Download ribosomal defects in a mutant deficient in the yajl homolog of the

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Fermentation and Alternative Respiration Compensate for NADH
Dehydrogenase Deficiency in a Prokaryotic Model of DJ-1 Associated
Parkinsonism.
Nadia Messaoudi1, Valérie Gautier1, Julien Dairou3, Mouhad Mihoub1, Gaëlle Lelandais1, Philippe
Bouloc2, Ahmed Landoulsi4 and Gilbert Richarme1*
1Stress
molecules, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75005 Paris, France.
de Signalisation et Réseaux de Régulations bactériens, UMR8621, Institut de Génétique et
Microbiologie, Orsay, France.
3Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative (BFA) UMR
8251 CNRS, Bioprofiler facility, F-75205, Paris, France.
4Laboratoire de Biochimie et Biologie Moléculaire 03/UR/0902, Faculté des Sciences de Bizerte, Zarzouna
7021, Bizerte, Tunisia.
2Laboratoire
Tel: 33 01 57 27 80 57; Fax: 33 01 44 27 57 16; Email: [email protected]
*Corresponding author
The two first authors contributed equally to the work.
Running title : Respirofermentative metabolism in yajL mutant.
Keywords : YajL / NADH dehydrogenase 1/ alternative dehydrogenases/ pyruvate oxidase/
NADH/NAD ratio.
Abbreviations: MPTP: 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine, RMSD: root mean square
deviation.
SUMMARY
YajL is the closest prokaryotic homolog of Parkinsonism-associated DJ-1, a protein of
undefined function involved in the oxidative stress response. We reported recently that YajL
and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as
covalent chaperones for the thiol proteome, including the NuoG subunit of NADH
dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant.
We report here that this mutant compensates for low NADH dehydrogenase activity by
utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB
and D-aminoacid dehydrogenase DadA, and mixed acid aerobic fermentations characterized
by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate
energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations
over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation
of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory
pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino
acids over Krebs cycle-related ones, and thus the yajL mutant utilizes pyruvate-centred
respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect, and
constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies,
especially those associated with Alzheimer and Parkinson’s diseases.
INTRODUCTION
YajL belongs to the PfpI/Hsp31/DJ-1 superfamily which includes chaperones (Quigley et al., 2003 ;
Sastry et al., 2002), peptidases (Malki et al., 2005) and the Parkinson’s disease-associated protein
DJ-1 (Canet-Aviles et al., 2004 ; Cookson, 2005). The crystal structures of YajL and DJ-1 are
strikingly similar (Wilson et al., 2003 ; Wilson et al., 2005), and their backbone structures are
essentially identical (0.9 A C RMSD), suggesting that they have similar functions.
DJ-1 is involved in the cellular response to oxidative stress, and has been suggested to function as a
weak protease (Lee et al., 2003), an oxidative stress activated chaperone (Le et al., 2012 ;
Shendelman et al., 2004 ; Zhou et al., 2006), an atypical peroxiredoxin-like peroxidase (AndresMateos et al., 2007 ; Canet-Aviles et al., 2004), a stabilizer of the antioxidant transcriptional
regulator Nrf2 (Clements et al., 2006), an apoptosis inhibitor via interaction with Daxx (Junn et al.,
2005), a transcriptional or translational regulator of gene expression (Cookson, 2005 ; van der Brug
et al., 2008) and a regulator of uncoupling protein expression affecting the production of reactive
oxygen species (Guzman et al., 2010).
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Like DJ-1, YajL protects cells against oxidative stress and oxidative stress-induced protein
aggregation, possibly through its chaperone function and control of gene expression (Kthiri et al.,
2010a ; Kthiri et al., 2010b). We reported recently that YajL and DJ-1 function as covalent
chaperones that, upon oxidative stress, form mixed disulfides with proteins containing essential
cysteines for catalysis, such as glyceraldehyde-3-phosphate dehydrogenase and peroxidases, or FeS
cluster binding, such as aconitases and the NuoG subunit of NADH dehydrogenase 1 (Le et al.,
2012). Moreover, YajL and DJ-1 preferentially form mixed disulfides with sulfenylated proteins and
protect cells against protein sulfenylation (Gautier et al., 2012). The specific interaction of YajL
with the NuoG subunit of NADH dehydrogenase is likely responsible for the NADH dehydrogenase
1 defect of the yajL mutant, which can be rescued by YajL and DJ-1 overproducing plasmids (Le et
al., 2012). These results suggest that DJ-1 may be involved in biogenesis and stress protection of
mitochondrial complex I (Danielson & Andersen, 2008). Respiratory chain abnormalities in cells of
patients with Parkinson’s disease have been reported (Bindoff et al., 1989), and complex I inhibitors
such as rotenone and MPTP produce a Parkinson-like syndrome in both human and primate,
identifying complex I as a major target in Parkinson’s disease. Moreover, the genetic causes of
Parkinson’s disease highlight the importance of mitochondrial dysfunction: parkin, PINK1, DJ-1,
LRRK2 and HTRA2 localize to the mitochondria (Schapira & Gegg, 2011) and their deficiency
induces mitochondrial defects that contribute to oxidative stress-induced cell death (Irrcher et al.,
2010).
The importance of complex I deficiencies in the etiology of Parkinson’s disease, the specific
involvement of YajL and DJ-1 in its biogenesis, and the scarcity of results describing the metabolic
characteristics of DJ-1-deficient cells led us to investigate the metabolism of the yajL mutant to
understand how cells compensate for NADH dehydrogenase 1 deficiency.
METHODS
Bacterial strains and plasmids. The yajL-deficient strain was constructed by Dr. H. Mori (Nara
Institute of Sciences and technology, Japan). It contains yajL disrupted by λRed in the E. coli strain
BW25113 (lacIq rrnB3 ∆lacZ4787 hsdR514 ∆(araBAD)567 ∆(rhaBAD)568 rph-1), leading to strain
JW5067 (Baba et al., 2006). We transduced the yajL mutation into strain MG1655 as described in
(Messaoudi et al., 2013). For complementation experiments, the yajL mutant was transformed by
plasmid pCA24N-yajL as described in (Le et al., 2012). Cells were grown aerobically in Luria-Bertani
medium at 37°C.
NAD, NADH, NADP, NADPH, ATP, ADP and AMP pools. Pools of metabolites were measured as
described in (Richarme, 1987), and calculations were done by assuming 130 µg of cell protein/ml
bacteria at an A600 of 1, and 5 µl of cell volume / mg of protein. The results are the mean values of three
experiments (S.E. was less than 15%).
NADH dehydrogenase 1 and 2 activities. NADH dehydrogenases 1 and 2 activities in membrane
extracts (prepared from exponential phase bacteria harvested at OD 600= 0.4) were assayed by measuring
the oxidation of 2,6 dichlorophenolindophenol at 600 nm, with NADH (NADH dehydrogenase 1 +
NADH dehydrogenase 2) or deamino-NADH (NADH dehydrogenase 1) as substrates (Cahloum &
Gennis, 1993). The assay mixture contained the following in a final volume of 1 ml: 0.1 M Tris, pH
7.5, 100 µM NADH or deamino-NADH, 30 mM KCN and 10 µg membranes prepared by the French
Press procedure. An activity of 100 represents 0.18 µmol of NADH/min/mg protein The results are the
mean values of three experiments.
NADH, succinate, pyruvate, D-alanine, -D-glycerolphosphate and formate oxidase activities.
Oxidases present in membrane extracts were estimated by measuring the rate of oxygen uptake using a
Clarke-type oxygen electrode. The reaction mixtures contained membranes (3 mg protein) and
substrates (0.6 mM NADH, 20 mM succinate, 4 mM lactate, 20 mM a-glycerophosphate) in 100 mM
Tris pH 7.5, 20 mM magnesium acetate and 0.25 M sucrose (Wallace & Young, 1977). Oxygen uptake
due to the presence of endogenous substrates was allowed to reach a steady state level before substrates
were added, except for the estimations for succinate oxidase in which case succinate was added to the
reaction mixture before the membranes (Wallace & Young, 1977). The concentration of oxygen
dissolved in the reaction buffer at 30°C was taken to be 225 nmol per ml. The results are the mean
values of three experiments.
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Analysis of culture supernatants. Bacteria were grown in LB medium (100 ml bacteria in a 1 l
Fernbach) to exponential phase (OD600= 0.8)(both strains displayed exponential growth with identical
doubling time until OD600= 1.6), and culture supernatants were analyzed enzymatically for the
determination of acetate, lactate, ethanol, succinate and formate. These determinations were made by
using the corresponding Megazyme assay kits for acetic acid, lactic acid, ethanol, acetic acid and formic
acid, following instructions of the manufacturer (Megazyme International Ireland Limited). The results
are the mean values of three experiments.
DNA microarray measurements. The yajL mutant and its parental strain MG1655 were grown under
aeration to exponential phase (OD600= 0.3) in LB rich medium (Kthiri et al., 2010). Total RNAs were
extracted and treated twice with Dnase I (Brumaghim et al., 2003; Collier et al., 2004). RNA quality
was monitored with a 2100 Bioanalyzer (Agilent, Santa Claran CA, USA). Transcriptome experiments
were performed using E. coli Affimetrix DNA chips (Santa Clara, CA, USA) by Cogenics (Newton,
MA, USA) according to the standard manufacturer’s instructions. Hybrized arrays were stained using
Affimetrix protocol. Samples were duplicated biologically, and we calculated the average of gene
expression ratios from both experiments; genes were considered to be clearly induced if the absolute
value of the expression ratio was higher than 2, and genes displaying a too low signal intensity were
discarded from the analysis.
Microarray analysis and data processing. After image quantification and global signal correction
(following the manufacturer’s instructions), the ratios between intensity values measured in yajL mutant
and its parent MG1655 were calculated and normalized using the standard Lowess procedure
(Messaoudi et al., 2013). This statistical correction has the advantage to remove intensity-dependent
effects in the observed log2(ratio) values and is thus well-adapted to correct systematic bias related to
low-intensity measures. As an additional filter, probes with low intensity signals in the yajL mutant
(<250) compared to background distribution were excluded from subsequent analyses. Reproducibility
between results obtained with different microarray replicates was verified and used to eliminate genes
with artifactual signals. Finally, to assess whether any of the cellular functions associated to the genes
identified as up-regulated in the yajL mutant were observed at a frequency greater than that expected by
chance, p-values were calculated as described (Messaoudi et al., 2013) (hypergeometric distribution).
Note that this approach was previously successfully applied (Messaoudi et al., 2013). The microarray
data were deposited at Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/),
Accession No GSE42702.
Materials. All chemicals were reagent grade and were obtained from Sigma.
RESULTS
Increased NADH/NAD ratio. NADH is produced primarily by catabolic reactions, including
glycolysis and the Krebs cycle, and is reoxidized by the respiratory chains and/or fermentations,
whereas NADPH, which is produced primarily by the pentose phosphate pathway and isocitrate
dehydrogenase, drives anabolic reactions (Brumaghim et al., 2003 ; Holm et al.,2010 ; Lin et al.,
2004) and acts as a major reductant during oxidative stress (Holm et al., 2010). We measured the
nucleotide pools of aerobic bacteria grown in LB medium during the exponential phase and found
that the NADH/NAD ratio of the parental strain (NADH/NAD ratio=0.22) was similar to that
reported by others (Auriol et al., 2011)), whereas that of the yajL mutant (NADH/NAD ratio=0.72)
was similar to ratios observed in NADH dehydrogenase I mutants (Pruss et al., 1994) and anaerobic
bacteria (de Graef et al., 1999) (Figure 1A). We also measured the NADH/NAD ratio of the yajL
mutant transformed with the YajL-overproducing plasmid pCA24N-yajL, and found that it displayed
a ratio of 0.28, close to that of the wild-type strain (not shown). The high NADH/NAD ratio of the
yajL mutant likely results from its NADH dehydrogenase defect (see below) and is probably
responsible for respiro-fermentative metabolism described below.
Increased NADPH/NADP ratio The yajL mutant displayed a higher NADPH/NADP ratio
(NADPH/NADP ratio=2.0) than the parental strain (NADPH/NADP ratio=1.4) and other wild-type
E. coli strains grown under similar conditions (Andersen & von Meyenburg, 1977) (Figure 1B).
This high NADPH/NADP ratio may be important for oxidative stress resistance.
Decreased adenylate energy charge. Intracellular ATP, ADP and AMP concentrations were 1.2 ±
0.16, 0.92 ± 0.12 and 0.42 ± 0.06 mM, respectively, in the yajL mutant, versus 2.2 ± 0.28 mM, 0.75
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± 0.09 mM and 0.21 ± 0.04 mM, respectively, in the parental strain (Figure 1C). Because the
mutant displayed higher ADP/ATP and AMP/ATP ratios than the parental strain, its adenylate
energy charge (0.58) was lower than that of the parental strain (0.78). This low adenylate energy
charge likely favours glycolytic flux (Koebmann et al., 2002), since adenyl nucleotides are allosteric
regulators (with AMP and ADP as activators and ATP an inhibitor) of phosphofructokinase and
pyruvate kinase.
NADH dehydrogenase defect. E. coli possesses two NADH dehydrogenases, namely, NADH
dehydrogenase 1, composed of the nuoA-N gene products (a homolog of mitochondrial respiratory
complex I) and NADH dehydrogenase 2, the ndh gene product. We measured the oxidation of
dichlorophenolindophenol with NADH (NADH dehydrogenase 1 + NADH dehydrogenase 2) or
deamino-NADH (NADH dehydrogenase 1). The wild-type strain and the yajL mutant oxidized
NADH at relative rates of 100 and 29, respectively, and deamino-NADH at relative rates of 41 and
5.3, respectively (Figure 1D); thus NADH dehydrogenase I was almost inactive in the mutant (13%
of the parental strain activity, as reported previously (Le et al., 2012) (we reported previously that
the YajL-overproducing plasmid efficiently rescued (up to 86%) the NADH dehydrogenase defect
of the yajL mutant (Le et al., 2012)), whereas NADH dehydrogenase 2 was 41% active. The weak
global NADH dehydrogenase activity of the yajL mutant may be responsible for the high
NADH/NAD ratio, fermentative metabolism and the induction of alternative respiratory pathways
(see below).
Overexpression of alternative respiratory dehydrogenases. The major respiratory
dehydrogenases of E. coli are NADH dehydrogenase 1, NADH dehydrogenase 2 and succinate
dehydrogenase. Other aerobic dehydrogenases include pyruvate oxidase PoxB, D-amino acid
dehydrogenase DadA, glycerol-3-phosphate dehydrogenase GlpD, formate dehydrogenase FdoGHI,
acyl-CoA dehydrogenase FadE, and proline oxidase PutA, all of which transfer electrons from
substrates to quinones without using NAD/NADH (Figure 2).
The expression levels of mRNAs coding for respiratory dehydrogenases were determined by DNA
array experiments (Figure 3A). Mutant/parental strain gene expression ratios were between 0.4 and
0.8 for nuoA-N, and were 0.75 for ndh and 1.3 for sdhABCD. By contrast, the yajL mutant
overexpressed several aerobic alternative dehydrogenases, including pyruvate oxidase PoxB (9fold), D-alanine dehydrogenase DadA (counterpart of eukaryotic D-aminoacid dehydrogenases; 4fold), glycerophosphate dehydrogenase GlpD (prokaryotic counterpart of eukaryotic
glycerophosphate dehydrogenase GPD2; 12-fold), formate dehydrogenase FdoGHI (3-fold)), acylCoA dehydrogenase FadE (counterpart of eukaryotic acyl-CoA dehydrogenases; 2-fold) and proline
dehydrogenase PutA (the counterpart of eukaryotic proline oxidase; 2-fold).
Using RT-PCR, we measured the expression of pyruvate oxidase and alanine dehydrogenase (which
play a major role in respiration in the yajL mutant, as described below) and glycerol-phosphate
dehydrogenase and found that expression of pyruvate oxidase and D-alanine dehydrogenase was
increased by 3-fold in the mutant, whereas glycerol-phosphate dehydrogenase was not significantly
overexpressed (not shown). As expected for aerobically grown cells, anaerobic dehydrogenases,
such as glycerophosphate dehydrogenase GlpABC, formate dehydrogenase FdnGHI, and
hydrogenases A and B, were expresssed at low levels in both the yajL mutant and the parental strain
(not shown); thus, whereas NADH dehydrogenases and succinate dehydrogenase constitute the
major dehydrogenases active in wild-type cells, alternative dehydrogenases are overexpressed in the
yajL mutant to compensate for NADH dehydrogenase deficiency.
Moderate changes in the expression of quinol oxidases. The major E. coli quinol oxidases
comprise CyoABCD, which transfers electrons from quinol to oxygen, and thus performs the
functions of mitochondrial complexes 3 and 4, and CydAB, which has a higher affinity for oxygen,
and is usually expressed in microaerobiosis (Unden, 1997)).
The genes cyoABCD were expressed at similar levels in both strains, whereas expression of cydAB
was increased by 1.9-fold in the yajL mutant, up to a level approaching that of cyoABCD (Figure
3B) (the expression of genes cbdABX coding for the second bd-type oxidase was negligible in both
strains (not shown)). As expected for aerobic cells, genes coding for anaerobic nitrate, nitrite,
DMSO, TMNAO reductases and hydrogenase C were not significantly expressed in either of the
two strains (Figure 3B, not shown). However, expression of frdABCD, the genes coding for
fumarate reductase, which transfers electrons from quinols to fumarate and is usually repressed in
aerobiosis was increased by 1.8-fold in the mutant, suggesting that oxaloacetate-succinate
interconversion occurs in the oxaloacetate-succinate direction (in accordance with its high
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NADH/NAD ratio and succinate excretion, as described below). Therefore, in contrast to its
impressive overexpression of multiple alternative dehydrogenases, the yajL mutant expressed quinol
oxidase CyoABCD at normal levels, and displayed only a 2-fold overexpression of microaerobic
quinol oxidase CydAB (Noda et al., 2006) and fumarate reductase.
Increased activities of alternative respiratory chains. To confirm the increased expression of
alternative dehydrogenases in the yajL mutant, we investigated its dehydrogenase activities. We
prepared membranes from the mutant and parental strains by the French Press procedure, incubated
them in an oxygraph cell in the presence of respiratory substrates, and measured oxygen
consumptions as a function of time (Figure 4) (Wallace & Young, 1977). The mutant/wild-type
ratios of NADH and succinate oxidase activities were 0.24 and 0.8, respectively; thus, the yajL
mutant displayed low NADH oxidase activity (in accordance with its low NADH dehydrogenase
activity), and a succinate oxidase activity slightly lower than that of the parental strain.
Pyruvate oxidase and D-alanine oxidase activities were 3-fold higher in the yajL mutant than in the
parental strain, suggesting that they compensate for the NADH dehydrogenase deficiency.
Replacement of pyruvate dehydrogenase by pyruvate oxidase constitutes a major metabolic shift
known as overflow metabolism (see below), in which pyruvate is directly converted into acetate in a
reaction that is not limited by high NADH levels, bypassing pyruvate dehydrogenase, the Krebs
cycle and NADH dehydrogenases (pyruvate oxidase transfers electrons directly from pyruvate to
quinones, and acetate is excreted). Glycerol-phosphate dehydrogenase activities were low in both
the yajL mutant and the parental strain.
Degradation of D-alanine by D-alanine oxidase produces pyruvate, the pyruvate oxidase substrate,
so alanine likely constitutes an important metabolite for the yajL mutant. L-alanine is also
metabolized after racemisation by alanine racemase DadX, mRNA levels of which are increased by
10-fold in the yajL mutant (not shown). The increased activities of pyruvate and alanine oxidases in
the yajL mutant are consistent with the increased excretion of acetate (see below); thus, the yajL
mutant displays increased pyruvate and alanine oxidase activities to overcome NADH
dehydrogenase deficiency. Alternative oxidases constitute an energetically favourable alternative to
fermentations, because, although PoxB and DadA do not expel protons by themselves (Unden,
1998), a proton motive force is generated during electron transfer through cytochrome oxidases.
Fermentative metabolism. Compared to the parental strain, the yajL mutant expressed 4-fold more
fermentative lactate dehydrogenase LdhA, 3-fold more acetaldehyde/alcohol dehydrogenase AdhE
and 2-fold more pyruvate decarboxylase FrsA (which degrades pyruvate to acetaldehyde) (Lee et
al., 2012) (Figure 5A). Accordingly, the yajL mutant excreted (as early as in the exponential phase)
higher amounts of acetate (2-fold), lactate (8-fold), ethanol (2.6-fold) and succinate (2-fold) than the
parental strain (Figure 5B).
Therefore, the yajL mutant exhibits active fermentative metabolism in aerobiosis, a characteristic of
cells displaying high NADH/NAD ratios (Berrios-Rivera et al., 2004 ; Holm et al., 2010). The
observed increase in excretion of acetate, succinate, lactate and ethanol is in accordance with
overexpression of pyruvate oxidase, fumarate reductase, lactate dehydrogenase and alcohol
dehydrogenase, respectively.
Overexpression of glycolytic and pentose phosphate pathway enzymes, and of PEP
carboxykinase and malic enzymes. Bacteria grown in LB medium metabolize both carbohydrates
and peptides (Baev et al., 2006 ; Yohannes et al., 2004). Glycolytic enzymes were overexpressed in
the yajL mutant, especially those acting downstream of aldolase (i.e., after convergence of the
glycolytic and pentose phosphate pathways), such as triose phosphate isomerase (tpiA gene product;
2.8-fold), glyceraldehyde-3-phosphate dehydrogenase (gap gene product; 2.1-fold), 3phosphoglycerate kinase (pgk gene product; 2.4-fold), enolase (eno gene product; 1.8-fold) and
pyruvate kinase (pyk gene product; 1.7-fold). The expression of pentose phosphate pathway
enzymes was also increased, including glucose-6-phosphate dehydrogenase (zwf gene product; 1.8
fold), transaldolase B (aldB gene product; 5 fold) and transketolase (tkt gene product; 1.7-fold),
suggesting that this pathway constitutes a privileged route for conversion of hexose to triose and is
responsible for the high NADPH/NADP ratio of the mutant (Figure 6A).
PEP carboxykinase and malic enzymes were also overexpressed, and may be involved in converting
metabolites derived from aspartate and asparagine (fumarate, malate and oxaloacetate) into PEP and
pyruvate, thereby feeding the pyruvate oxidase and fermentative pathways. By contrast, PEP
carboxylase (ppc gene product), which catalyzes the conversion of PEP into oxaloacetate for
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replenishment of the Krebs cycle, was underexpressed in the yajL mutant (not shown), in accordance
with depressed Krebs cycle activity, as discussed below)).
These results suggest that metabolism is increased in the yajL mutant through glycolytic and pentose
phosphate pathways, in accordance with the observed lower adenylate energy charge, pyruvatebased overflow metabolism, fermentative metabolism and NADPH requirement for combating
oxidative stress (Koebmann et al., 2002). The expression levels of PEP carboxykinase (pck gene
product) and of a malic enzyme (maeA gene product), which replenish PEP and pyruvate pools from
oxaloacetate and malate, are also increased (Figure 6A).
Overflow metabolism. In aerobic bacteria, the major route for pyruvate degradation is via pyruvate
dehydrogenase, the Krebs cycle and respiratory chains. In E. coli, pyruvate is also metabolized into
acetate via either the sequential action of pyruvate dehydrogenase, phosphotransacetylase and
acetate kinase, or via the activity of pyruvate oxidase (Eiteman & Altman, 2006). Pyruvate oxidase
was overexpressed by several-fold in the yajL mutant, which suggests that the mutant preferentially
uses pyruvate oxidase for acetate production. Moreover, pyruvate dehydrogenase is likely inhibited
in the mutant by the high NADH/NAD ratio. In the mutant, pyruvate is also degraded into lactate
and ethanol, and the pyruvate-alanine interconversion likely functions in the alanine-pyruvate
direction via the alanine dehydrogenase DadA and thus contributes to pyruvate formation.
Therefore, overflow metabolism is active in the yajL mutant, with a significant fraction of pyruvate
being metabolized into acetate via pyruvate oxidase and into lactate and ethanol by lactate and
alcohol dehydrogenases.
Depressed Krebs cycle. In aerobic wild-type bacteria, substrates are completely oxidized via the
Krebs cycle, and the NADH that is produced is oxidized by respiratory chains. Several findings
suggest that the Krebs cycle is weakly active in the yajL mutant, as follows: i) a high NADH/NAD
ratio is known to inhibit pyruvate dehydrogenase, citrate synthase, ketoglutarate and malate
dehydrogenases; ii) NADH dehydrogenases, which oxidize Krebs cycle NADH are weakly active;
iii) alternative dehydrogenases utilize Krebs cycle-independent substrates (mainly pyruvate and
alanine; iv) the mutant overexpresses fumarate dehydrogenase and excretes succinate, which
suggests that the oxaloacetate/succinate interconversion occurs in the oxaloacetate-succinate
direction, and vi) the ketoglutarate pool is 10-fold smaller than that of the parental strain (11 µM
versus 110 µM; not shown), suggesting that ketoglutarate dehydrogenase activity is negligible
(Kmketoglutarate=140 µM). Therefore, the Krebs cycle is likely depressed in the yajL mutant, and
pyruvate-dependent metabolism is characterized by fermentation and pyruvate-related respiration
involving D-alanine dehydrogenase and pyruvate oxidase.
Defective aminoacid metabolism. Because the yajL mutant displays reduced Krebs cycle activity
and contains low ketoglutarate levels, growth on mixed amino acids is affected, and degradation of
pyruvate-forming, transaminase-independent amino acids is favoured, over that of Krebs cyclerelated, transaminase-dependent amino acids (aminoacid degradation via transaminases requires
alpha-ketoglutarate as a substrate). We cultured the yajL mutant and the parental strain in tryptone
broth (a casein hydrolysate lacking carbohydrates), monitored growth by optical density, and
removed aliquots of the medium for amino acid analysis at appropriate intervals. The yajL mutant
grew more slowly than the parental strain in tryptone broth, whereas both strains grew at similar
rates in LB medium (which contains sugars and peptides) (not shown), suggesting that it harbours an
amino acid utilization defect. Both strains consumed alanine, serine, glycine, aspartate, asparagine
and tryptophan rapidly, as reported by others for wild-type strains (Pruss et al., 1994) (Figure 6B).
The efficient consumption of alanine, serine, glycine and tryptophan by the mutant can be explained
by their degradation into pyruvate without transamination. The yajL mutant also actively consumed
asparagine and aspartate, which are mainly degraded into fumarate by aspartase (AspA), with
fumarate being converted either (via oxaloacetate) into PEP by PEP carboxykinase, or (via malate)
into pyruvate by malic enzymes.
By contrast, the yajL mutant consumed glutamate, glutamine, arginine, phenylalanine, tyrosine,
methionine, valine, isoleucine and leucine less efficiently than the parental strain. These amino acids
are degraded via Krebs cycle intermediates (oxoglutarate, succinyl CoA and fumarate) and require a
transamination, so their decreased utilization may be explained by the depressed Krebs cycle activity
of the mutant and its low ketoglutarate pools; thus, the yajL mutant preferentially degrades pyruvateforming amino acids, that do not require transamination for their degradation.
DISCUSSION
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We show in this study that a mutant defective in YajL, the prokaryotic homolog of the
Parkinsonism-associated protein DJ-1, displays impressive metabolic changes, including NADH
dehydrogenase deficiency, a high NADH/NAD ratio, decreased adenylate energy charge, and
pyruvate-centred metabolism involving alternative dehydrogenases and fermentation (Figure 7).
High NADH/NAD and NADPH/NADP ratios. The high NADH/NAD ratio of the yajL mutant is
similar to those of NADH dehydrogenase I mutants (Husain et al., 2008) and anaerobic bacteria (de
Graef et al., 1999). High NADH/NAD ratios reduce the flow of material through pyruvate
dehydrogenase and the Krebs cycle, and favour fermentation and overflow metabolism. The
NADPH/NADP ratio in the yajL mutant is higher than in the parental strain. NADPH acts as a
reductant for peroxides and protein disulfides during oxidative stress, and might be protective
against endogenous oxidative stress in the mutant (Husain et al., 2008).
Decreased adenylate energy charge. The low adenylate energy charge of the yajL mutant likely
triggers high glycolytic flux (Koebmann et al., 2002), since ADP and AMP are allosteric activators
of phosphofructokinase and pyruvate kinase. The low adenylate energy charge probably results from
fermentative metabolism (Unden, 1998) and alternative respiratory pathways which are less efficient
than NADH dehydrogenase in generating a protonmotive force (Unden & Bongaerts, 1997). Less
tightly coupled respiratory chains may allow the yajL mutant to escape to the back pressure from the
proton motive force and achieve efficient overflow metabolism (Unden, 1998). Moreover, loosely
coupled respiratory chains are advantageous for reducing the formation of reactive oxygen species
by respiratory chains. Whereas in bacteria, a decrease in the proton motive force occurs by the use of
alternative respiratory chains, in eukaryotic cells this is mediated by uncoupling proteins.
Interestingly, underexpression of uncoupling proteins 4 and 5 in DJ-1 deficient cells results in
defective uncoupling and the overproduction of reactive oxygen species (Guzman et al., 2010).
NADH dehydrogenase 1 deficiency. The NADH dehydrogenase 1 activity of the yajL mutant is 7fold lower than that of the parental strain. Since the NuoG subunit of NADH dehydrogenase 1 is a
YajL substrate (Le et al., 2012), the low NADH dehydrogenase 1 activity of the mutant can be
explained by its YajL deficiency. The relationship between Parkinson’s disease and complex I
inhibition has been extensively studied (Danielson & Andersen, 2008). In the yajL mutant, NADH
dehydrogenase 2 is not able to restore a normal NADH/NAD ratio, so the mutant excretes
fermentation products and induces NADH-independent alternative dehydrogenases. Human cells do
not possess the second NADH dehydrogenase found in bacteria, yeasts and plants, that confers
significant flexibility to their respiratory chains. Transkingdom gene therapy, however, is a
promising strategy for remedying complex I deficiency in humans (Schiff et al., 2012).
Overexpression of alternative respiratory dehydrogenases. Alternative dehydrogenases are
overexpressed in the yajL mutant and several display increased activity, including pyruvate oxidase
PoxB and D-alanine dehydrogenase DadA. In contrast to NADH dehydrogenase 1 which contains
many subunits and several FeS clusters, and is sensitive to dysregulated biogenesis and
environnemental stresses, the alternative dehydrogenases PoxB, GlpD and DadA each consist of a
single protein devoid of FeS clusters. The overexpression of alternative dehydrogenases in the
mutant could be induced by the stress regulator sigma S, which is a known inducer of pyruvate
oxidase PoxB and alanine dehydrogenase DadA, and is overexpressed in the mutant in the
exponential phase (Messaoudi et al., 2013).
Several NADH-independent dehydrogenases described in this study have an eukaryotic counterpart,
including succinate dehydrogenase (eukaryotic complex II), glycerophosphate dehydrogenase GlpD
(mitochondrial glycerol-3-phosphate dehydrogenase GPD2), D-alanine dehydrogenase DadA (Damino acid oxidases), acyl-CoA dehydrogenase FadE (fatty acyl-CoA dehydrogenases) and proline
oxidase PutA (mitochondrial proline oxidase ProDH). Succinate dehydrogenase likely displays
reduced activity in complex I-deficient eukaryotic cells because of inhibition of the Krebs cycle by
the high NADH/NAD ratio. Glycerol-3-phosphate dehydrogenase GPD2, which is part of the
NADH shuttle that transfers the reducing power of cytoplasmic NADH to mitochondrial quinones,
is interesting because in contrast to the malate/aspartate shuttle, it circumvents mitochondrial NADH
production and may bypass NADH dehydrogenase defects. Eukaryotic D-amino acid oxidases
(which are restricted to D-amino acid detoxification) are of limited metabolic interest. Eukaryotic
acyl-CoA dehydrogenases are involved in the first step of fatty acyl-CoA degradation, and transfer
hydrogens to quinones; however, the next oxidoreduction step of -oxidation and acetyl CoA
oxidation in the Krebs cycle produce NADH, so they would be of little use to overcome NADH
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dehydrogenase defects (Abdallah et al., 2007; Bindoff et al., 1989; Cahloun & Gennis, 1993;
Koebmann et al., 2002; Kthiri et al., 2012; Schapira & Gegg, 2011). Proline oxidase catalyzes the
conversion of proline to 1-pyrroline-5-carboxylate and transfers hydrogens to quinones via FAD,
whereas the reverse reaction, catalyzed by 1-pyrroline-5-carboxylate reductase uses NAD(P)H as a
cofactor: consequently, both reactions produce a cycle of proline synthesis and degradation that can
transfer redox potential between cellular compartments, and might contribute to bypassing NADH
dehydrogenase 1 deficiency.
Increase in expression of microaerobic quinol oxidase and fumarate reductase. The yajL mutant
overexpresses the microaerobic ubiquinol oxidase CydAB (Unden, 1998), and also fumarate
reductase which catalyzes the reverse reaction of succinate dehydrogenase, suggesting that the
oxaloacetate/succinate branch of the Krebs cycle functions in the reverse direction, as favoured by
the high NADH/NAD ratio, and in accordance with increased excretion of succinate.
Fermentative metabolism. The yajL mutant displays mixed acid fermentation in accordance with
its high NADH/NAD ratio. Human cells utilize lactic fermentations, especially when respiration is
unable to cope with high metabolic flux: this occurs when oxygen availability is low during intense
exercise, or in respiratory chain diseases and cancer. In respiratory chain diseases, fermentation
increases as a consequence of the respiratory chain defect, whereas in cancer cells, a high rate of
glucose metabolism (aerobic glycolysis, known as the Warburg effect) sustains anabolic reactions
(DNA and RNA synthesis), and fermentative metabolism protects cells from intermittent hypoxia.
Amino acid degradation. The yajL mutant favours degradation of pyruvate-forming, transaminaseindependent amino acids over that of Krebs cycle-related, transaminase-dependent amino acids.
Such a metabolic status might occur in many metabolic diseases, including cancer, mitochondrial
respiratory chain diseases and Parkinson’s disease, in which respiratory activities are compromised
and/or fermentative metabolism is accelerated. In these cases, adequate amino acid supplementation
of patients, considering the two sets of amino acids described above, might be of benefit.
Finally, the present study exemplifies the consequences of a single gene deficiency on cellular
metabolism and constitutes an interesting model for the study of DJ-1 deficiencies and Parkinson’s
disease, as well as respiratory chain deficiencies. The remarkable ability of bacteria to cope with
NADH dehydrogenase deficiency resulting from YajL deficiency may influence our thinking with
regard to the way in which we treat respiratory chain deficiencies in eukaryotic cells by taking into
accounts the differences and similarities between prokaryotic and eukaryotic cells.
ACKNOWLEDGEMENTS. NM was supported by a grant from EGIDE.
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FIGURE LEGENDS.
Figure 1. Nucleotide pools and NADH dehydrogenase activities. A) NAD (black bars) and NADH
(grey bars) pools, and B) NADP (black bars) and NADPH (grey bars) pools were measured as described
in Methods. C) ATP (black bars), ADP (dark grey bars) and AMP (light grey bars) pools were
measured as described in Methods. D) The activities of NADH dehydrogenases 1 (black) and 2 (grey)
in membrane extracts were assayed by measuring the oxidation of 2,6 dichlorophenol indophenol at 600
nm with NADH (NADH dehydrogenase 1 + NADH dehydrogenase 2) or deamino-NADH (NADH
dehydrogenase 1) as substrates. An activity of 100 represents 0.18 µmol of NADH/min/mg protein.
Figure 2. Topology and arrangement of aerobic and anaerobic respiration enzymes in E. coli.
Topology and membrane insertion was determined from biochemical data or from protein sequence
properties such as hydrophobicity or signal sequence. Q and QH 2 stand for quinones and reduced
quinones, respectively. The orientation and topology of the subunits were derived from the E. coli
database and experimental evidence. This figure is taken from Unden & Bongaerts, 1997 with
permission.
Figure 3. Expression of respiratory chains. mRNAs expression values are taken from microarray
data; A) Expression of respiratory dehydrogenases in the yajL mutant (grey bars) and the parental strain
(black bars); B) Expression of ubiquinol oxidases and reductases in the yajL mutant (grey bars) and the
parental strain (black bars).
Figure 4. NADH, succinate, pyruvate, D-alanine and -D-glycerolphosphate oxidase activities. A)
Oxidases present in membrane extracts of the yajL mutant (grey bars) and the parental strain (black
bars) were estimated by measuring the rate of oxygen uptake using a Clarke-type oxygen electrode.
Reaction mixtures contained membranes (3 mg protein) and substrates (0.6 mM NADH, 20 mM
succinate, 4 mM lactate, 20 mM a-glycerophosphate) in a solution of 100 mM Tris (pH 7.5), 20 mM
magnesium acetate and 0.25 M sucrose.
Figure 5. Fermentation. A) Expression of fermentative enzymes in the yajL mutant (grey bars) and the
parental strain (black bars). B) Excretion of acetate, lactate, ethanol, succinate and formate from the
yajL mutant (grey bars) and the parental strain (black bars). Bacteria were grown in LB medium at
37°C, and culture supernatants were tested at different times for the presence of fermentation products
as described in Methods. “1” represents a concentration of 1 mM in the culture medium for all products,
except acetate for which it represents a concentration of 10 mM.
Figure 6. Expression of intermediary metabolism enzymes and amino acid catabolism. A) Ratios
of mRNA expression values (taken from microarray data) for several metabolic enzymes involved in
glycolysis, the HMP pathway and neoglucogenesis. B) Amino acid utilization efficiencies (ratio of the
yajL mutant/parental strain) by bacteria during exponential growth on casein hydrolysate medium
during the second hour after dilution from overnight cultures.
Figure 7. Metabolic scheme proposed for intermediary metabolism in the yajL mutant. The width
of the arrows is proportional to the flux through metabolic pathways. Hollow arrows represent amino
acid degradation pathways, and elongated grey arrows fermentative pathways.