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Journal of Bacteriology, March 1999, p. 1698-1702, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Correlation of Activity Regulation and Substrate
Recognition of the ADP-Ribosyltransferase That Regulates Nitrogenase
Activity in Rhodospirillum rubrum
Kitai
Kim,1,2
Yaoping
Zhang,1,2,3 and
Gary P.
Roberts1,2,*
Departments of
Bacteriology1 and
Biochemistry3 and The Center for
the Study of Nitrogen Fixation,2 University
of Wisconsin
Madison, Madison, Wisconsin 53706
Received 23 February 1998/Accepted 20 December 1998
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ABSTRACT |
In Rhodospirillum rubrum, nitrogenase activity is
regulated posttranslationally through the ADP-ribosylation of
dinitrogenase reductase by dinitrogenase reductase
ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered
both in the posttranslational regulation of DRAT activity and in the
ability to recognize variants of dinitrogenase reductase have been
found. This correlation suggests that these two properties are
biochemically connected.
| |
TEXT |
The posttranslational
ADP-ribosylation of proteins is the result of the enzymatic transfer of
ADP-ribose from NAD, typically resulting in alteration of the
functional properties of the respective proteins. The interest in the
field has concentrated on two types of phenomena: poly-ADP-ribosylation
of nuclear proteins in eukaryotes in the process of DNA excision repair
(26, 27) and mono-ADP-ribosylation by bacterial enzymes as
the mechanism of diphtheria, cholera, and pertussis toxins in
eukaryotic host cells (3, 20). However, the reversible
regulation of metabolic functions by ADP-ribosylation in animal tissues
(8, 19, 31) and bacterial cells (16, 17, 25) is
also physiologically important, and the regulation of the dinitrogenase
reductase from the nitrogen fixation system in photosynthetic bacteria
provides an attractive model system. In Rhodospirillum
rubrum, the posttranslational regulation of the nitrogenase by the
ADP-ribosylation has been well characterized. Nitrogenase is a protein
complex of two components: dinitrogenase (an
2
2 tetramer of the nifD and
nifK gene products) contains the active site of dinitrogen
reduction, and dinitrogenase reductase (an 2 dimer of
the nifH gene product) transfers electrons to dinitrogenase.
The posttranslational regulation of the complex involves reversible
mono-ADP-ribosylation of dinitrogenase reductase at R101
(22).
Two enzymes have been found to perform this reversible regulation in
R. rubrum. Under certain conditions, dinitrogenase reductase ADP-ribosyltransferase (DRAT, the gene product of draT)
transfers an ADP-ribosyl group from NAD to one subunit of the
dinitrogenase reductase dimer, and the ADP-ribosylated dinitrogenase
reductase is no longer competent to transfer electrons to dinitrogenase (13, 14). The ADP-ribosyl group on the inactivated
dinitrogenase reductase can be removed by the dinitrogenase
reductase-activating glycohydrolase (DRAG, the gene product of
draG), thus recovering dinitrogenase reductase activity
(5, 15-17, 24).
Presumably to avoid the possibility of futile cycling of
ADP-ribosylation, the activities of both DRAT and DRAG are themselves regulated posttranslationally (12, 29). Under conditions
appropriate for nitrogen fixation (energy sufficiency and a deficiency
in fixed nitrogen), DRAG is active, causing dinitrogenase reductase to
be in the active form. However, following an environmental shift that
makes nitrogenase activity undesirable, such as the depletion of the
energy or introduction of a good source of fixed nitrogen, DRAG loses
its activity and DRAT becomes active. This results in the
ADP-ribosylation of a fraction of the dinitrogenase reductase.
Surprisingly, it has been found that DRAT activation is only transient,
so that some time after the environmental shift, neither DRAT nor DRAG
is active (28). This typically results in a plateau of
nitrogenase activity, reflecting the amount of dinitrogenase reductase
that was not ADP-ribosylated by DRAT during its active stage. When
conditions change again to those favorable to nitrogenase activity,
DRAG recovers its activity and restores nitrogenase activity by
removing the ADP-ribosyl group from dinitrogenase reductase. The
central issue in understanding this regulatory system has therefore
become the nature of the posttranslational regulation of DRAT and DRAG.
Isolation and characterization of mutants with altered
dinitrogenase reductase unable to be regulated by DRAT.
To better
understand the behavior of this posttranslational regulatory system, a
screen was developed for mutants whose nitrogenase activity was no
longer regulated by DRAT. A strain (UR484) that lacks DRAG but has
excess DRAT was created; the chromosomal copy of draG was
mutated, and multiple copies of draT were provided on a
replicating plasmid (Table 1). This
strain displayed low nitrogenase activity under all conditions (see
Table 2), which resulted in poor growth on nitrogen-free medium and
allowed us to seek mutants that grew better because they possessed
higher nitrogenase activity. UR484 (excess DRAT) was then mutagenized with
N-methyl-N'-nitro-N-nitrosoguanidine
(NTG) by a modification of a published protocol (1). Thirty
independent mutagenized samples were inoculated into 10 ml of
MN
(nitrogen-free) medium (culture media are described in
references 4, 10, and 11,
cultured for 10 days under a regimen of 30-min dark-90-min light
cycles to increase the population of the desired mutants, then diluted
and plated on MN medium and screened under the same
light-dark regimen. After 7 days, 100 fast-growing candidates were
chosen, individually inoculated in liquid medium, and analyzed for
nitrogenase activity under different conditions.
Three independent mutants (UR592, 594, and 595) were chosen for direct
examination of their ADP-ribosylation based on the
criteria that they
displayed relatively high nitrogenase activity
under nitrogen-fixing
conditions and little or no decrease in
that activity in response to
darkness or ammonium (Table
2).
In vitro
DRAT activity assays (
14) were performed on extracts
of the
fast-growing mutants and controls, with the mutants showing
93 to 104%
of the DRAT activity seen in the parent strain, UR484
(data not shown).
The
nifH regions from all the mutants were each
PCR
amplified and sequenced, and the results showed that all three
mutants
possessed the identical mutation in
nifH, resulting in
an
E112K substitution. This mutation was moved into an otherwise
wild-type
background and caused the same phenotype, indicating
that this mutation
was indeed causative of the phenotype of good
growth in the presence of
elevated levels of DRAT. The mutation
was given the allele number
nifH1073, and the protein product
is referred below as
NifH-E112K.
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TABLE 2.
Behavior of derivatives of UR484 chosen for high
nitrogenase activity in the presence of high levels of DRAT
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Based on the structure of dinitrogenase reductase,
Azotobacter
vinelandii (
21) E112 lies on the same face of
dinitrogenase
reductase as the R101 residue that is ADP-ribosylated by
DRAT,
so it is not surprising that an alteration at this site might
perturb DRAT interaction with dinitrogenase
reductase.
Isolation of DRAT variants that are capable of regulating
NifH-E112K.
To explore the nature of interaction between DRAT and
dinitrogenase reductase, mutants with altered DRAT that would be
capable of regulating the activity of NifH-E112K were sought. A library of plasmids carrying a mutagenized draT (with random PCR
mutagenesis) was introduced into strain UR662 (NifH-E112K and no DRAG),
creating strain UR663. Mutagenesis of the
BamHI-EcoRI fragment of draT was
performed by a PCR procedure (30), and the resulting strains were screened for poor growth on nitrogen-free medium. Approximately 5,000 colonies were screened, and 100 slowly growing colonies were
picked and retested in liquid medium. The nitrogenase activities in
UR663 and in two of the slowly growing mutants are shown in Table
3. UR663 has a fairly high nitrogenase
activity under nitrogen-fixing conditions and no loss of activity after
a shift to the dark. The mutants (UR666 and 668) showed rather lower
nitrogenase activity initially, which is presumably the basis for the
poor growth, and a further loss of activity upon a shift to the dark,
consistent with the ability of the altered DRAT in these strains to
modify NifH-E112K.
The
draT regions from the plasmids in these mutants were
sequenced and, where necessary, reconstructed. Plasmids pKT114 and
pKT115 (strain UR666) had identical causative mutations, creating
a
K103E substitution, while plasmid pKT143 (UR668) had a causative
mutation creating a Q81R
substitution.
Altered substrate recognition in DRAT correlates with altered
posttranslational regulation of DRAT activity.
Concurrent with
these experiments, a related project involved the screening of strains
with PCR-mutagenized draT-containing plasmids for any
conferring constitutive DRAT activity. This procedure had identified
two substitutions, DRAT-K103E and DRAT-N248D, that caused significant
ADP-ribosylation of wild-type dinitrogenase reductase under
nitrogen-fixing conditions. A mutant with DRAT-K103E has just been
described in the preceding section as having the capability of
regulating NifH-E112K, so the isolation of the DRAT-K103E substitution
from this very different screen was striking, and we wondered if the
two properties of "constitutive DRAT activity" with the wild-type
dinitrogenase reductase and ability to regulate the NifH-E112K were
actually related. DRAT-N248D was therefore examined for altered
substrate recognition, and DRAT-Q81R was tested for constitutive DRAT activity.
The data in Table
3 show that DRAT-N248D was generally similar to
DRAT-K103E and DRAT-Q81R in its ability to regulate NifH-E112K,
indicating that the N248D substitution also confers altered substrate
recognition on DRAT. The results from the activity analysis of
DRAT-Q81R are shown in Fig.
1. Based on
both detectable ADP-ribosylation
(Fig.
1A) and activity (Fig.
1B),
DRAT-Q81R possesses constitutive
DRAT activity, albeit at a lower level
than that seen with DRAT-K103E.
Because independent mutants, isolated
for different phenotypes,
show both altered substrate recognition and
altered regulation,
it seems likely that there is a common biochemical
interaction
shared by these processes, as rationalized at the end of
this
report.
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FIG. 1.
Detection of constitutive DRAT activity in strains with
DRAT-Q81R and DRAT-K103E by ADP-ribosylation of dinitrogenase reductase
and by effects on nitrogenase activity. (A) Immunoblot of dinitrogenase
reductase on one-dimensional SDS PAGE of UR2 (wild-type DRAT and DRAG)
(lanes 1 and 2), UR462 (with elevated levels of wild-type DRAT and low
levels of normal DRAG) (lane 3), UR508 (with elevated levels of
DRAT-K103E) (lane 4), and UR670 (with DRAT-Q81R) (lane 5). Samples were
precipitated with trichloroacetic acid (28), analyzed on
low-cross-linker SDS-PAGE (ratio of acrylamide to bisacrylamide, 172:1)
(10), immunoblotted (9), and detected by an ECL
detection reagent (Amersham). (B) In vivo nitrogenase activity assay of
UR2, UR462, UR508, and UR670. a, WT stands for wild-type
nifH or draT, as appropriate.
b, 1× refers to normal levels of DRAT due to a
single gene copy; 10× refers to a level approximately 10-fold higher
due to the plasmid location of draT. c, 1×
refers to normal levels of DRAG; 0.03× refers to the greatly reduced
levels of DRAG resulting from a polar insertion upstream of
draG in draT. d, nitrogenase activity
(in nanomoles of ethylene per milliliter times hours) was determined as
in reference 2 and was normalized to an optical density at 600 nm
(OD600) of 1. The variability of the nitrogenase activity
was about 10%. The data were represented by at least three individual
runs.
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What is the basis for the observed regulation of nitrogenase
activity in strains with NifH-E112K and the constitutively active DRAT
variants?
The correlation between altered substrate recognition
and altered regulation was interesting, but it did not resolve the
paradox that we were detecting regulation of the activity of NifH-E112K by the constitutive DRAT variants, but without the detection of an
ADP-ribosylated dinitrogenase reductase on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To test the
possibility that the constitutively active DRAT was actually modifying
some other protein in the nitrogenase system (an electron carrier to
dinitrogenase reductase, for example), the constitutively active DRAT
variants were examined in vivo with NifH-R101Y; this substitution
eliminates the site of ADP-ribosylation on dinitrogenase reductase and
therefore also eliminates DRAT-DRAG regulation but allows low
nitrogenase activity (18). The constitutive DRAT variants
had no effect on the nitrogenase activity in this background (data not
shown), showing that their ability to regulate NifH-E112K involved a
specific recognition of this form of dinitrogenase reductase.
The results shown in Fig.
2 resolve the
paradox by demonstrating that different DRAT variants ADP-ribosylate
NifH-E112K but
that the ADP-ribosylated form of NifH-E112K fails to
show a detectable
shift in the SDS dimension. Figure
2A shows the
behavior of the
wild type, with a loss of nitrogenase activity and the
appearance
of an ADP-ribosylated spot (the "new" spot higher and to
the right)
upon a shift of the culture to darkness. Strain UR666
(NifH-E112K
and excess DRAT-K103E) shows several differences (Fig.
2B)
from
the wild type. Dinitrogenase reductase runs as two spots
(ADP-ribosylated
and nonribosylated) that are both shifted two charge
positions
to the left (basic side) because of the E112K substitution.
There
is ADP-ribosylated dinitrogenase reductase under all conditions
due to the high level of DRAT and the absence of DRAG, but there
is a
significant loss of nitrogenase activity upon a shift to
darkness. The
shift in the proportions of unmodified and ADP-ribosylated
spots is
difficult to detect, as the dinitrogenase reductase is
already
substantially modified, as indicated by the nitrogenase
activity assay.
To make a more compelling case that DRAT-K103E
actually becomes active
after a shift to darkness, we created
a new strain, UR673, with
NifH-E112K, normal levels of DRAT-K103E,
and no active DRAG. This
strain shows a very clear increase in
the proportion of dinitrogenase
reductase after a shift to darkness
and a substantial drop in detected
nitrogenase activity in vivo
(Fig.
2C). In contrast to the strain in
Fig.
2B, this strain shows
little modified subunit in the light,
consistent with low DRAT
activity under these conditions. Taken
together, the results in
Fig.
2B and C suggest that DRAT-K103E has a
very low activity
under nitrogen-fixing conditions when NifH-E112K is
the substrate
but is activated after a shift to the dark. This is in
contrast
to the behavior of DRAT-K103E with the wild-type dinitrogenase
reductase as a substrate (Fig.
1), where it has a substantial
level of
constitutive activity.
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FIG. 2.
Regulation of nitrogenase activity and two-dimensional
(2D) analysis of dinitrogenase reductase modification status. A
nitrogenase activity assay and immunoblotting of two-dimensional PAGE
results were performed as described in the text. "1×" refers to
normal levels of DRAT or DRAG due to a single gene copy on the
chromosome; "11×" refers to the approximately 11-fold-higher DRAT
level due to both plasmid and chromosomal copies of draT;
the dash refers to the absence of DRAT or DRAG activity. Nitrogenase
activity (in nanomoles of ethylene per milliliter times hours) was
normalized to an OD600 of 1. The variability of the
nitrogenase activity was about 10%. The data were represented by at
least three individual runs. Two-dimensional PAGE (23)
results were immunoblotted (9), and the protein spots on the
film were quantitated by scanning with a densitometer. Arrows indicate
the positions of wild-type unmodified dinitrogenase reductase. The left
side of each panel is the basic side of the gel, while the right side
is the acidic side. Light, before darkness treatment; Darkness, after
60 min of darkness; WT, wild-type nifH or draT,
as appropriate.
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Strain UR663 (NifH-E112K, elevated levels of wild-type DRAT, but no
DRAG) displays a significant amount of ADP-ribosylation
under both
growth conditions (Fig.
2D) but no apparent regulation
in response to
darkness. In designing our selections, we had assumed,
based on the
absence of an upper band on SDS, that NifH-E112K
could not be modified
by wild-type DRAT, but this is clearly incorrect.
However, most of the
dinitrogenase reductase is active in this
strain, providing high
nitrogenase activity, and that was the
basis for the selective
conditions that we used. A comparison
of the nitrogenase activities in
Fig.
2B and D shows that DRAT-K103E,
but not wild-type DRAT,
ADP-ribosylates NifH-E112K after a shift
to darkness. When NifH-E112K
was examined with normal levels of
wild-type DRAT, little
ADP-ribosylation and no activity regulation
was seen (compare Fig.
2E
to
2C).
As a final demonstration that the identification of the left and right
spots in Fig.
2 as unmodified and ADP-ribosylated dinitrogenase
reductase was correct, strain UR671 (NifH-E112K, but without DRAT
or
DRAG) was analyzed (Fig.
2F). Neither regulation of nitrogenase
activity nor the right-hand (acidic) spot was
detected.
These results suggest that while wild-type DRAT can modify NifH-E112K,
it does so poorly under all conditions and DRAT activity
is not
stimulated upon a shift to darkness as it is in the presence
of the
wild-type dinitrogenase reductase. It is probably not correct
to
suggest that wild-type DRAT is "constitutively active" with
NifH-E112K, as we see a rather similar low-level modification
of
wild-type dinitrogenase reductase under similar conditions
when there
is no active DRAG. As noted previously, there is always
a low level of
ADP-ribosylated dinitrogenase reductase detectable
in strains that lack
any active DRAG, as the posttranslational
regulation of DRAT activity
is not complete (
12).
Conclusions.
The results of the present work demonstrate that
both protein partners in this complex are important for proper
regulation and that the "inappropriate" complexes alter the
regulation of DRAT activity. A summary of the regulation of the various
combinations of DRAT and dinitrogenase reductase is shown in Table
4.
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TABLE 4.
Summary of the effects of various combinations of DRAT
and dinitrogenase reductase on the regulation of DRAT activity
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Two results are particularly important for the model of a
DRAT-dinitrogenase complex as the target for regulation. First,
the
nature of the substrate protein can clearly alter the regulation
of
DRAT activity. This is clear from the constitutive activity
of
DRAT-K103E in the presence of wild-type dinitrogenase reductase
and
from the failure of wild-type DRAT to become active in the
presence of
NifH-E112K. Second, the fact that DRAT variants capable
of regulating
NifH-E112K (DRAT-K103E, -N248D, and -Q81R) are uniformly
perturbed in
the regulation of their activity in the presence
of wild-type
dinitrogenase reductase demonstrates a striking correlation
between
substrate recognition and DRAT regulation. These results
are
reminiscent of the observation that the wild-type DRAT of
R. rubrum has different requirements for optimal in vitro activity
when a dinitrogenase reductase from another organism is used as
a
substrate, although the physiological significance of that observation
had been unclear (
13).
Taken together, these results strongly suggest that the
posttranslational regulation of DRAT activity is a function of DRAT
and
its substrate. Cross-linking analysis has demonstrated that
DRAT forms
a complex with dinitrogenase reductase, even in the
absence of
ADP-ribosylation (
7). This result has been interpreted
to
mean that the DRAT-dinitrogenase reductase complex might be
the
predominant form of DRAT in the cell and represents the form
that is
actually subject to posttranslational regulation (
7).
It is
our working hypothesis that only this complex is competent
to receive
some presently unknown signal that modulates DRAT activity.
Such a
hypothesis has been presented previously (
6,
29),
based on
the observation that overexpressed DRAT decreased nitrogenase
activity
in vivo without ADP-ribosylation, which suggested that
a complex
between the two proteins existed even under conditions
where
modification was not occurring. We are continuing to test
this
hypothesis through the biochemical analysis of the DRAT variants
described in this
report.
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ACKNOWLEDGMENTS |
This work was supported by the College of Agricultural and Life
Sciences at the University of WisconsinMadison and by USDA grant
96-35305-3696 and Hatch project 4024 to G.P.R.
We thank Paul Ludden and members of his laboratory for suggestions and assistance.
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FOOTNOTES |
*
Corresponding author. Mailing address: University of
WisconsinMadison, Department of Bacteriology, 106A E. B. Fred
Hall, 1550 Linden Dr., Madison, WI 53706-1567. Phone: (608) 262-3567. Fax: (608) 262-9865. E-mail: groberts{at}bact.wisc.edu.
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Journal of Bacteriology, March 1999, p. 1698-1702, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
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Zhang, Y., Kim, K., Ludden, P. W., Roberts, G. P.
(2001). Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum. Microbiology
147: 193-202
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