Fachgebiet Technische Biochemie, Institut für Biotechnologie der Technischen Universität Berlin, Seestraße 13, D-13353 Berlin, Germany1
Author for correspondence: Helmut Görisch. Tel: +49 30 314 27582. Fax: +49 30 314 27581. e-mail: Goerisch{at}lb.TU-Berlin.de
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ABSTRACT |
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Keywords: ethanol oxidation, malate conversion, pyruvate carboxylase, malic enzyme
Abbreviations: MDH, malate dehydrogenase; ME, malic enzyme; MQO, malate:quinone oxidoreductase; PC, pyruvate carboxylase; PEP, phosphoenolpyruvate; PEP-C, PEP carboxylase; PQQ, pyrroloquinoline quinone
The GenBank accession number for the Pseudomonas aeruginosa ATCC 17933 mqo sequence reported in this work is AY129296.
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INTRODUCTION |
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MQO is a FAD-dependent membrane-associated protein that catalyses the oxidation of malate to oxaloacetate. The electrons are donated to quinones of the electron transfer chain and NAD will not be accepted as electron acceptor (Kather et al., 2000 ). MQO is involved in the citric acid cycle/glyoxylate cycle (Fig. 1
). ORFs encoding all of the enzymes required in both metabolic pathways are found in the P. aeruginosa PAO1 genome.
MQO activity in P. aeruginosa PAO1 has already been described by Mizuno & Kageyama (1978) as a membrane-associated malate dehydrogenase (MDH) activity that uses dichlorophenolindophenol and phenazine methosulfate as electron acceptors. MQO has previously been studied in Corynebacterium glutamicum (Molenaar et al., 1998
), Helicobacter pylori (Kather et al., 2000
) and Escherichia coli (van der Rest et al., 2000
). In C. glutamicum and E. coli, both a cytoplasmic NAD-dependent MDH and a membrane-associated MQO are present. In C. glutamicum, a deletion in mqo resulted in a mutant whose growth defects could be eliminated by growth in the presence of nicotinamide; apparently, the NAD-dependent MDH was able to take over the function of MQO (Molenaar et al., 2000
). In E. coli, the deletion of mqo did not result in an observable phenotype (van der Rest et al., 2000
).
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METHODS |
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Construction of an mqo::Kmr mutant.
A pUC19 derivative, pTB4116, containing a 3·1 kb fragment from pTB4112 with the kanamycin-resistance gene of Tn5 in mqo, was used to transform P. aeruginosa ATCC 17933 by electroporation (Smith & Iglewski, 1989 ). Potential site-directed double-crossover mutants with a kanamycin-resistant phenotype were selected for loss of ampicillin resistance.
Preparation of cell-free extracts and membrane fractions.
All operations were done at 4 °C. Bacteria were grown to late-exponential phase, harvested, washed twice with ice-cold 0·1 M Tris/HCl buffer (pH 7·4) containing 10 mM MgSO4, 10% glycerol and 5 mM ß-mercaptoethanol (Saeki et al., 1999 ), and resuspended in the same buffer. After cell disruption by sonication, cell debris was removed by centrifugation for 30 min at 6000 g. Part of the supernatant was used as the cell-free extract; the other part was used to obtain the membrane fraction. The supernatant was centrifuged for 1 h at 100000 g; the resulting membrane-rich pellet was washed with ice-cold 50 mM HEPES buffer (pH 7·5) containing 10 mM potassium acetate, 10 mM CaCl2 and 5 mM MgCl2 (Molenaar et al., 1998
), before being resuspended in a small volume of the HEPES buffer. Cell-free extracts and membrane fractions were split into aliquots and either assayed immediately for enzymic activity or stored at -20 °C. The protein content of the cell-free extracts was between 2 and 5 mg protein ml-1, while the membrane fractions contained between 9 and 20 mg protein ml-1.
PQQ determination.
PQQ in the supernatants of P. aeruginosa cultures was determined according to Geiger & Görisch (1987) . Crude membranes of E. coli containing the apoprotein of quinoprotein glucose dehydrogenase were used to reconstitute holoenzyme activity.
Enzyme assays.
All assays were carried out at 25 °C in a total volume of 1 ml. The MQO activity was assayed with the membrane fraction. The activity was determined by measuring the decrease in absorbance of 2,6-dichlorophenol indophenol (DCPIP) at 600 nm, assuming an absorption coefficient of 22 cm-1 mM-1 (Armstrong, 1964 ). The test of OBrien & Taylor (1977)
was modified and contained, in a total volume of 1 ml, 50 mM Tris/HCl, 0·05 mM DCPIP, 0·1 mM phenazine methosulfate, 5 mM ethylamine, 5 mM KCN, 10 µM FAD and 1 mM L-malate. All other enzyme activities were determined in cell-free extracts. NAD-dependent MDH activity was determined using oxaloacetate as the substrate in a mixture containing 0·1 M Tris/HCl buffer (pH 7·5, with 0·06% BSA), 15 mM KCN, 0·2 mM NADH and 1 mM oxaloacetate. Catalytic activity was calculated from the decrease in absorbance of NADH at 340 nm, using an absorption coefficient of 6·22 cm-1 mM-1 (Horecker & Kornberg, 1948
). Pyruvate carboxylase (PC) and PEP carboxylase (PEP-C) activities were measured according to Saeki et al. (1999)
by the decrease in absorbance of NADH at 340 nm in a coupled test with MDH. The reaction was started by the addition of pyruvate or phosphoenolpyruvate (PEP). The activity of malic enzyme (ME) was determined by the increase in absorbance due to the reduction of NAD. The method of Bergmeyer & Graßl (1987)
was used with some modifications. The reaction mixture contained 0·1 M Tris/HCl buffer (pH 7·5), 2 mM NAD (or NADP), 5 mM L-malate and 4 mM MnCl2. The reaction was started by the addition of MnCl2. Specific activities of all of the enzymes assayed were expressed as µmol product formed min-1 (mg protein)-1. Using the same sample, standard deviations of the enzyme assays were calculated as ±5·6% for MQO, ±4·2% for PC, ±2·6% for ME (NAD) and ±15·5% for ME (NADP). Protein concentrations were determined using the method of Groves et al. (1968)
.
Internet tools.
BLAST was used for DNA or protein database searches (Altschul et al., 1997 ), the advanced BLAST2 server service of the NCBI (http://www.ncbi.nlm.nih.gov/) was used for comparing the sequence of mqo from P. aeruginosa ATCC 17933 to mqoB from P. aeruginosa PAO1, and the Pseudomonas aeruginosa Genome Database (http://pseudomonas.bit.uq.edu.au/; version from 21 November 2001) was used to obtain the DNA sequence of the PAO1 mqoB gene.
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RESULTS AND DISCUSSION |
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A clone carrying only the intact ORF1 from strain ATCC 17933 was obtained by PCR. In pTB4111, ORF1 was oriented linear with the lac promoter of pUCP20T, and complementation of mutant MS7 with this clone restored growth of this organism on ethanol. To confirm that complementation occurred in trans and was not a result of homologous recombination, plasmid DNA was prepared from complemented mutant strains and used to transform E. coli JM109. The resulting transformants were used again in triparental matings and successfully complemented the original mutant, MS7.
pTB4110 was used for sequencing both strands of the putative mqo gene of strain ATCC 17933. The gene was 1524 bp long, and its deduced amino acid sequence showed high similarity (67%) to the MQOs from C. glutamicum (Molenaar et al., 1998 ), E. coli (van der Rest et al., 2000
) and Neisseria meningitidis (Leighton et al., 2001
). A putative FAD-binding site, DMLLVGAG, near the N-terminal region of the protein was also found. This sequence was almost identical to the conserved motif DhhhhG(A/G)G found in C. glutamicum and E. coli MQOs (Molenaar et al., 1998
), where h stands for the amino acid I, V or L. In addition, the N terminus of the putative protein contained the motif GXGXXG (GTGHAG) of the ADP-binding ß
ß fold, which was found in the MQO of H. pylori (Kather et al., 2000
) and in 16 other flavin-binding enzymes (Austin & Larson, 1991
) but not in the MQO of C. glutamicum (Molenaar et al., 1998
).
A putative ShineDalgarno sequence (Rothmel et al., 1991 ), GGAGA, was found 7 bp upstream of the mqo start codon ATG. A putative rho-independent transcription terminator was found 11 bp downstream of the translation stop codon TGA. The inverted repeat had a 12 bp stem. The gene corresponding to ORF1 from strain ATCC 17933 in the P. aeruginosa PAO1 genome database (PA4640) is designated mqoB. A comparison of the putative mqoB gene with the ORF1 sequence from P. aeruginosa ATCC 17933 revealed there to be only five base pairs different between the two sequences (0·3% divergence). These changes did not cause differences between the deduced amino acid sequences of the two Mqo proteins.
Inactivation of mqo
The product of mqo is essential for growth of P. aeruginosa on ethanol or acetate, as demonstrated by site-directed mutagenesis using pTB4116. A kanamycin-resistant insertion mutant (UK2) was isolated, as described in Methods. Mutant UK2, like mutant MS7, showed a severe growth defect on ethanol and was unable to grow on 2,3-butanediol or acetate. PCR with the genomic DNA of mutant UK2 confirmed the presence of the kanamycin-resistance cassette in mqo (mqo::Kmr) (data not shown). The kanamycin-resistance gene was transcribed in the same orientation as mqo. The mqo::Kmr allele in mutant UK2 did not express a toxic variant of MQO, since complementation with pTB4111, carrying only mqo, restored wild-type growth on ethanol, acetate and succinate (Table 2).
Characteristics of mutant UK2
Mutant UK2 (mqo::Kmr) did not grow on ethanol within 10 h incubation, whereas wild-type cells and UK2 cells complemented with pTB4111 reached stationary phase after 89 h incubation (Fig. 2). However, after 15 h incubation mutant UK2 started to grow very slowly. After 33 h incubation, mutant UK2 reached an optical density close to that observed for the wild-type after 9 h incubation. The observed growth was not caused by spontaneous revertants, because when this UK2 culture was used as the inoculum for a subsequent cultivation the long lag phase and very slow growth were observed again.
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Growth of mutant UK2 on glucose, lactate and pyruvate was almost comparable to that of the wild-type. Also, succinate, fumarate and malate supported growth of mutant UK2, but its lag phase and generation time were increased with respect to the wild-type (Table 2; Fig. 2
).
Activities of malate-converting and oxaloacetate-forming enzymes
Wild-type P. aeruginosa, as well as mutant UK2, was grown on different carbon sources. Cells were collected by centrifugation at the late-exponential phase and disrupted by sonication. The soluble cytoplasmic and periplasmic fractions and the supernatant after removal of membrane fragments by ultracentrifugation at 100000 g, as well as the membrane fraction, were tested for their MQO, MDH, ME, PC and PEP-C activities. The MQO activity was associated with the membrane fraction, indicating that it is a membrane-bound or membrane-associated enzyme. ME, PC and PEP-C appeared to be soluble enzymes. The activities of the different enzymes are shown in Table 3.
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In contrast to P. aeruginosa, MQO activity in C. glutamicum and E. coli is regulated in response to the carbon source used, with the activity of the enzyme being about fourfold higher on acetate than on glucose. MQO activity in C. glutamicum varied between 110 mU (mg protein)-1 on glucose and 370 mU (mg protein)-1 on acetate (Molenaar et al., 2000 ), while in E. coli on glucose 12 mU (mg protein)-1 were found compared to 47 mU (mg protein)-1 on acetate (van der Rest et al., 2000
).
A probable L-MDH (EC 1.1.1.37) gene is recorded in the P. aeruginosa PAO1 genome database (gene PA1252). However, neither wild-type nor mutant UK2 cells of P. aeruginosa ATCC 17933 showed any NAD-dependent L-MDH activity under any of the conditions tested. Also, no L-MDH activity was found with NADP. Commercial L-MDH (EC 1.1.1.37) and cell-free extracts from Rhodococcus strain DTB (Hauck et al., 2001 ) were used as a positive control for enzyme activity. Hopper et al. (1970)
have already noted the absence of any NAD-or NADP-dependent L-MDH activity in a strain of Pseudomonas; in addition, these authors also found a dye-dependent, membrane-bound L-MDH activity in the same strain.
It appears that P. aeruginosa 17933 contains two MEs one that lacks an absolute coenzyme specificity but is more active with NAD than with NADP (EC 1.1.1.39; MDH, decarboxylating), and one that is highly specific for NADP (EC 1.1.1.40; MDH, oxaloacetate-decarboxylating). This is in agreement with the P. aeruginosa PAO1 genome database, where a gene encoding ME (PA5046) and a gene encoding a probable ME (PA3471) are recorded. We found an inhibition of the NAD-dependent ME by acetyl-CoA. With 0·2 mM acetyl-CoA, the initial activity was reduced to 70%, and to 40 and 20%, respectively, with 2 and 10 mM acetyl-CoA. In contrast, PC was not inhibited by 0·210 mM acetate nor by 0·52·5 mM acetyl-CoA. Also, the NADP-dependent ME activity was not inhibited by acetyl-CoA up to a concentration of 4 mM, and acetate did not show any regulatory effect.
The reaction catalysed by the NADP-dependent enzyme was reversible, since a reductive carboxylation of pyruvate could be detected (data not shown). NAD- and NADP-dependent ME activities did not vary significantly with different carbon sources. Mutant UK2 showed a small increase in NAD- and NADP-dependent ME activities.
PC activity (EC 6.4.1.1) in wild-type cells was strongly influenced by the carbon source used for growth. On glucose and lactate, the specific activity of this enzyme was induced three- to fourfold, compared to growth on ethanol, acetate or succinate. In mutant UK2, the specific activity of PC increased four- to eightfold on all carbon sources tested, except for on glucose and lactate, where it increased about 1·5-fold when compared to the wild-type.
A substrate-inducible PC, with highest activity observed in cells grown on glucose or lactate, has already been described for Pseudomonas citronellolis (OBrien et al., 1977 ). Roehl et al. (1983)
also reported a PC, and the gene encoding this protein, in P. aeruginosa PAO1.
PC is an inducible, anaplerotic enzyme that is essential for the utilization of all carbohydrates. In C. glutamicum, besides PC, PEP-C is also needed for growth on lactate or glucose to replenish the TCA cycle with precursors for biosynthesis. In this bacterium, the formation of PC is regulated by the carbon source used and the activity of the enzyme is inhibited by 0·1 mM acetyl-CoA (Peters-Wendisch et al., 1997 ; Wendisch et al., 2000
).
In P. aeruginosa ATCC 17933, only low constitutive PEP-C (EC 4.1.1.31) activity could be detected on all of the carbon sources tested. The enzyme appeared to be uninducible (Table 3).
Proposed alternative pathway for malate oxidation
Oxaloacetate is an important precursor for gluconeogenesis and the synthesis of amino acids. It is also the substrate of citrate synthase in the citric acid cycle. Since in the wild-type and the mutant UK2 no NAD-dependent MDH activity could be detected, MQO must be the enzyme responsible for malate oxidation. However, the mutant lacking the mqo gene product was able to grow on glucose and lactate at levels similar to those of the wild-type; it was also able to grow on succinate, fumarate and malate, albeit with a prolonged lag phase and a reduced growth rate. We assume that in mutant UK2 an alternative pathway formed by the collective actions of ME, generating pyruvate, and PC, generating oxaloacetate, takes over the function of MQO (Fig. 1). Indeed, PC activity was induced about fivefold in the mutant (Table 3
).
Also, for B. subtilis, which possesses an MDH and lacks MQO, a pyruvate shunt has been described (Diesterhaft & Freese, 1973 ). ME and PC form a bypass to the citric acid cycle during growth of this organism on malate, to allow the production of sufficient oxaloacetate for gluconeogenesis and biosynthesis. In contrast to the situation in P. aeruginosa, the activity of PC was not dependent on the growth medium and the enzyme was strongly activated by acetyl-CoA (Diesterhaft & Freese, 1973
). In E. coli, which contains MQO and MDH, MQO partly takes over the function of MDH in an mdh mutant. However, an mqo mutant did not show a specific phenotypic defect (van der Rest et al., 2000
).
The effects of growth substrates on the specific activities of MEs and the response of enzyme activity to effector molecules reported in the literature differ between different organisms. For instance, in Rhizobium meliloti, which contains two MEs, the NAD(P)-dependent ME showed allosteric regulation by acetyl-CoA. The activity of this enzyme was reduced to 2% by 0·1 mM acetyl-CoA. A strictly NADP-specific ME was not subjected to regulatory mechanisms (Voegele et al., 1999 ).
In our mqo mutant, UK2, we assumed that a bypass for the oxidation of malate to oxaloacetate was operating through the activities of a NAD-dependent ME and a NADP-dependent ME, together with a higher expression of PC activity. With glucose, lactate or pyruvate as the carbon source, growth of the mutant UK2 was only slightly impaired with respect to the wild-type. Pyruvate is available in abundance and with the increased activity of PC enough oxaloacetate can be synthesized for biosynthetic purposes, and for gluconeogenesis in the case of either lactate or pyruvate as the carbon source. Under these growth conditions, the activities of the MEs are only necessary to close the citric acid cycle for generating energy.
When mutant UK2 was grown on succinate or malate, the complete bypass of the citric acid cycle with ME and PC activities was needed to obtain energy, as well as to provide oxaloacetate for gluconeogenesis and amino acid biosynthesis. Under these conditions, the bypass seemed to operate at its limits, since growth of mutant UK2 was strongly delayed and the growth rate was reduced compared to that of the wild-type.
Growth of wild-type cells on ethanol or acetate is a strenuous exercise, because more ATP is required for the synthesis of cell material than when the same cells are grown on succinate or glucose. Under these conditions, even for wild-type cells the lag phase is increased and the growth rate is reduced compared to that on the two other carbon sources (Fig. 2). Mutant UK2 does not grow at all on ethanol or acetate within 10 h incubation. On these carbon sources, the bypass of the citric acid cycle formed by ME and PC activities would again be needed to allow operation of the citric acid cycle for the generation of energy; this bypass would also be required to supply intermediates for both gluconeogenesis and biosynthetic purposes. Apparently, the capacity of the bypass is not sufficient to support growth on ethanol or acetate, resulting in energetic limitations or a limitation in a biosynthetic precursor. In addition, growth on ethanol or acetate requires all of the carbon used in metabolism to be provided as acetyl-CoA. We assume that under these conditions the acetyl-CoA concentration would be raised to levels higher than those of mutant UK2 grown on malate or glucose; hence, the activity of the NAD-dependent ME would be inhibited, lowering the capacity of the bypass even further.
However, to prove the operation of the proposed pathway, the generation of mutants with defects in ME, PC and PEP carboxykinase, PEP-C or PEP synthase and in combination with a defective MQO will be necessary, together with enzymic studies with respect to the induction and regulatory properties of the various enzymes. These studies are under way in our laboratory.
Concluding remarks
The reported results demonstrate that the putative mqo gene of P. aeruginosa ATCC 17933 encodes an MQO activity. This gene corresponds to mqoB (PA4640) of the P. aeruginosa PAO1 genome database. The enzyme, which catalyses the conversion of malate to oxaloacetate in the citric acid cycle, has so far not been studied in P. aeruginosa. A sufficient level of MQO activity is essential for growth of P. aeruginosa on ethanol or acetate. However, the mqo mutant UK2 can grow on glucose, lactate and pyruvate, and also on fumarate, succinate and malate, apparently by the collective actions of ME and PC, which bypass the MQO-catalysed conversion of malate to oxaloacetate.
The residual low MQO activity found in the membrane fraction of mutant UK2, in which mqo was interrupted by a kanamycin-resistance cassette, may have been caused by a second putative mqo gene, mqoA (PA3452), found in the genome of P. aeruginosa PAO1.
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ACKNOWLEDGEMENTS |
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Received 5 July 2002;
revised 12 August 2002;
accepted 11 September 2002.