mM ß mercaptoethanol (pH 7.4) and frozen at –80°C. Protein concentrations were determined with the Bio-Rad protein assay, using bovine serum albumin as a standard.
Pyruvate assay. Samples for pyruvate analysis were prepared as described for the isolation of the cytosolic fraction. Briefly, cultures of L. lactis of 50 ml were grown in M17 to an OD545 of 0.5. Cultures were chilled in ice water for 5 min and harvested by centrifugation for 20 min at 5,000 x g. The pelleted cells were washed twice with cold M17 medium. 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. A 1/10 volume of cold 1 M perchloric acid was immediately added to the cell homogenate and the samples kept on ice for 10 min. Precipitated proteins and cell debris were removed by centrifugation for 10 min at 2,000 x g. To obtain the final extract, the pH of the supernatant was adjusted to 7.5 with 3.75 M triethanolamine. Pyruvic acid concentrations were determined spectrophotometrically in a total volume of 1 ml by adding 25 µM NADH and 27 U of L-lactate dehydrogenase (Sigma) to 980 µl of extract and measuring NADH consumption at 340 nm. L-Lactate was used as a standard.
Lactate oxidase assay. Oxygen consumption by purified lactate oxidase was measured with a Clark-type oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH) at 30°C in 3 ml of air-saturated buffer containing 100 mM KCl, 50 mM Na-MOPS (morpholinepropanesulfonic acid), 1 mM EGTA, 5 mM NaPi, 27 µM Na-L-lactate (pH 5.7). The reaction was started by the addition of 100 µg of LctO, and O2 consumption was recorded. When the reaction reached steady state, 400 U of catalase (Fluka Biochemica, Buchs, Switzerland) was added to the reaction to determine the amount of hydrogen peroxide produced in the reaction.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
Inspection of the genomic DNA region encoding LctO led us to conclude that the lctO gene was not annotated correctly. According to the gene bank entries NP_267408 and AAK05350, the lctO gene encodes a protein of 383 amino acids. However, in the sequence deposited in the database, there is no recognizable ribosome binding site upstream of the proposed start of the protein, while there is a clear L. lactis ribosome binding site, GGAG, 41 base pairs into the assigned lctO gene, with an optimal spacing of 7 nucleotides to the next possible ATG start codon. This suggests that LctO translation starts at Met-18, encoding a protein of 366 amino acids and a calculated molecular mass of 39 kDa. This protein would have a calculated pI of 6.0, compared to 6.87 for the annotated protein. The location of LctO spots on 2D gels is clearly in favor of a pI of 6 (Fig. 1), further supporting the proposed translation product. Interestingly, there is a perfect cop box upstream of the proposed GGAG ribosome binding site. cop boxes are short, inverted repeats of sequence motif TACAnnTGTA and are the consensus binding motif of CopY-type repressors of firmicutes (16, 17). CopY-type repressors are copper inducible and regulate the expression of the copper ATPases in Enterococcus hirae (26) and L. lactis (17) and probably also in related bacteria. 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 test this hypothesis, the homologous CopR protein of L. lactis was purified as previously described (17), and its interaction with an oligonucleotide containing the cop box of the lctO gene was analyzed by EMSA. Figure 3 shows that CopR indeed interacted with a 30-bp oligonucleotide containing this cop box, while it did not interact with a similar control oligonucleotide. The CopR repressor-DNA interaction was abolished by copper, providing a molecular basis for the induction of LctO by copper. It would thus appear that the lctO gene is regulated in trans by the CopR repressor of the copRZA operon, which is located distantly on the L. lactis genome. It remains unexplained at present why LctO is not induced under anaerobic conditions, and this aspect deserves further investigation. In any case, the interaction of CopR with the cop box provides further support for the start of the lctO gene at Met-18.
|
-hydroxy-acid oxidase flavoenzymes. Figure 4 shows an alignment of LctO of L. lactis as proposed here with known lactate oxidases. It exhibits 42% and 46% sequence identity with the lactate oxidases of Streptococcus iniae and Aerococcus viridans, respectively. LctO of L. lactis belongs to the class of NAD-independent lactate oxidases. This family of enzymes has received considerably less attention than NAD-dependent lactate oxidases. The cloned gene encodes the six functionally important residues which are typically conserved in this class of enzymes, namely, Y-51, Y-157, D-185, K-252, H-276, and R-279 (11). LctO of L. lactis has, to our knowledge, never been studied directly, and its assigned function rests on sequence similarity. Lactate oxidase of A. viridans is well characterized, and its structure has been solved at a resolution of 2.1 Å (8). The enzyme contains flavin mononucleotide (FMN) and is a homotetramer. It uses molecular oxygen to catalyze the oxidation of L-lactate to pyruvate under generation of hydrogen peroxide. To determine the nature and catalytic activity of LctO of L. lactis, the gene was cloned in an Escherichia coli expression vector and purified by means of a His6 affinity tag by Ni-NTA chromatography.
|
Figure 5 shows the purification of LctO to greater than 90% purity in a single affinity purification step. The cloned protein was composed of an N-terminal His6-thioredoxin tag and a C-terminal V5 epitope. The purified product from this construct had the expected molecular mass of 46 kDa and exhibited a typical FMN absorption spectrum (Fig. 6). Based on an extinction coefficient of 12,700 M–1 cm–1, the FMN concentration in the sample was 6.3 µM, while the LctO monomer concentration based on protein determination was 11 µM. This falls a bit short of the expected stoichiometry of one FMN per protein monomer and is probably due to some loss of the FMN cofactor during purification.
|
|
pyruvate + H2O2. To verify the generation of hydrogen peroxide, catalase was added to the reaction mixture, which catalyzes the conversion of hydrogen peroxide back to oxygen according to the following reaction: 2H2O2 2H2O + O2. Clearly, LctO can act as a lactate oxidase. The estimated specific activity based on the FMN content was 2 µmol/min/mg and thus was 5- to 10-fold lower than that reported for other lactate oxidases. The reduced activity of our purified enzyme may be due to the addition of N- and C-terminal tags.
|
To obtain more information about the function of LctO, an lctO knockout strain was constructed by homologous, Campbell-like recombination (4). The lctO knockout strain or the lctO knockout strain complemented with the wild-type lctO gene did not display significantly changed resistance to copper, cadmium, or silver, arguing against a direct role of LctO in heavy metal resistance (not shown). Conceivably, the beneficial effect of the induction of LctO by copper lies in the consumption of molecular oxygen, which can promote radical formation in Fenton-type reactions.
Taken together, our results suggest a different annotation for the lctO gene and show that it encodes an NAD-independent lactate oxidase. The gene appears to be under the control of copper via the unlinked CopR repressor. Copper induction of LctO led to higher cytoplasmic pyruvate levels, demonstrating increased enzyme activity in vivo. Conceivably, LctO induction by copper represents a mechanism for the scavenging of molecular oxygen. Proof of this concept will require further investigations.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The presence of a cop box with the consensus TACAnnTGTA sequence in the promoter region of lctO suggested that the gene is under the control of a CopY-type repressor. In Enterococcus hirae, CopY regulates the expression of the copYZAB operon. In conditions of low cytoplasmic copper levels, CopY is in the zinc form (Zn2+-CopY). When cytoplasmic copper becomes excessive, the zinc ion of CopY is replaced by two Cu+ ions to form Cu+2-CopY, which is released from the DNA and allows transcription of the downstream genes to proceed (1, 2). CopY-type repressors are ubiquitous in firmicutes and we have previously shown that the purified CopY-type repressors of Enterococcus hirae, Streptococcus mutans, and L. lactis all bind to the same cop box-containing promoters with similar affinity and that binding is abolished by copper (16, 17). In agreement with this general concept, purified CopR of L. lactis was found to bind to the cop box present in the promoter region of the lctO gene, and this interaction was abolished by copper. This strongly suggests that LctO is under the control of CopR, which would provide the molecular basis for the control of LctO by copper. The (fortuitous) induction of LctO by Cd2+ and Ag+ is in line with the observation that these metal ions also induce genes under the control of CopY of E. hirae (13).
The copR gene of L. lactis is part of the copRZA operon, which is involved in copper homeostasis (Magnani and Solioz, unpublished data) and is distant from lctO on the L. lactis genome. Regulation of LctO by CopR would thus be a trans regulatory mechanism. It would be predicted that disruption of the copR gene would lead to constitutive expression of LctO. Due to the small size of this gene (453 base pairs), attempts at targeted disruption by homologous recombination have not met with success.
Based on the sequence similarity of LctO of L. lactis to known lactate oxidases, it appeared likely that the function of LctO is that of an NAD-independent lactate oxidase. It possesses the typical functional residues conserved in this type of enzymes (11) (Fig. 4). 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. Spectral analysis showed that it contains an FMN prosthetic group, and determination of the reaction catalyzed by purified LctO proved its activity to be the oxidation of lactic acid to pyruvate, with use of molecular oxygen and production of hydrogen peroxide. While NAD-dependent lactate oxidases enable bacteria to use lactate as an energy source, the function of NAD-independent lactate oxidases is less clear. It has been proposed for Streptococcus iniae that this type of enzyme serves in the removal of L-lactate, which accumulates and could become toxic late in the growth phase (5). However, L. lactis is not sensitive to even 0.5% L-lactate in the growth medium (5), making this an unlikely physiological mechanism.
Could the induction of LctO serve to reduce the acidification of the growth medium? Copper added to growth medium is, to a large extent, bound to medium components. Lowering of the medium pH can induce the release of bound copper, thus raising the effective copper concentration. It has been shown with Rhizobium leguminosarum and Sinorhizobium meliloti that acidification of chemically defined growth media increased the free copper concentration, which in turn induced actP, a gene encoding a putative copper ATPase. Transposon insertion into the actP gene resulted in an acid-sensitive phenotype, which was not apparent when copper was omitted from the growth medium (19). This documents that medium acidification can cause copper stress. In a more recent study, it was shown that adaptation of Lactobacillus bulgaricus to acidic conditions lead to the induction of three CPx-type ATPases which presumably are involved in copper homeostasis (15). However, omission of copper from the chemically defined growth medium did not significantly change the acid induction of these ATPases. Based on sequence data, a CopY-type repressor controls the expression of these genes, and it was hypothesized that factors other than copper could induce genes under the control of such repressors.
We found that the medium pH of the L. lactis lctO knockout strain decreased more rapidly than that of the wild type, reaching a maximal difference of half a pH unit (pH 5.5 versus 5) in late log phase. 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). It is not clear if the level of free copper is significantly raised by low pH in the complex growth medium we used. The transient nature of the pH effect and the observation that the lctO knockout strain did not display a significant change in copper sensitivity relative to that of the wild type argue against a function of LctO in pH buffering.
We showed that the induction of LctO by copper resulted in a 2.5-fold increase of cytoplasmic pyruvate, reaching 73 µM. Thus, further metabolism of lactate to acetate or formate takes place at a low rate, if at all. Pyruvate can also nonenzymatically react with hydrogen peroxide, resulting in the generation of acetate, carbon dioxide, and water. Pyruvate accumulation to 93 µM has also been shown in L. lactis ATCC 19435, and it has been suggested that this represents a protective mechanism against hydrogen peroxide toxicity in this organism (29). However, the production of pyruvate by LctO produces equimolar amounts of hydrogen peroxide and would thus be a futile cycle for hydrogen peroxide removal.
A "paradoxical" increase in hydrogen peroxide appears to be common in lactic acid bacteria (12, 21), and many species, including L. lactis IL1403, do not possess catalase or superoxide dismutase for its removal. Hydrogen peroxide per se may in fact not be as toxic as has been surmised. For one thing, it is diffusible and can leave the cell through the cytoplasmic membrane. Toxic hydroxyl radicals can, however, be generated by hydrogen peroxide in a Fenton-type reaction with copper and other redox-active metal ions. These reactions require molecular oxygen, and it may be a priority of cells to scavenge residual oxygen, rather than to keep hydrogen peroxide levels down. 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. 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. They could in fact represent alternative means to the same goal, namely, the scavenging of molecular oxygen. Indeed, an oxygen-consuming NADH oxidase in Lactobacillus delbrueckii subsp. bulgaricus was concluded to probably serve in eliminating oxygen (12). The beneficial effect of the induction of LctO by copper in L. lactis may serve the same function but may only come to bear under "real-life" conditions, such as in fermentation processes.
Taking our findings together, we here show that the lctO gene of L. lactis IL1403 is probably not annotated correctly and that it encodes a NAD-independent lactate oxidase. LctO appears to be under the control of the unlinked copper-responsive CopR repressor, which interacts with a cop box in the lctO promoter. We suggest that induction of LctO by copper serves in the scavenging of molecular oxygen to prevent toxic Fenton chemistry. Clearly, the elucidation of the physiological significance of this novel copper stress response will require further, detailed metabolic studies. The present work showing the function and regulation of LctO sets the stage for further such investigations.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Published ahead of print on 8 June 2007.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |
