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Journal of Bacteriology, December 2001, p. 6869-6874, Vol. 183, No. 23
Department of Bioenergetics, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State
University, Moscow 119899, Russia
Received 29 May 2001/Accepted 16 August 2001
The gene encoding the noncoupled NADH:ubiquinone oxidoreductase
(NDH II) from Azotobacter vinelandii was cloned, sequenced, and used to construct an NDH II-deficient mutant strain. Compared to
the wild type, this strain showed a marked decrease in respiratory activity. It was unable to grow diazotrophically at high aeration, while it was fully capable of growth at low aeration or in the presence
of NH4+. This result suggests that the role of
NDH II is as a vital component of the respiratory protection mechanism
of the nitrogenase complex in A. vinelandii. It was also
found that the oxidation of NADPH in the A. vinelandii
respiratory chain is catalyzed solely by NDH II.
Molecular nitrogen reduction is
mediated by the nitrogenase complex, an enzyme known to be highly
vulnerable to oxygen damage. Thus, the majority of bacteria are capable
of reducing N2 only in anaerobic or microaerobic
conditions. Contrary to this, Azotobacter vinelandii is an
obligate aerobe capable of fixing molecular nitrogen even at very high
ambient O2 concentrations. The purified nitrogenase complex
of this bacterium was shown to be as susceptible to oxygen damage as
those in other microorganisms (26). Dalton and Postgate hypothesized that the concentration of O2 in the cytoplasm
of Azotobacter is effectively reduced below
nitrogenase-damaging levels by the extremely active respiration
characteristic of this bacterium (5, 6). The existence of
such a mechanism, termed respiratory protection, was later
substantiated in a large number of studies. In particular, it was found
that the A. vinelandii bd-type quinol oxidase is essential
for respiratory protection (15, 21). It was also shown
that respiration mediated by the bd-type oxidase is dominant
in cells growing diazotrophically or at high oxygen concentrations
(18, 24). Mutant strains with disruptions in the gene
encoding the bd-type oxidase failed to fix N2 at
high ambient oxygen concentrations (15).
It would be logical to assume that in order to match the
above-mentioned highly rapid oxygen consumption at the bd
terminus of the A. vinelandii respiratory chain, there must
exist a mechanism at the initial segment that feeds electrons into the
chain just as effectively. Moreover, this putative enzyme should
utilize a major respiratory substrate as well as have no coupling with proton translocation in order to avoid limitations imposed by the
generated transmembrane proton potential.
In our previous study (3), it was shown that NADH
oxidation in A. vinelandii is mediated by two distinct
NADH:quinone oxidoreductases; one is coupled to the generation of the
transmembrane proton potential (NDH I), and the other is not (NDH II).
We also showed that the expression of NDH II, in contrast to that of
NDH I, is induced by high O2 concentration and by switching
of the bacterial culture to diazotrophic growth. Induction of NDH II
activity was observed under the same conditions in which induction of
the bd-type oxidase was shown to take place
(3). It is known that the latter effect is mediated by a
regulatory A. vinelandii protein, CydR (homologous to
FNR in Escherichia coli) (24, 25). It was shown
that the A. vinelandii strains lacking this protein
overproduce the bd-type oxidase even during growth at
lowered O2 concentrations. Later it was found that the
cydR mutation also leads to induction of NDH II and
repression of NDH I (3). Thus, at all growth conditions NADH is oxidized in the respiratory chain of this mutant almost entirely via NDH II.
The properties of the noncoupled NADH dehydrogenase (NDH II) and the
conditions of its induction in A. vinelandii suggest the
possible role of this enzyme in respiratory protection. To prove this
assumption, in the current work we constructed an NDH II-deficient
strain of A. vinelandii and tested its nitrogen-fixing capabilities at high ambient oxygen concentrations.
Bacterial strains, plasmids, growth, and medium
compositions.
The A. vinelandii and E. coli
strains used in this study are listed in Table
1. A. vinelandii cells were
grown in modified Burke's media BS and BSN (BS with ammonia salts
added) (7). The cells were grown in a rotary shaker at 250 rpm and 30°C. Antibiotics (where used) were added to the following
final concentrations: rifampicin, 10 µg/ml; kanamycin, 1 µg/ml;
ampicillin, 50 µg/ml; tetracycline, 10 µg/ml. E. coli
cells were routinely grown in Luria-Bertani (LB) medium.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6869-6874.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Noncoupled NADH:Ubiquinone Oxidoreductase of
Azotobacter vinelandii Is Required for Diazotrophic
Growth at High Oxygen Concentrations
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
KL
.
The oxygen transfer
coefficient (KL) was determined by the
sulfite method as described previously (10) with some
modifications: 100 g of KI/liter and 63.5 g of
I2/liter were used to prepare a 0.25 M iodine solution, and
Na2S2O3 was used instead of
Na2S2O4 for titration. To establish
high aeration (measured KL = 14 mmol of
O2 liter
1 h1), 100 ml of growth
medium was shaken in 1-liter flasks. Decreased aeration
(KL = 1.4 mmol of O2
liter1 h1) was established by shaking 200 ml of medium in 350-ml flasks.
SBPs. Subbacterial particles (SBPs) from A. vinelandii cells were prepared as described previously (2).
(i) Respiration of A. vinelandii cells and SBPs. Respiration of A. vinelandii cells and SBPs was monitored using a standard Clark-type electrode at 30°C. The following buffer was used as the respiration medium for SBPs: 60 mM KCl-2 mM MgSO4-20 mM HEPES-KOH (pH 7.5). Respiration of whole A. vinelandii cells was measured in BSN medium. Logarithmic cells containing to 30 to 60 µg of overall protein were sampled from the growth medium and directly injected into a polarograph chamber.
(ii) Oxidation rates of NADH, NADPH, and reduced nicotinamide hypoxanthine dinucleotide (dNADH) by A. vinelandii SBPs. Oxidation rates were monitored by means of a Hitachi-557 spectrophotometer as described previously (3). All reduced pyridine dinucleotides were used at a final concentration of 150 µM.
Estimation of Km values of A. vinelandii NDH I and NDH II for reduced pyridine nucleotide
oxidation.
Estimation of Km values for NDH
I and NDH II were carried out using SBPs from A. vinelandii
strains DN165 and MK8, respectively. NADH and dNADH oxidation was
monitored by registering the decrease in optical density at 340 nm
(OD340) (for both NADH and dNADH, 
340 = 6.22 mM1 cm1 was used). The rates of NADH
and dNADH oxidation at different concentrations of these reduced
pyridine dinucleotides were determined by analyzing the first
derivative of the (d)NADH oxidation progress curves. Briefly, the
(d)NADH concentration can be calculated from a direct
OD340-versus-time curve, while the values of the first derivative of the direct curve are proportional to the rates of the
(d)NADH oxidation. The data obtained were fitted to the
Michaelis-Menten equation using the nonlinear regression analysis method.
Construction of an NDH II-deficient A. vinelandii strain by site-directed mutagenesis. Amplification of the A. vinelandii ndh fragment was carried out using the PCR with degenerative primers ndhb1a [5'-CACCT(G/C)TTCCAGCCGCTGCT] and ndhb2a [5'-CCGGT(G/C)GGGCC(A/C)GC(G/C)CCGAC] (17). The amplified ndh fragment was cloned into pGEM-T vector, resulting in plasmid pND7.
Construction of the ndh::
Tc strain of A. vinelandii was carried out as follows. A tetracycline resistance
cassette from pHP45Tc was ligated into the SmaI site of
pND7, and a Tc-containing plasmid (pNDT6) bearing the ndh
gene together with the unidirectionally transcribing tetracycline
resistance cassette was selected. Competent A. vinelandii
cells were transformed by the pNDT6 plasmid. This transformation
resulted in a series of tetracycline-resistant clones. Screening for
both tetracycline and ampicillin resistance led to selection of an
Amps Tcr clone (DN165) characteristic of a
double-crossover introduced mutation. A single recombinant
Ampr Tcr clone (N24) was also selected and
later used for ndh cloning by the plasmid rescue technique.
Segregation of the ndh mutation in A. vinelandii. In order to obtain an A. vinelandii strain with a mutated ndh in all copies of the chromosome, A. vinelandii DN165 obtained on selective medium with 10 µg of tetracycline per ml was plated on solid medium supplied with 30 µg of tetracycline per ml. An individual clone was selected and plated on solid medium supplied with 60 µg of tetracycline per ml.
Based on the determined primary sequence of the cloned ndh fragment, a p_ndh (5'-CGGCGACGAACTGAACTA) and r_ndh (5'-GTGGGCACGCAAGTAGTG) primer pair was selected. With these primers, a unique 300-bp PCR product from wild-type A. vinelandii DNA was formed. It was employed as a specific marker to monitor the segregation process. PCR analysis of DNA from the segregated mutant clone showed the presence of the major 2.4-kbp fragment only (comprising the 2.1- and 0.3-kbp components corresponding to the tetracycline resistance cassette and the ndh fragment, respectively). The 0.3-kbp wild-type fragment was not present.Sequencing of the whole ndh gene from A. vinelandii. The 3' part of ndh was cloned by the plasmid rescue technique (8). Four micrograms of chromosomal DNA of the single recombinant clone, N24, was incubated with the AatII restrictase. The restriction mixture was treated with T4 DNA ligase in a 300-µl volume and used for E. coli transformation. As a result, an ampicillin-resistant clone bearing the pGEM plasmid with an ~4-kbp insert of A. vinelandii genomic DNA was obtained. This plasmid was used for sequencing of the 3' part of the ndh gene.
The 5' part of ndh was cloned by an inverse PCR technique (19). Two micrograms of A. vinelandii UW136 DNA was incubated with the PstI restrictase. The restriction mixture was treated with T4 DNA ligase in a 150-µl volume. Subsequently, 1 µl of the ligase mixture was used as the template for PCR with the rev_ph (5'-GGTCGAGTTCAGCGAGC) and rev_mdh (5'-CACTACTTGCGTGCCCAC) primers. A unique ~2.8-kbp PCR product was obtained and later used for sequencing of the 5' part of ndh. Nucleotide sequence was analyzed with double-stranded templates (both strands were sequenced) using synthesized oligonucleotide primers. This analysis was carried out by the sequencing group of the Engelhard Institute of Molecular Biology using an Applied Biosystems ABI 373A DNA sequencer. Competent A. vinelandii cells were obtained as described by Page and von Tigerstrom (20). Transformation of A. vinelandii cells was carried out as described by Glick et al. (11). Protein concentration was measured by means of the bicinchoninic acid method with bovine serum albumin (Serva, type V) as the standard.Nucleotide sequence accession number. The novel DNA sequence reported here has been deposited into the GenBank database and is available under accession number AF346487.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of an NDH II-deficient A. vinelandii strain. (i) Cloning of a fragment of the ndh gene from A. vinelandii. An ndh gene fragment from the A. vinelandii DNA was amplified by means of a PCR technique using degenerative primers (17). PCR with these primers and the A. vinelandii DNA as a template resulted in amplification of a variety of fragments of different-lengths. The PCR products were separated using gel electrophoresis. In accordance with expected size of the ndh gene, the amplified product was searched for in the 400-bp band. This band was isolated, cloned into a pGEM-T vector (resulting in plasmid pND7), and sequenced. A BLASTX analysis of the acquired fragment (with the exclusion of primer sequences) showed that the putative peptide corresponding to this nucleotide fragment is homologous to NDH II from a wide variety of bacteria. The highest homologies were was found with the sequence of NDH II from Pseudomonas aeruginosa (79%) and Pseudomonas fluorescence (77%). Thus, the ndh fragment of A. vinelandii DNA was cloned.
(ii) Construction of an ndh::Tc strain of
A. vinelandii.
The sequence data for the cloned
ndh fragment revealed the presence of a unique
SmaI site located 263 bp away from the 5' end of the PCR
product. Subsequently, a tetracycline resistance cassette from
pHP45Tc was ligated into this SmaI site of plasmid pND7,
resulting in plasmid pNDT6. In order to construct a strain of A. vinelandii with the insertion-inactivated fragment replacing the
wild-type sequence, competent A. vinelandii cells were
transformed using plasmid pNDT6 (this plasmid is unstable in A. vinelandii because of its ColE1 replicon). An Amps
Tcr clone (DN165) characteristic of a double-crossover
introduced mutation was selected. Localization of this mutation in the
ndh gene of the acquired strain was verified by PCR analysis.
Properties of the NDH II-deficient A. vinelandii strain. (i) Respiration rates of whole A. vinelandii cells and subbacterial particles. As experiments showed, the DN165 mutant cells have a much lower respiratory activity than the wild type. Generally, the wild-type cells grown in highly aerated BSN medium consumed 2.1 ± 0.6 µg-atom of oxygen per min per mg of protein, whereas the figure for DN165 was as low as 0.4 ± 0.1 (average from three independent experiments).
NADH- and dNADH-oxidizing activities of the A. vinelandii DN165 SBPs were also tested (Table 2). It is well known that NDH I can oxidize either NADH or its analog dNADH, while NDH II utilizes only NADH and not dNADH (3). SBPs from wild-type A. vinelandii oxidized NADH at much higher rates than dNADH due to operation of both NADH dehydrogenases (Table 2). The difference between NADH and dNADH oxidase rates is much more profound in the MK8 (
cydR) strain, which contains very
low levels of NDH I (3). On the other hand, in the
constructed NDH II-deficient mutant strain DN165, NADH and dNADH
oxidation rates were equal and low (Table 2). This confirms that
the DN165 mutant does not possess significant NDH II activity.
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(ii) Participation of the A. vinelandii NDH II in
respiratory protection.
Growth of three A. vinelandii
strains was tested at different aeration levels in media with (BSN) and
without (BS) ammonium acetate. As seen from Fig.
1, at high aeration
(KL = 14 mmol of O2
liter1 h1) the DN165 strain lacking NDH II
is capable of growing in the presence of NH4+
at a rate comparable to that of the wild-type strain (doubling time,
150 and 140 min, respectively), but contrary to the wild type, it
failed to grow at a high oxygen concentration if N2
fixation was necessary. For comparison, Fig. 1 also includes growth
curves for A. vinelandii MK8, which was shown to most
preferably oxidize NADH via NDH II (3). As seen from Fig.
1C, the absence of NDH I activity (in contrast to NDH II) does not lead
to any increase in O2 sensitivity of the
N2-fixing bacterial culture.
|
) and BS minimal
medium (
) (see Materials and Methods for details). (A) A. vinelandii UW 136 (wild type); (B) A. vinelandii DN165
( = 1.4 mmol of
O2 liter1 h1) were similar both
in the presence and in the absence of NH4+. An
assumption can be drawn that NDH II is vital for the protection of the
nitrogenase complex against oxygen insult, i.e., during diazotrophic
growth at a high ambient oxygen concentration.
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(iii) Role of NDH II in the NADPH oxidation activity of A. vinelandii. Earlier studies (13) have shown that A. vinelandii membranes are capable of oxidizing NADPH. Ackrell and coworkers (1) have postulated the presence of a specific enzyme mediating this reaction. In the present study, we tested the effect of ndh mutation on the NADPH-oxidizing activity of A. vinelandii. Our data show (Table 2) that SBPs from A. vinelandii strain DN165 completely lack the ability to oxidize NADPH. This provides proof that NDH II is solely responsible for the NADPH dehydrogenase activity in the respiratory chain of A. vinelandii. It is noteworthy that a similar conclusion has been recently drawn for NDH II of Corynebacterium glutamicum (17).
(iv) Estimation of Km values of A. vinelandii NDH I and NDH II for reduced pyridine nucleotide
oxidation.
Using A. vinelandii strains that respire
almost solely via NDH I (MK8 [3]) or entirely via NDH II
(DN165), we measured the Km values of these
enzymes for various respiratory substrates (Table
3). Tables 2 and 3 show that NDH I is
capable of oxidizing both NADH and dNADH, but not NADPH, while NDH II
oxidizes NADH and NADPH but cannot utilize dNADH. The affinity of NDH
II for NADPH is very poor, which indicates that it is not a major
substrate under physiological conditions. When comparing
Km values for NADH, we expected to see higher
values for NDH II then for NDH I, as in theory this would render NDH II
efficient only when NDH I is either substrate saturated or limited by
respiratory control. Test results showed that the
Km value of NDH II for NADH turned out to be
twice that of NDH I (Table 3). It is possible, however, that the
electron flow through the two different NADH dehydrogenases is
downregulated at the level of quinone, i.e., via their different affinities for ubiquinone-8. Investigation of this speculation, however, is left for future research.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The current work clearly indicates that NDH II in A. vinelandii is vital for diazotrophic growth at high ambient oxygen concentrations. Thus, the result may suggest that the role of NDH II is as a key component of the oxygen protection mechanism in this bacterium. In earlier work (15, 21), a similar function has been assigned to the bd-type terminal oxidase from the same organism. Studies by our group (3) found NDH II induction by increased ambient oxygen concentration or by the absence of NH4+ in the medium, i.e., under the same conditions that induce cytochrome bd (18, 24). Such coordinated expression of both of these enzymes is achieved through their joint induction by the CydR regulator protein (3, 24, 25).
Taking into account the results of previous work (2, 3, 15, 16,
22), the A. vinelandii respiratory chain may be described by the scheme shown in Fig. 3.
It is assumed that A. vinelandii cells possess at least two
respiratory chains differing in enzyme composition and physiological
function. One of them is completely coupled (thin arrows); it includes
NDH I (3), the bc1-complex
(2), cytochromes c4 and
c5 (22), and the o-type
oxidase (16). The other chain (thick arrows) is much simpler and includes the noncoupled NDH II (3) and the
bd-type quinol oxidase (15). The
energy-conserving efficiency of this chain
(H+/e = 1 [H+/e is the number of H+ ions
pumped across the membrane for every one electron passed down the
respiratory chain]) is much lower than that of the coupled one
(H+/e = 5) (2, 3). This
allows avoidance of limitations of oxygen consumption rates by systems
utilizing transmembrane electrochemical proton potential
(µH+).
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It is also worth noting that both NDH II and the bd oxidase
possess unusually high specific activities (more so for NDH II), i.e.,
560 µg-atom of oxygen min1 mg of
protein1 (14) and 106 mmol of NADH
min1 mg of protein1 (4) for
the bd-type quinol oxidase and NDH II, respectively. Such
high rates may be due to the absence of the proton-translocating function for these enzymes (the function of the bd-type
oxidase does in fact generate µH+ but solely due to
scalar proton effects [2, 12]). Exceptionally high
turnover of NDH II should be favorable for optimally efficient utilization of the membrane-occupied space which can limit overall rates of oxygen consumption in a cell. Such a high turnover rate of the
respiratory protection chain is what makes it possible to decrease the
intracellular oxygen level required for the operation of the nitrogenase.
While the respiratory chain in the inner membrane of animal mitochondria possesses a single NADH-oxidizing enzyme (complex I, analogous to bacterial NDH I) (23), most bacteria also harbor a second enzyme (i.e., NDH II) the physiological role of which has not yet been revealed. The analysis of the A. vinelandii NDH II sequence indicates that this protein is very similar to the NDH II of microorganisms from the gamma subdivision of proteobacteria. This suggests that NDH II from A. vinelandii is a typical representative of this class of enzymes and that in other organisms harboring NDH II the enzyme may also be employed similarly, i.e., for very fast quinone reduction (or very fast NADH oxidation). The possible physiological role(s) of NDH II in bacteria other than Azotobacter is left for future research.
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ACKNOWLEDGMENTS |
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This work was supported in part by RFBR grant 99-04-49161. Y.V.B. and A.V.B. are indebted to the RFBR for fellowships (no. 01-04-06480 and 01-04-06481).
We thank D. Molenaar for generously providing the degenerative primers, R. K. Poole for kindly providing A. vinelandii strains, I. V. Elanskaya for help in the cloning experiments and for fruitful discussion, and A. I. Shestopalov for help with preparation of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Bioenergetics, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia. Phone: (7) 095 939 55 30. Fax: (7) 095 939 03 38. E-mail: skulach{at}head.genebee.msu.su.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, December 2001, p. 6869-6874, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6869-6874.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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