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Journal of Bacteriology, April 1999, p. 1994-2000, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in
Rhodobacter capsulatus Is Correlated with
Cellular Nitrogen Status
Alexander F.
Yakunin,1,2
Tatyana V.
Laurinavichene,2
Anatoly A.
Tsygankov,2 and
Patrick C.
Hallenbeck1,*
Département de Microbiologie et
Immunologie, Université de Montréal, Succursale
Centre-ville, Montréal, Québec H3C 3J7,
Canada,1 and Institute of Basic
Biological Problems, Russian Academy of Sciences, Pushchino, Moscow
Region, Russia2
Received 23 October 1998/Accepted 25 January 1999
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ABSTRACT |
The photosynthetic bacterium Rhodobacter
capsulatus has been shown to regulate its nitrogenase by covalent
modification via the reversible ADP-ribosylation of Fe protein in
response to darkness or the addition of external
NH4+. Here we demonstrate the presence of
ADP-ribosylated Fe protein under a variety of steady-state growth
conditions. We examined the modification of Fe protein and nitrogenase
activity under three different growth conditions that establish
different levels of cellular nitrogen: batch growth with limiting
NH4+, where the nitrogen status is externally
controlled; batch growth on relatively poor nitrogen sources, where
the nitrogen status is internally controlled by
assimilatory processes; and continuous culture. When cultures
were grown to stationary phase with different limiting
concentrations of NH4+, the ADP-ribosylation
state of Fe protein was found to correlate with cellular nitrogen
status. Additionally, actively growing cultures (grown with
N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in
the modified state. The correlation between cellular nitrogen status
and ADP-ribosylation state was corroborated with continuous cultures
grown under various degrees of nitrogen limitation. These results show
that in R. capsulatus the modification
system that ADP-ribosylates nitrogenase in the short term in response
to abrupt changes in the environment is also capable of modifying
nitrogenase in accordance with long-term cellular conditions.
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INTRODUCTION |
The reduction of dinitrogen to
ammonia is catalyzed by the nitrogenase complex, which is composed of
two electron-transferring proteins: an iron protein (Fe protein,
dinitrogenase reductase) and a molybdenum-iron protein (MoFe protein,
dinitrogenase). This reaction is an energy-demanding process which
consumes 20 to 30 ATP molecules for each N2 reduced, and
N2-fixing microorganisms have evolved efficient mechanisms
to control both nitrogenase synthesis and its activity. Several
nitrogen-fixing bacteria have been shown to regulate nitrogenase in the
short term by posttranslational covalent modification via reversible
ADP-ribosylation of the Fe protein in response to different
environmental stimuli: ammonium addition, darkness, and the
absence of oxygen (21, 22). This process is catalyzed by two
non-nif-specific enzymes: dinitrogenase reductase
ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase (19, 28) and has been particularly well
characterized for the photosynthetic bacterium Rhodospirillum
rubrum (4, 18, 20). In Rhodospirillum rubrum
dinitrogenase reductase ADP-ribosyltransferase catalyzes the transfer
of ADP-ribose from NAD to the Arg 101 residue of one subunit of the Fe
protein homodimer in response to ammonium addition, darkness, or the
presence of O2 (22). This modification is
accompanied by the loss of nitrogenase activity. After effector
exhaustion or removal, dinitrogenase reductase-activating
glycohydrolase removes ADP-ribose from modified Fe protein, restoring
nitrogenase activity (22). Thus ADP-ribosylation of Fe
protein provides one molecular basis for the fast and reversible inhibition of nitrogenase activity (nitrogenase switch-off) seen in
vivo upon the addition of NH4+.
Previously, this ADP-ribosylation process has been viewed as a
transitory response to abrupt changes in culture conditions (22). The possible presence of ADP-ribosylated Fe protein in different cultures has received little or no attention. For example, numerous studies have shown that crude extracts of glutamate- or
N2-grown cells of Rhodospirillum rubrum,
Rhodobacter capsulatus, and
Rhodopseudomonas palustris contain modified
(ADP-ribosylated) Fe protein (3, 10, 36, 37, 39). This
was surprising, since glutamate-grown cultures exhibit high
nitrogenase activity and are fully capable of regulating this
activity in response to added NH4+ (i.e.,
nitrogenase switch-off) (1, 10). It has been assumed that
the observed modification is due to darkness, shown to induce rapid Fe
protein ADP-ribosylation, during postculture manipulations (12). During a recent study of the regulation of nitrogenase activity in R. capsulatus (34), where
we used a fast and sensitive immunochemical method for the analysis of
the modification state of Fe protein, which avoids artifactual changes
in modification state, we noticed that, depending upon the growth
conditions, some culture types contained a proportion of the Fe protein
as the ADP-ribosylated form without having been subjected to
experimental manipulation. These observations led us to hypothesize
that cells may naturally contain ADP-ribosylated Fe protein whose
relative proportion depends upon cellular nitrogen status and to
systematically examine the effects of culture conditions, particularly
nitrogen status, on the cellular content of ADP-ribosylated Fe protein.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Experiments were
performed with R. capsulatus SB1003 (35) and
strains JP23 and JP28, which are derivatives of strain RcM1 (
nifHDK) containing the conjugative plasmids pJP23 and
pJP28, respectively. These plasmids bear the genes that encode
wild-type MoFe protein and Fe protein with amino acid replacements of
Arg 102 by Tyr (JP23) or by Lys (JP28) (27). Strains W107I
and W107II are draTG deletion derivatives of B10S containing
the gentamicin resistance interposon in different orientations
(23). Cultures were grown essentially as previously
described (34) in liquid RCV medium (32)
containing 36 mM lactate and twice the normal concentration of
K-phosphate (19.1 mM) to avoid a pH shift observed with concentrated
cultures. Cultures from late exponential growth phase grown on RCV
medium with NH4+ excess (30 mM) were used as
inocula (5% vol/vol). Batch cultures were grown in long tubes (1.6 by
20.5 cm) filled with 20 ml of RCV medium, which were sealed with rubber
stoppers (Suba seal) with needles inserted for gas sparging (argon or
N2; 5 to 10 ml/min). Seven millimolar glutamate or
N2 or limiting amounts of NH4+
(concentrations as indicated in the figures) were used as nitrogen sources. During growth, culture aliquots were withdrawn for the determination of cell density (A660), in vivo
nitrogenase activity was determined, and immunoblot analysis of
nitrogenase proteins was carried out.
Continuous cultivation of R. capsulatus was performed
with a photobioreactor specially designed for the continuous
cultivation of photosynthetic microorganisms (30). Cultures
were grown under photoheterotrophic conditions on the medium of Ormerod
et al. (25) with 45 mM lactate at pH 7.0 and at 30°C with
a light intensity of 81.4 W/m2.
(NH4)2SO4 was used as the nitrogen
source. Concentrations of 10 and 2 mM were used for turbidostat
conditions and chemostat conditions, respectively. Cultures were
continuously sparged with 98% argon plus 2% CO2 (50 ml/min). All assays were performed under steady-state conditions after
at least five culture doublings.
Determination of nitrogenase activity and Fe protein modification
status.
Analysis of in vivo nitrogenase activity was performed by
the acetylene reduction method (7) with 1- or 2-ml culture
samples. Immunoblotting with chemiluminescence detection (33,
34) was used to monitor the modification state of Fe protein in
R. capsulatus cells under different conditions. Samples
(25 to 50 µl) were removed at the times indicated in the figures,
brought to a 1× sodium dodecyl sulfate (SDS) sample buffer
concentration (16), and immediately incubated in a
boiling-water bath for 2 min. This method of sample preparation and
subsequent SDS-polyacrylamide gel electrophoresis and immunoblot
analysis present an accurate picture of the degree of in vivo
nitrogenase modification since (i) samples that showed no
treatment-induced ADP-ribosylation could be prepared by this method and
(ii) increasing the time in the boiling-water bath did not change the
amount of ADP-ribosylated subunit (as determined quantitatively by
densitometric scanning). In our hands the SDS extraction method
produced results equivalent to those of the previously described
trichloroacetic acid precipitation procedure (38), but the
latter method is more time-consuming and laborious.
SDS samples prepared as described above were analyzed by Laemmli
SDS-polyacrylamide gel electrophoresis (12.5% of total acrylamide)
with low cross-linker gels
(acrylamide-
N,
N'-methylenebis(acrylamide),
30:0.2) and an increased duration of electrophoresis to obtain
increased resolution of Fe protein subunits (
12). Equal
amounts
of total protein (1 mg/well) were loaded onto all gels. In this
system,
R. capsulatus Fe protein showed apparent
molecular masses
of 34.9 kDa for the unmodified subunit and 38.7 kDa
for the ADP-ribosylated
subunit. The slower-migrating band was assumed
to be ADP-ribosylated
Fe protein based on its recognition by
anti-Fe-protein antibody,
its appearance in response to the appropriate
stimuli, its apparent
molecular mass (38.7 kDa), and its absence in
draT mutants as
well as in strains containing an
unmodifiable Fe protein (
23,
27). Since only one of the two
Fe protein subunits of the Fe
protein dimer was modified, 100%
modification of Fe protein dimers
corresponds to two equal-intensity
bands. The occasional occurrence
of additional immunoreactive bands of
Fe protein is due to the
transient formation of incompletely denatured
intermediates (results
not shown). All bands identified and quantitated
as either modified
or unmodified were shown by control experiments
(with, for example,
culture extracts made from strains containing
unmodifiable Fe
protein) to be assignable to the appropriate category.
For Fe
protein quantitation, the X-ray films were scanned with a
Molecular
Dynamics personal densitometer and the percentages of
unmodified
and modified Fe protein dimers were calculated as described
previously
(
20). The protein concentrations of culture
samples were determined
after 2 min of sonication by the Bradford
method (
2) with bovine
serum albumin as the standard. The
NH
4+ concentrations in culture supernatants
were measured by the phenol-hypochlorite
method (
29).
Determination of intracellular glutamine concentrations.
Glutamine levels were measured by high-performance liquid
chromatography analysis of o-phthalaldehyde-derivatized
amino acid pools extracted from bacterial cultures by modifications of
a previously described method (8). Samples were obtained
with a no-harvest protocol with methanol extraction, which prevents metabolic depletion of the glutamine pool (8). Typically,
0.2 to 0.5 ml of culture was directly pipetted into ice-cold methanol to give a final concentration of 80% and the mixture was dried with a
vacuum concentrator and stored at 
70°C. Immediately prior to the
assay, samples were dissolved in 100 µl of water and clarified by
centrifuging them for 15 min at 4°C. Ten microliters of the o-phthalaldehyde-derivatized sample (8) was
injected onto a C18 Partisphere column (Whatman), which was
developed with gradient elution (1 ml/min) with buffer A (90 mM sodium
acetate [pH 7.2], 0.5% tetrahydrofuran) and buffer B (100% methanol
[high-performance liquid chromatography grade]). The program was as
follows: 0 to 5 min with 100% buffer A, 5 to 35 min with 0 to 30%
buffer B, 35 to 45 min with 30 to 80% buffer B, and 45 to 50 min with
80 to 0% buffer B. A Shimadzu RF-551 fluorescence detector was used, with excitation at 340 nm and emission at 455 nm. Under these conditions, glutamate and glutamine eluted at 12.5 and 27.7 min, respectively.
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RESULTS |
We examined nitrogenase activity and the modification state of Fe
protein using three types of culture conditions that differ in how
nitrogen limitation is achieved. With ammonia-limited cultures, the
nitrogen status is directly established by the experimentally set ratio
of ammonia to carbon source (lactate). With either N2- or
glutamate-grown cultures, the nitrogen status is indirectly established
by the assimilatory mechanisms of the cell. With continuous cultures,
the nitrogen status of ammonia-limited cultures is controlled by the
dilution rate (D).
Batch growth with limiting NH4+ as the
nitrogen source.
The possible relationship between the degree of
cellular nitrogen limitation and Fe protein modification state was
checked by varying the initial concentration of ammonium (0 to 25 mM
NH4+) in the growth media of batch cultures,
producing cultures with different severities of nitrogen limitation
(Fig. 1). In the initial NH4+ concentration range of 0 to 11.4 mM, the
final culture density was proportional to the initial
NH4+ concentration and the cells did not
contain any ADP-ribosylated Fe protein. These cultures can be
considered to be purely N limited. However, when the initial
NH4+ concentration in the medium was higher
than 11.4 mM, the final culture density did not increase proportionally
with an increase in ammonium. Under these conditions, the cultures
appeared to be under dual limitation: they were still N limited up to
22.8 mM, since the nitrogenase proteins were still expressed, and, additionally, were light limited, since incubation at a higher light
intensity resulted in a higher final cell density (e.g., growth of a
17.1 mM NH4-limited culture at a higher light intensity gave a 10 to 15% increase in final optical density). A variety of
genetic and molecular studies have described a complex regulatory machinery that ensures that nitrogenase is synthesized in R. capsulatus only under conditions of nitrogen limitation
(15). Thus, we take the presence of nitrogenase proteins in
cultures as indicative of nitrogen limitation. Dual limitation, by
light and fixed nitrogen, of continuous cultures of R. capsulatus has been previously demonstrated (31). The
final concentrations of extracellular ammonium were very low (10 to 20 µM) with 1.9 to 11.1 mM initial NH4+,
slightly higher (60 to 250 µM) with initial
NH4+ in the range 13.3 to 19.0 mM, and
appreciably higher (
0.7 mM) with initial
NH4+ exceeding 19 mM.
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FIG. 1.
Growth (final culture density), extracellular
NH4+, and Western immunoblot analysis of
nitrogenase Fe protein in R. capsulatus batch cultures
grown with different initial concentrations of
NH4+. Cultures were grown
photoheterotrophically on RCV medium containing various
NH4+ concentrations (as indicated). After
40 h of growth the final culture densities
(A660) and extracellular
NH4+ concentrations were measured and samples
were prepared for immunoblot analysis of Fe protein as described in
Materials and Methods. (A) Final culture densities
(A660),
extracellular-NH4+ concentrations
(NH4+), and the proportions of ADP-ribosylated
Fe protein, calculated from the scan of the immunoblot presented in
panel B. (B) Immunoblot analysis of nitrogenase Fe protein in samples
from cultures grown at the indicated initial concentrations of
NH4+ (results for 0 to 9.5 mM
NH4+ are not shown). One hundred percent
modification of Fe protein dimers corresponds to two equal-intensity
bands.
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Immunoblot analysis of these cultures showed that some
ADP-ribosylated Fe protein was present in cells grown on
media containing
13.3 mM and higher concentrations of initial
ammonium (until complete
nitrogenase repression occurred at 24.7 mM
NH
4+). The amount of ADP-ribosylated Fe protein
was directly proportional
to the initial NH
4+
concentration, with there being almost 90% modification in cells
grown
with 22.8 mM NH
4+. While the light limitation
experienced by the weakly nitrogen-limited
(high-density) cultures may
have had some effect on the overall
modification status, it is more
likely that light limitation had
an indirect effect. Indeed, that the
intracellular nitrogen pool
plays a more direct role than light
intensity in establishing
the ADP-ribosylation status can be seen in
experiments where the
nitrogen status of
NH
4+-limited cells was varied, under conditions
of a fixed concentration
of NH
4+ (and therefore
with a fixed cell density) and a static intensity
of light, by varying
the initial lactate concentration (15 to
35 mM). Immunoblot analysis
(Fig.
2) clearly demonstrated the
presence of ADP-ribosylated Fe protein in cells grown with a reduced
amount of lactate (reduced nitrogen limitation) at an
NH
4+ concentration where growth was not light
limited. That these
cultures were not light limited can be seen by a
comparison of
the results with the results presented in Fig.
1. At the
limiting
NH
4+ concentration used here (9.5 mM),
the culture in Fig.
1 had a
final
A600 of 6.0 whereas the 20 mM lactate culture shown in Fig.
2 had a final
A600 of 3.8.
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FIG. 2.
Nitrogenase Fe protein immunoblot analysis of
NH4+-limited R. capsulatus
cells grown with various lactate concentrations. Batch cultures were
grown photoheterotrophically on RCV medium containing 9.5 mM
NH4+ as the nitrogen source and the indicated
concentrations of lactate. After 40 h of growth the modification
state of Fe protein was determined as described in Materials and
Methods.
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Assimilation of nitrogen requires the synthesis of only two central
intermediates, glutamine and glutamate, from which other
compounds
derive nitrogen by secondary transfers. In the enteric
bacteria
Salmonella typhimurium and
Klebsiella pneumoniae,
the
size of the intracellular glutamine pool appears to
reflect the
cellular nitrogen status, with external nitrogen limitation
causing
a drop in this pool (
8). Therefore, we determined
the levels
of intracellular glutamine and glutamate in
R. capsulatus cells
grown with different initial concentrations of
NH
4+ in the medium. As can be seen from the
data presented in Fig.
3, the pool of
intracellular glutamine was low (1 to 2 nmol/mg
of cell protein)
in cultures grown with initial concentrations
of
NH
4+ of 0 to 11.4 mM (no Fe protein
modification) and increased (4.5
to 20 nmol/mg of cell protein) in
cells grown with higher concentrations
of ammonium in the medium
and containing modified Fe protein.
Comparison of Fig.
1 and
3
suggests that Fe protein ADP-ribosylation
is more closely correlated
with intracellular glutamine (nitrogen
status) than with intracellular
glutamate or the external concentration
of residual
NH
4+ in the medium.
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FIG. 3.
Levels of intracellular glutamine and glutamate in
R. capsulatus batch cultures grown with different
initial concentrations of NH4+. Cultures were
grown (40 h) photoheterotrophically on RCV medium containing various
NH4+ concentrations as indicated, and samples
were prepared and analyzed for intracellular glutamine and glutamate as
described in Materials and Methods. Values averages of results from at
least five independent determinations, with standard deviations
indicated by error bars.
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Growth with N2 or glutamate.
With
NH4+-limited cultures, nitrogenase is expressed
only when the externally supplied ammonia is nearly exhausted,
which at moderately to highly limiting concentrations of
NH4+ occurs at the beginning of the stationary
growth phase (results not shown). Therefore, to determine the
possible presence of modified Fe protein in actively growing cultures,
we monitored the nitrogenase activity and the ADP-ribosylation status
of the Fe protein during batch growth of R. capsulatus
with N2 or glutamate as the nitrogen source. With
N2 as the sole nitrogen source, in vivo nitrogenase activity reached a maximum in the middle of the exponential phase of
growth and subsequently rapidly declined to a very low level (Fig.
4). Similar results have previously been
reported for N2-grown cultures of Rhodospirillum
rubrum (24). In contrast to these dramatic effects the
effects of changes in the content of the nitrogenase proteins (MoFe
protein and Fe protein) were much less marked (not shown).
Immunoblot analysis of the modification state of the Fe protein in
these cells revealed that the ADP-ribosylated form of this protein was
present, at various levels, throughout cultivation (Fig. 4B).
Scanning densitometry showed that the content of ADP-ribosylated Fe
protein dimers increased simultaneously with the increase of
nitrogenase activity, reaching a maximum at approximately the same time
(Fig. 4C, right y axis). In fact, after 20 to 25 h of
cultivation, an N2-grown R. capsulatus
culture exhibited the highest nitrogenase-specific activity at the same time that 80 to 90% of the Fe protein dimers were in the
ADP-ribosylated form. Analysis of NH4+ in
culture supernatants showed that there were only very low levels of
extracellular NH4+ (9 to 15 µM)
throughout the period of growth examined. While at first glance it may
seem surprising that cultures actively synthesize Fe protein and then
immediately modify it, this may be a consequence of different rates of
response of the controls for transcription and modification with
respect to intracellular nitrogen status. Thus, a culture which
has already synthesized an appreciable amount of nitrogenase proteins
will rapidly assimilate N2, leading to modification of
preformed and newly synthesized Fe protein before transcription and
synthesis can be shut off.
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FIG. 4.
In vivo nitrogenase activity and the content of
nitrogenase proteins in R. capsulatus cells during
growth with N2 as the nitrogen source. RCV medium
containing no added nitrogen was inoculated (5%, vol/vol) with
R. capsulatus cells grown on medium containing an
excess of NH4+ (30 mM) to give an initial
culture density of 0.2 (A660). At the times
indicated, aliquots were removed for the determination of culture
density (A660), whole-cell nitrogenase activity,
nitrogenase protein content, and Fe protein modification state as
described in Materials and Methods. (A) Growth
(A660) and specific in vivo nitrogenase
(N2ase) activity. (B) Immunoblot analysis of nitrogenase
proteins. (C) Changes in the content of nitrogenase proteins
(calculated from the scan of the immunoblot presented in panel B) and
in in vivo nitrogenase activity (from the graph in panel A). One
hundred percent corresponds to (read from the left y axis,
hatched bars) in vivo N2ase activity (61 nmol of
C2H4 · min 1 · mg of
protein1), MoFe protein (29.1 µg/mg of cell protein),
unmodified Fe protein (3.1 µg/mg of cell protein), and (read from the
right y axis, cross-hatched bars) ADP-ribosylated Fe protein
(percentage of total Fe protein dimers present at the indicated time
points, where bar numbers correspond to lane numbers in panel B). Each
bar represents an average of results from at least three replicate
assays.
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The changes in the nitrogenase activities of these cultures appeared
not to be correlated with the ADP-ribosylation status
of the Fe
protein. These results were corroborated by examining
changes in
nitrogenase activity during growth on N
2 of
R. capsulatus draT draG mutants (W107I
and W107II) which are unable to modify
Fe protein. Changes in the
nitrogenase activities of cultures
of these strains were very similar
to changes observed with the
wild type (results not shown), with a very
low level of activity
occurring in the stationary phase of growth
(about 5% of the maximal
activity obtained in the mid-exponential
phase). Obviously, the
low level of final activity could not be
attributed to ADP-ribosylation
of the Fe protein and could only
partially be explained by a modest
decrease in the relative contents of
nitrogenase proteins (20
to 30%).
We also examined the nitrogenase activities and the ADP-ribosylation
states of the Fe protein of cultures growing on the relatively
poor
nitrogen source glutamate. Glutamate-grown cultures of
R. capsulatus demonstrated two to five times greater
nitrogenase
specific activity than N
2-fixing cells (Fig.
5). There was a significant
rate of
C
2H
2 reduction even in the stationary
phase of growth.
A broadly maximal nitrogenase activity was observed
after 20 to
30 h of cultivation, and the decline in nitrogenase
activity in
the stationary phase was significantly greater than was
warranted
by the modest decrease in levels of nitrogenase proteins.
Western
immunoblot analysis of Fe protein in these glutamate-grown
cultures
again demonstrated the presence, at various levels, of
ADP-ribosylated
Fe protein throughout the period of cultivation (Fig.
5B). Scanning
densitometry clearly showed that the proportion of this
form varied
over a twofold range, reaching a maximum shortly after
maximal
nitrogenase activity was reached, at which point it consisted
of 40% of the total Fe protein dimers (Fig.
5C, right
y
axis).
The presence of ADP-ribosylated Fe protein in actively growing
glutamate cultures can be rationalized in a number of ways. As
is
evident from the results presented here, regulation of transcription
and regulation of modification appear to respond differently to
the
intracellular nitrogen status. In addition, while regulation
of
transcription might control the overall amounts of nitrogenase
proteins
available at any given time, the modification system
may provide a
mechanism to fine-tune the amount of active nitrogenase.
Again, the
ADP-ribosylation state of Fe protein appeared to be
correlated with the
nitrogen status of the cells, since the analysis
of N
2- and
glutamate-grown cultures revealed the presence of increased
levels of
intracellular glutamine in these cells, with N
2-grown
cells
(greater Fe protein ADP-ribosylation) showing higher
levels
of glutamine (7.3 to 11.2 nmol/mg of cell protein)
than those
in glutamate-grown cells (3.5 to 6.1 nmol/mg of cell
protein).
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FIG. 5.
In vivo nitrogenase activity and the content of
nitrogenase proteins in R. capsulatus cells during
growth with glutamate as the nitrogen source. RCV medium containing 7 mM glutamate was inoculated as described in the legend to Fig. 4. At
the times indicated, aliquots were removed for the determination of
culture density (A660), whole-cell nitrogenase
activity, nitrogenase protein content, and Fe protein modification
state as described in Materials and Methods. (A) Growth
(A660) and specific in vivo nitrogenase activity
(N2ase). (B) Immunoblot analysis of nitrogenase proteins.
(C) Changes in the content of nitrogenase proteins (calculated from the
immunoblot in panel B) and in in vivo nitrogenase activity (from the
graph in panel A). One hundred percent corresponds to (read from the
left y axis, hatched bars) in vivo N2ase
activity (280 nmol C2H4 · min1 · mg of protein1), MoFe protein
(29.1 µg/mg of cell protein), unmodified Fe protein (14.9 µg/mg of
cell protein), and (read from the right y axis,
cross-hatched bars) ADP-ribosylated Fe protein (percentage of total Fe
protein dimers present at the indicated time points, where bar numbers
correspond to lane numbers in panel B). Each bar represents an average
of results from at least three replicate assays.
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Continuous (chemostat) cultivation under
NH4+ limitation.
In order to directly test
the role of the cellular nitrogen status in the regulation of
nitrogenase in R. capsulatus, we checked the effects of
different dilution rates on nitrogenase activity and the Fe protein
modification states of cells growing in
NH4+-limited chemostat cultures. Continuous
cultivation makes it possible to vary the degree of culture limitation
by nitrogen while keeping other parameters (carbon supply, light, pH,
etc.) at constant levels. In an NH4+-limited
chemostat, an increase in D reduces the degree of nitrogen limitation and, conversely, a reduction in D increases the
degree of nitrogen limitation. As presented in Fig.
6, R. capsulatus cells
growing in an NH4+-limited chemostat culture
expressed nitrogenase activity within a wide range of dilution rates
(0.01 to 0.21 h1), with a broad maximum at 0.04 to 0.08 h1. At higher rates of dilution nitrogenase activity was
decreased due to a gradual suppression of nitrogenase synthesis and
activity by an increased supply of nitrogen, and at lower rates of
dilution it was decreased because of the effect of severe nitrogen
limitation on cell metabolism and nitrogenase synthesis and activity. A
similar effect of dilution rate on the nitrogenase activity of
R. capsulatus cells grown in
NH4+-limited chemostat cultures has also been
observed by other investigators (9).
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FIG. 6.
In vivo nitrogenase activity and the content of
ADP-ribosylated Fe protein dimers in R. capsulatus
cells grown in an NH4+-limited continuous
(chemostat) culture at different flow rates (D). Inflowing
medium contained 1 mM (NH4)2SO4.
After the culture had reached steady state at a given flow rate,
aliquots were removed for the analysis of in vivo nitrogenase
(N2ase) activity and Fe protein modification state as
described in Materials and Methods.
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Immunoblot analysis demonstrated the absence of ADP-ribosylated Fe
protein at low rates of dilution (0.01 to 0.02 h
1; strong
nitrogen limitation) and the presence of increasing amounts
of this
form at higher rates of dilution (decreased nitrogen limitation)
(Fig.
6). The presence of ADP-ribosylated Fe protein at high rates
of
dilution is not due to nitrogenase switch-off by
NH
4+ being added with the fresh medium since
even at maximal rates
of dilution (0.21 h
1) the
steady-state concentration of NH
4+ in the
photobioreactor was less than 5 µM (as ascertained with
an
NH
4+ electrode), which is below the threshold
necessary for the nitrogenase
switch-off response. In the
D
range of 0.02 to 0.06 h
1 the increase in the content of
ADP-ribosylated Fe protein occurred
simultaneously with an
increase in nitrogenase activity. A possible
role of light intensity in
Fe protein modification in
R. capsulatus cells was
checked with an NH
4+-limited chemostat culture
(
D = 0.02 h
1) grown under decreasing
light intensities (from 226.5 to 2.7
W/m
2). The culture
became purely light limited at 6.6 W/m
2, as judged by our
inability to detect nitrogenase component proteins
(data not shown).
Since no ADP-ribosylated Fe protein was observed
in this experiment, it
appears that the cellular nitrogen status
(degree of nitrogen
limitation) was the main factor controlling
the modification state of
the Fe protein, which therefore supports
the conclusion reached from
the experiments with batch cultures
reported
here.
| |
DISCUSSION |
The short-term regulation of nitrogenase, termed nitrogenase
switch-off, in photosynthetic bacteria has been extensively
investigated over the past 2 decades. With R. capsulatus two different, presumably independent, control
mechanisms have been demonstrated. Like Rhodospirillum rubrum, R. capsulatus contains draT and
draG (23) and carries out the reversible
ADP-ribosylation of its Fe protein in response to the addition of
NH4+ or darkness (6). The enzymatic
system that carries out this process appears to be highly similar in
most respects to that of Rhodospirillum rubrum (6,
11). Nevertheless, much remains to be learned about the signal
transduction processes for both organisms. Moreover, in addition to
covalent modification, R. capsulatus has been shown to
carry out an ADP-ribosylation-independent regulation of
nitrogenase activity since strains carrying Fe protein mutant
alleles that are unable to be ADP-ribosylated (26) or strains with mutations in draT or draG
(34) are nonetheless capable of the switch-off of
nitrogenase activity. The basis of this response is obscure, although
it has been suggested that it results from the gating of electron flow
to nitrogenase (26, 34).
It has been widely assumed that the modification of Fe
protein observed with N2- or glutamate-grown cultures of
photosynthetic bacteria is due to the darkness associated with cell
concentration by centrifugation (12, 21), and the present
model of nitrogenase regulation in photosynthetic bacteria assumes
that only the addition or removal of external
NH4+ or darkness can induce modification or
demodification of Fe protein (21, 22). However, here we have
shown that the system that ADP-ribosylates nitrogenase in the short
term in response to abrupt changes in the environment is also capable
of modifying nitrogenase in accordance with long-term cellular
conditions. We used a fast and sensitive immunochemical method for
the analysis of the modification state of the Fe protein in
R. capsulatus cells which avoids sample concentration
by centrifugation. Our results clearly demonstrate that a significant
proportion (30 to 90%) of nitrogenase Fe protein is present, in vivo,
as the ADP-ribosylated form in R. capsulatus cells
grown with different nitrogen sources. ADP-ribosylated Fe protein was
found in cells in both exponential (N2, glutamate) and
stationary (NH4+-limited) growth phases.
Analysis of the glutamine contents of the various culture types
indicated that the modification state of Fe protein is directly
proportional to the level of intracellular glutamine and therefore is
presumably a function of the cellular nitrogen status. Here we used
glutamine levels as an indicator of cellular nitrogen status
(8). The role for glutamine as a controller of the Fe
protein ADP-ribosylation process is presently unclear. Previously it
has been suggested, on the basis of short-term incubation experiments
using another photosynthetic bacterium, Rhodospirillum
rubrum, that the intracellular glutamine pool may serve as
the signal for nitrogenase modification (17). However, other researchers have found that the glutamine concentration varied independently of Fe protein ADP-ribosylation (13).
That the modification status of the Fe protein is correlated with
cellular nitrogen status was directly demonstrated here with chemostat cultures of R. capsulatus where ADP-ribosylation was
shown to be inversely proportional to the severity of nitrogen limitation.
In this study, under some conditions substantial nitrogenase activity
was observed at the same time that a significant proportion of Fe
protein was found to be ADP-ribosylated. In fact, all experimental conditions tested showed wide variations in levels of nitrogenase activity with small or no changes in the levels of both nitrogenase proteins (determined by immunoblotting). While some of this disparity might be due to differing ADP-ribosylation states of the Fe protein in
cultures of the wild-type strain, this is clearly not the case with
draT G mutant strains, and the contents of
unmodified Fe protein dimers in both the wild type and the
draT G mutant strains did not correspond to the
level of nitrogenase activity. These results strongly suggest that in
vivo nitrogenase activity is not limited by the concentration of
catalytically active nitrogenase proteins. This conclusion was
corroborated by determining the in vitro nitrogenase activities of
extracts of cells grown under conditions where there was a wide
variation in in vivo activity. For example, N2-grown
cultures showed a 100-fold variation in in vivo activity but only a
20% decline in in vitro activity (results not shown). A similar,
although less drastic variation has been reported for Azotobacter
vinelandii, which is incapable of Fe protein ADP-ribosylation,
with a twofold increase in nitrogenase activity without an
increase in the content of nitrogenase proteins (14). These
observations suggest that at least under some conditions whole-cell
nitrogenase activity may be determined mainly by electron flow to
nitrogenase and/or ATP supply and not be the levels of nitrogenase
proteins or the proportion of unmodified Fe protein. This
regulation may be due to the ADP-ribosylation-independent response
previously noted in studies of the short-term regulation of nitrogenase.
Thus, both regulatory systems may be operative under conditions where
no changes in the external milieu are taking place. These two levels of
control may thus mirror elements previously shown to be involved in the
short-term switch-off of nitrogenase. Both systems may be necessary to
fine-tune the nitrogenase system in response to internal cellular
conditions. Thus, nitrogenase in R. capsulatus is
subject to both a short-term regulation of activity in response to
sudden environmental changes and a long-term adaptation of the activity
of preformed enzyme to specific growth conditions.
| |
ACKNOWLEDGMENTS |
This research was carried out within the framework of the
international project Regulation of Nitrogen Fixation in Photosynthetic Microorganisms and was supported by grants from the Natural Sciences and Engineering Research Council of Canada (OGP0036584) and the Russian
Foundation for Basic Research.
W. Klipp and G. Roberts are thanked for their generous supply of strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Département de Microbiologie et Immunologie, Université de
Montréal, C.P. 6128, Succursale Centre-ville, Montréal,
Québec H3C 3J7, Canada. Phone: (514) 343-6278. Fax: (514)
343-5701. E-mail: patrick.hallenbeck{at}umontreal.ca.
| |
REFERENCES |
| 1.
|
Alef, K.,
D. J. Arp, and W. G. Zumft.
1981.
Nitrogenase switch-off by ammonia in Rhodopseudomonas palustris: loss under nitrogen deficiency and independence from the adenylylation state of glutamine synthetase.
Arch. Microbiol.
130:138-142.
|
| 2.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 3.
|
Carithers, R. P.,
D. C. Yoch, and D. I. Arnon.
1979.
Two forms of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.
J. Bacteriol.
137:779-789[Abstract/Free Full Text].
|
| 4.
|
Fitzmaurice, W. P.,
L. L. Saari,
R. G. Lowery,
P. W. Ludden, and G. P. Roberts.
1989.
Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum.
Mol. Gen. Genet.
218:340-347[Medline].
|
| 5.
|
Garvey, J. S.,
N. W. Cremer, and D. H. Sussdorf.
1977.
Methods in immunology, p. 1-38.
W. A. Benjamin, Inc., Reading, Mass.
|
| 6.
|
Hallenbeck, P. C.
1992.
Mutations affecting nitrogenase switch-off in Rhodobacter capsulatus.
Biochim. Biophys. Acta
1118:161-168[Medline].
|
| 7.
|
Hallenbeck, P. C.,
C. M. Meyer, and P. M. Vignais.
1982.
Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties.
J. Bacteriol.
149:708-717[Abstract/Free Full Text].
|
| 8.
|
Ikeda, T. P.,
A. E. Shanger, and S. Kustu.
1996.
Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation.
J. Mol. Biol.
259:589-607[Medline].
|
| 9.
|
Jouanneau, Y.,
S. Lebecque, and P. M. Vignais.
1984.
Ammonia and light effect on nitrogenase activity in nitrogen-limited continuous cultures of Rhodopseudomonas capsulata. Role of glutamine synthetase.
Arch. Microbiol.
139:326-331.
|
| 10.
|
Jouanneau, Y.,
C. M. Meyer, and P. M. Vignais.
1983.
Regulation of nitrogenase activity through iron protein interconversion into an active and inactive form in Rhodopseudomonas capsulata.
Biochim. Biophys. Acta
749:318-328.
|
| 11.
|
Jouanneau, Y.,
C. Roby,
C. M. Meyer, and P. M. Vignais.
1989.
ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus.
Biochemistry
28:6524-6530.
|
| 12.
|
Kanemoto, R. H., and P. W. Ludden.
1984.
Effect of ammonia, darkness, and phenazine methosulfate on whole-cell nitrogenase activity and Fe-protein modification in Rhodospirillum rubrum.
J. Bacteriol.
158:713-720[Abstract/Free Full Text].
|
| 13.
|
Kanemoto, R. H., and P. W. Ludden.
1987.
Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity.
J. Bacteriol.
169:3035-3043[Abstract/Free Full Text].
|
| 14.
|
Klugkist, J.,
H. Haaker,
H. Wassink, and C. Veeger.
1985.
The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells.
Eur. J. Biochem.
146:509-515[Medline].
|
| 15.
|
Kranz, R. G., and P. J. Cullen.
1995.
Regulation of nitrogen fixation genes, p. 1191-1208.
In
R. E. Blankenship, et al. (ed.), Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
|
| 16.
|
Laemmli, U. K., and M. Favre.
1973.
Maturation of the head of bacteriophage T4. I. DNA packaging events.
J. Mol. Biol.
80:575-599[Medline].
|
| 17.
|
Li, J.,
C.-Z. Hu, and D. C. Yoch.
1987.
Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness.
J. Bacteriol.
169:231-237[Abstract/Free Full Text].
|
| 18.
|
Liang, J.,
G. M. Nielsen,
D. P. Lies,
R. H. Burris,
G. P. Roberts, and P. W. Ludden.
1991.
Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation.
J. Bacteriol.
173:6903-6909[Abstract/Free Full Text].
|
| 19.
|
Lowery, R. G., and P. W. Ludden.
1988.
Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum.
J. Biol. Chem.
263:16714-16719[Abstract/Free Full Text].
|
| 20.
|
Lowery, R. G.,
L. L. Saari, and P. W. Ludden.
1986.
Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro.
J. Bacteriol.
166:513-518[Abstract/Free Full Text].
|
| 21.
|
Ludden, P. W., and G. P. Roberts.
1989.
Regulation of nitrogenase activity by reversible ADP-ribosylation.
Curr. Top. Cell. Regul.
30:23-56[Medline].
|
| 22.
|
Ludden, P. W., and G. P. Roberts.
1995.
The biochemistry and genetics of nitrogen fixation by photosynthetic bacteria, p. 929-947.
In
R. E. Blankenship, M. T. Madigan, and C. E. Bauer (ed.), Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
|
| 23.
|
Masepohl, B.,
R. Krey, and W. Klipp.
1993.
The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase.
J. Gen. Microbiol.
139:2667-2675[Abstract/Free Full Text].
|
| 24.
|
Neilson, A. H., and S. Nordlund.
1975.
Regulation of nitrogenase in intact cells of Rhodospirillum rubrum: inactivation of nitrogen fixation by ammonia, L-glutamine and L-asparagine.
J. Gen. Microbiol.
91:53-62[Abstract/Free Full Text].
|
| 25.
|
Ormerod, J. G.,
K. S. Ormerod, and H. Gest.
1961.
Light-dependent utilization of organic compounds and photo-production of hydrogen by photosynthetic bacteria: relationships with nitrogen metabolism.
Arch. Biochem. Biophys.
94:449-463.
|
| 26.
|
Pierrard, J.,
P. W. Ludden, and G. P. Roberts.
1993.
Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium.
J. Bacteriol.
175:1358-1366[Abstract/Free Full Text].
|
| 27.
|
Pierrard, J.,
J. C. Willison,
P. M. Vignais,
J. L. Gaspar,
P. W. Ludden, and G. P. Roberts.
1993.
Site-directed mutagenesis of the target arginine for ADP-ribosylation of nitrogenase component II in Rhodobacter capsulatus.
Biochem. Biophys. Res. Commun.
192:1223-1229[Medline].
|
| 28.
|
Saari, L. L.,
E. Triplett, and P. W. Ludden.
1984.
Purification and properties of the activating enzyme for iron protein of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.
J. Biol. Chem.
259:15502-15508[Abstract/Free Full Text].
|
| 29.
|
Solorzano, L.
1969.
Determination of ammonia in natural sea water by the phenolhypochlorite method.
Limnol. Oceanogr.
14:799-801.
|
| 30.
|
Tsygankov, A. A.,
T. V. Laurinavichene, and I. N. Gogotov.
1994.
Laboratory scale photobioreactor.
Biotechnol. Tech.
8:575-578.
|
| 31.
|
Tsygankov, A. A.,
T. V. Laurinavichene,
I. N. Gogotov,
Y. Asada, and J. Miyake.
1996.
Switching from light limitation to ammonium limitation in chemostat cultures of Rhodobacter capsulatus grown in different types of photobioreactor.
J. Mar. Biotechnol.
4:43-46.
|
| 32.
|
Weaver, P. F.,
J. D. Wall, and H. Gest.
1975.
Characterization of Rhodopseudomonas capsulata.
Arch. Microbiol.
105:207-216[Medline].
|
| 33.
|
Yakunin, A. F., and P. C. Hallenbeck.
1998.
A luminol/iodophenol chemiluminescent detection system for Western immunoblots.
Anal. Biochem.
258:146-149[Medline].
|
| 34.
|
Yakunin, A. F., and P. C. Hallenbeck.
1998.
Short-term nitrogenase regulation in Rhodobacter capsulatus: multiple in vivo nitrogenase responses to NH4+ addition.
J. Bacteriol.
180:6392-6395[Abstract/Free Full Text].
|
| 35.
|
Yen, H.-C., and B. Marrs.
1976.
Map of genes of carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata.
J. Bacteriol.
126:619-629[Abstract/Free Full Text].
|
| 36.
|
Yoch, D. C.
1980.
Regulation of nitrogenase A and R concentrations in Rhodopseudomonas capsulata by glutamine synthetase.
Biochem. J.
187:273-276[Medline].
|
| 37.
|
Yoch, D. C., and M. Cantu.
1980.
Changes in the regulatory form of Rhodospirillum rubrum nitrogenase as influenced by nutritional and environmental factors.
J. Bacteriol.
142:899-907[Abstract/Free Full Text].
|
| 38.
|
Zhang, Y.,
R. H. Burris,
P. W. Ludden, and G. P. Roberts.
1993.
Posttranslational regulation of nitrogenase activity by anaerobiosis and ammonium in Azospirillum brasilense.
J. Bacteriol.
175:6781-6788[Abstract/Free Full Text].
|
| 39.
|
Zumft, W. G.,
H. Körner,
D. J. Arp,
W. Klipp, and A. Pühler.
1985.
Nitrogen fixation in the anoxygenic phototrophic bacteria, p. 411-424.
In
Y. A. Ovchinnikov (ed.), Proceedings of the 16th FEBS Congress, part A. VNU Science Press, Utrecht, The Netherlands.
|
Journal of Bacteriology, April 1999, p. 1994-2000, Vol. 181, No. 7
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Top
Abstract
Introduction
