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Plant and Soil 137: 127-134, 1991.
© 1991 Kluwer Academic Publishers. Printed in the Netherlands.
Control of nitrogenase in
PLSO NL19
Azospirillum sp.
R O B E R T H. B U R R I S , A N T O N H A R T M A N N , Y A O P I N G Z H A N G and H A I A N FU
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA
Key words:
ammonium, Azospirillum sp., dinitrogenase reductase, 'switch off'
Abstract
Many N2-fixing organisms can turn off nitrogenase activity in the presence of N H ] and turn it on again
when the N H 4 is exhausted. One of the most interesting systems for accomplishing this is by covalent
modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase ( D R A T ) . The system can be reactivated when NH~- is exhausted, by dinitrogenase reductase
activating glycohydrolase ( D R A G ) which removes the inactivating group. It is fascinating that some
species of the genus Azospirillum possess the D R A T and D R A G systems (A. lip@rum and A.
brasilense), whereas A. amazonense in the same genus lacks D R A T and D R A G . A. amazonense
responds to N H 4 but does not exhibit modification of dinitrogenase reductase characteristic of the
action of D R A T . However, it has been possible to clone D R A T and D R A G and to introduce them into
A. amazonense, whereupon they become functional in this organism. The D R A T and D R A G system
does not appear to function in Acetobacter diazotrophicus, an organism isolated from sugar cane, that
fixes N 2 at a p H as low as 3.0. A. diazotrophicus does show a rather sluggish response to N H 4 . A level
of about 1 0 / z M N H 4 is required to 'switch off' the system. The response to N H 4 is influenced by the
dissolved oxygen concentration ( D O C ) as has been reported for Azospirillum sp. A D O C in
equilibrium with 0.1 to 0.2 kPa 0 2 seems optimal for the response in A. diazotrophicus.
Introduction
Nitrogen fixation is very energy demanding.
Chemical fixation via the Haber-Bosch process
achieves breakage of the N - N bond and reduction of N 2 to 2 N H 3 by employing high temperature and high pressure in the presence of a
catalyst. Biological fixation of N 2 faces the same
energy barriers but operates in a more subtle
fashion at ambient temperature and a pN 2 of
0.78 atm. by invoking the combined action of
dinitrogenase and dinitrogenase reductase. The
action of these enzymes is supplemented by
ferredoxin or flavodoxin as a reductant and
M g A T P as an energy source. When one provides
optimal conditions for nitrogenase activity it still
requires 16 M g A T P for the reduction of 8 H ÷ +
N 2 -+ 2 N H 3 + H 2. Under natural conditions, 20
to 30 M g A T P are used per N 2 reduced. Faced
with this energy demand, there have been evolutionary pressures for organisms possessing nitrogenase to develop a means to turn off nitrogenase when fixed nitrogen is available to supply the
organism's needs. These control systems vary
among N2-fixing organisms. Our interest has centered on the system that invokes covalent modification of one unit of the dimer of dinitrogenase
reductase.
When N H 4 enriched in 15N became available,
the 15N furnished a means for quickly establishing the initiation and cessation of N 2 fixation and
the influence of fixed nitrogen on the process of
biological N a fixation. When Azotobacter vinelandii was given the choice of 15N2 and N H 4 or
other nitrogenous compounds, Wilson et al.
(1943) found that the organisms showed a clear
preference for N H 4 over nitrate, nitrite and a
variety of amino acids. When N H 4 at 121 or
128
Burris et al.
315 ppm N was present, only 0.9% or 0.0%,
respectively, of the cell nitrogen was derived
from the I5N2. Burris and Wilson (1946) expanded on these observations and followed the time
course of utilization of NH 4 and nitrate by A.
vinelandii. Although switch-off of N 2 fixation of
A. vinelandii by NH 4 was demonstrated may
years ago, the detailed mechanism of switch-off
in A. vinelandii has not been elucidated.
In contrast to A. vinelandii, the control in
Rhodospirillum rubrum has been worked out in
considerable detail. Kamen and Gest (1949) and
Gest and Kamen (1949) demonstrated that
Rhodospirillum rubrum is capable of fixing N 2.
Although Schneider et al. (1960) reported that
they had prepared cell-free extracts from R.
rubrum that fixed N2, there was difficulty in
getting consistent N 2 fixation with extracts from
this organism. Munson and Burris (1969) found
that the time course of reductions catalyzed by
R. rubrum nitrogenase preparations was nonlinear and exhibited a lag phase. The inconsistencies arose because R. rubrum has a method
for quickly turning off its N 2 fixation system and
then turning it on again. Ludden and Burris
(1976) first reported the presence of an activating system for nitrogenase in R. rubrum
chromatophores. The factor could be separated
from the dinitrogenase and dinitrogenase reductase on a column of DEAE cellulose. ATP and
Mg 2+ or Mg 2÷ plus Mn 2+ were necessary for the
activation of dinitrogenase reductase. The activating factor if preincubated with R. rubrum
preparations abolished the lag. It was apparent
that the action of the activating factor was centered on dinitrogenase reductase and had no
influence on dinitrogenase.
Several students in the laboratory had problems obtaining active nitrogenase preparations
from R. rubrurn despite the initial success of
Schneider et al. (1960). Paul Ludden took up the
problem and made the crucial observation that
an extract from R. rubrurn chromatophores
would activate dinitrogenase reductase from the
organism. The responses suggested that an enzyme was activating the Fe protein of nitrogenase, and with incubation the activity increased.
The agent was designated the 'activating factor'
by Ludden and Burris (1976).
It is interesting that Nordlund et al. (1977) in
Sweden observed the factor independently. They
derived the activating agent from NaC1 extracts
of chromatophore membranes. They also reported that it was sensitive to oxygen, reacted
specifically with the Fe protein of nitrogenase
and that its activity required ATP and Mg 2+.
Ludden and Burris (1979) reported that when
inactive dinitrogenase reductase was activated it
released an 'adenine-like molecule'. ATP and a
divalent metal ion were required for activation of
the protein. Nordlund and Eriksson (1979) observed that preparations were not active in H 2
production until treated with the activating
factor.
Ludden and Burris (1976) reported that the
addition of Mn 2+ and Mg 2+ enhanced the activity of extracts from R. rubrum. Anaerobic collection of R. rubrum aided in recovering active
extracts from R. rubrum. In 1980 Ludden could
verify that the activating factor was oxygen labile
and sensitive to trypsin. It could by stabilized,
and it could be precipitated with 30% polyethylene glycol 4000. The extract could be
purified further on a column of DEAE cellulose.
The presence of NH 4 in the culture medium did
not suppress the production of activating factor.
The dinitrogenase reductase recovered from R.
rubrum had its full complement of Fe and S
whether it was active or inactive.
It proved possible to separate the inactive Fe
protein from R. rubrum into two components on
polyacrylamide gels (Ludden and Burris, 1978).
The inactive Fe protein carried phosphate, ribose and an 'adenine-like' unit. With an active Fe
protein available it was possible to purify both
dinitrogenase and dinitrogenase reductase from
R. rubrum, and the properties of both were
similar to those of other nitrogenase proteins.
The modifying group did not appear on dinitrogenase reductase isolated from other organisms.
Nordlund et al. (1977) had suggested that the
'activating factor' formed a stable complex with
dinitrogenase reductase to restore its catalytic
activity. Ludden (1980) in contrast presented
evidence that activity was restored when the
activating enzyme removed the adenine-ribosephosphate unit whose addition had inactivated
the enzyme. The process required the activating
enzyme, a divalent metal ion (Mg z+, Mn 2+,
Control of nitrogenase in Azospirillurn
Fe 2+) and ATP. He pointed out that upon activation the second band disappeared from a gel
used to separate components of inactive dinitrogenase reductase. So the picture emerged
that R. rubrum dinitrogenase reductase was inactivated in the dark or aerobically by adding a
unit with phosphate, ribose and adenine, and
that the dinitrogenase reductase could be reactivated by enzymatic removal of these blocking
units.
From this point the story has developed primarily with observations by Ludden and colleagues, and the observations and conclusions
have been summarized by Ludden (1980) and by
Ludden and Roberts (1989). They depict their
scheme for activation and inactivation of dinitrogenase reductase as shown in Figure 1. We
will define their terms and then develop the
rationale. Starting at the left, we have the active
form of dinitrogenase reductase with its shared
Fe/S center and with two equivalent protein
subunits. As illustrated at the top of the figure
the dinitrogenase reductase is altered by the
enzyme D R A T (dinitrogenase reductase ADPribosyltransferase) which with NAD and
MgADP, ADP-ribosylates one of the two equivalent protein subunits and thus inactivates dinitrogenase reductase. This is accompanied by
the release of nicotinamide. The inactivation oc-
129
curs by ADP-ribosylation of the arginine in position 101. Note that D R A T is coded for by draT
and that the inactivation is triggered by NH2 or
darkness.
The inactive complex can be reactivated by
D R A G (dinitrogenase reductase-activating glycohydrolase) acting with MgATP and Mn a+. ADP
ribose is removed from arginine-101 of the inactive dinitrogenase reductase and is released. The
D R A G is coded for by draG. Dinitrogenase
reductase is restored to its normal active state in
which the two subunits are equivalent.
The story is well told by Ludden and Roberts
(1989) of how the activation and inactivation of
dinitrogenase reductase was resolved and how
the various components in the system fit together. Ludden and Burris (1979) presented evidence that both adenine and ribose were covalently bound to inactive dinitrogenase reductase
and that both were removed when the dinitrogenase reductase was reactivated. Pope et al.
(1985a,b) purified the displaced adenine moiety
and demonstrated its identity as ADP-ribose by
NMR and mass spectrometric analysis. Jouanneau et al. (1988) found ADP ribosylation also
functioned in the inactivation of the nitrogenase
system in Rhodobacter capsulatus. The attachment of the ADP to ribose was established by
Pope et al. (1986).
MgADP
NAD
DRAT
nicotinamide
[ NHI
[darkness
I PMS
-
(active)
~ f
ADPR
IcccP
(-)
J DRAG
MgATP
MnZ*
(inactive)
Fig. i. Control of dinitrogenase reductase activity by ADP-ribosylation as depicted by Ludden and Roberts (1989). ADPR,
ADP-ribose; PMS, phenazine methosulfate; CCCP, chlorocarbonylcyanidephenylhydrazone.
130
Burris et al.
Experimental
We now shift to some of the specifics of our work
and the work of others on the control of nitrogenase in Azospirillum sp. and related organisms. Under proper conditions Azospirillum
species fix nitrogen vigorously. Characteristically
they thrive at a low partial pressure of oxygen,
and certain species have their N 2 fixation systems
turned off readily by low concentrations of NH 4 .
Azospirillum lip@rum is the most studied
species of the group.
A. lipoferum was first described by Beijerinck
(1925) as Spirillum lipoferum, reexamined by
Becking (1963), and brought into prominence
when D6bereiner and Day (1976) reported that
they had found it associated with the roots of
grasses that did not exhibit symptoms of nitrogen
deficiency whereas surrounding grass did. They
described how to culture the organism and pointed out that it was microaerobic. Since then the
organism has been renamed and studied extensively. Okon et al. (1976a) reported that S.
lipoferum grew vigorously on malate, lactate,
succinate and pyruvate and poorly on glucose. It
grows aerobically when supplied NH 4 , but when
it is fixing N 2 the optimal pO 2 is a bit below 0.01
atm. (1 kPa).
Okon, Houchins, Albrecht and Burris (1977)
reported that on NH 4 the doubling time of S.
lipoferum was 1 h, whereas on N 2 it was 5.5 to
7 h. They found that its dinitrogenase reductase
was activated by the activating factor from R.
rubrum. Ludden et al. (1978) separated S.
lipoferum extracts into three components: dinitrogenase, dinitrogenase reductase and an activating factor comparable to that recovered
from R. rubrum. The efficacy of the activating
factor was increased by added Mn 2+ and Mg 2+
The activation required ATP.
Hartmann and Burris (1987) reported that the
nitrogenase activity of A. brasilense and A.
lipoferum was completely inhibited by 2 kPa of
oxygen; some activity was recovered by transferring to 0.2 kPa of oxygen. There was no covalent
modification of dinitrogenase reductase comparable to that arising from switch-off by NH 4 as
evidenced by Western-blotting and 32p-labeling
experiments.
The question was posed by Ludden et al.
(1982), 'Why is the Fe protein inactive as
purified from R. rubrum grown on glutamate as
its nitrogen source'? Although the inactive Fe
protein proved capable of binding MgATP and
undergoing a conformational shift upon binding
MgATP, and it could undergo oxidation-reduction, it was incapable of transferring electrons to
dinitrogenase at a substantial rate. Hence, it
could not function in the reduction of N 2 or
acetylene.
Apparently the only reported purification of
the nitrogenase from the Azospirillum group is
that of Song et al. (1985). Purification from A.
amazonense was not unlike that for other nitrogen fixers and specific activities were comparable. They were unable to find an activating
enzyme for A. amazonense so the organism differed in this respect from A. lipoferum and A.
brasilense.
Vignais and coworkers (1987) reviewed information on regulation of N 2 fixation in the photosynthetic bacteria. They pointed out that it is
generally accepted that NH 4 itself is not the real
effector of switch-of of nitrogenase, and that
glutamine synthetase may be involved directly or
indirectly. However, they concluded that there is
no direct evidence that glutamine is the in vivo
effector of switch-off.
The switch-on-off process can be very rapid in
N2-fixing organisms. Sweet and Burris (1981)
followed nitrogenase activity by measuring H 2
production with a hydrogen electrode. This permitted continuous measurement, and rapid
changes could be observed. R. rubrum grown on
N 2 on a glutamate medium turned nitrogenase
off quickly when NH 4 was added and turned
production of H a back on in seconds after
exhaustion of the NH 4.
Cejudo et al. (1984) found that low levels of
NH~- turned off nitrogenase immediately in
Azotobacter chroococcum, and that the activity
returned when the NH 4 was exhausted. Inhibitors of NH 4 assimilation, such as methionine
sulfoximine, prevented NH 4 switch-off.
Hartmann et al. (1986) reversibly inhibited the
nitrogenase activity of A. brasilense, A.
lipoferum and A. amazonense. The interesting
observation was that methionine sulfoximine
abolished switch-off in A. lipoferum and A.
brasilense but not in A. amazonense. Obviously
Control of nitrogenase in Azospirillum
there was something different about the control
of nitrogenase among species of the same genus.
In A. amazonense, the in vitro nitrogenase activity of NH4-treated cells was not decreased,
and no evidence could be found for a modified
Fe protein band on electrophoretic examination.
Hartmann et al. (1988) noted further differences among species and strains of the azospirilla. A. brasilense grew poorly on glutamate, aspartate, serine or histidine, and these amino acid
even at concentrations of 10mM showed little
inhibition of N 2 fixation. In contrast, A.
lip@rum and A. amazonense grew well on these
amino acids as a source of carbon and nitrogen.
Azospirillum spp. thus differ substantially in
their metabolism of amino acids.
Herbaspirillum seropedicae, a N2-fixing organism isolated and characterized by D6bereiner
(1989), is microaerobic. Fu and Burris (1989)
found its optimal pO 2 is from 0.04 to 0.2 kPa for
N 2 fixation. NH 4 inhibited N 2 fixation only partially even at 20 mM concentration. Treatment
with NH 4 gave no detectable change in the
electrophoretic pattern of the nitrogenase components, so there is no evidence that ADPribosylation plays a part in turning off nitrogenase in this organism.
Control of nitrogenase by the DRAT and
D R A G system is by no means universal among
N2-fixing microorganisms. The question arises
whether one can transfer the system to other
microorganisms devoid of it. Fu, Wirt et al.
(1989) demonstrated that the DRAT system
could be moved to E. coli and KlebsieIla pneumoniae. When the DRAT was expressed in wildtype K. pneumoniae strains, they lost their ability to fix N 2. If the arginine 101 site on dinitrogenase reductase was eliminated, the dinitrogenase reductase could not be inactivated
by ADP-ribosylation.
The mechanism of NH 4 switch-on-off in A.
brasilense and A. lipoferum was investigated by
Fu, Hartmann et al. (1989), and they established
a correlation between in vivo regulation of nitrogenase activity by NH 4 or glutamine and the
reversible covalent modification of dinitrogenase
reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was found in extracts of A. brasilense with NAD serving as the
donor molecule. Dinitrogenase reductase-
131
activating glycohydrolase (DRAG) activity was
present in extracts of both A. lipoferum and R.
rubrum. The region homologous to R. rubrum
draT and draG was identified in the genomic
DNA of A. brasilense as a 12-kilobase EcoRI
fragment and in A. lip@rum as a 7-kilobase
EcoRI fragment. The authors concluded that a
posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A.
lipoferum, and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R.
rubrum.
The question of whether the regulatory system
characteristic of R. rubrum could be transferred
to a N2-fixing organism lacking it and whether it
would function there was posed by Fu, Burris
and Roberts (1990). They found that they could
transfer draT and draG, the genes coding for
DRAT and DRAG, from R. rubrum or A.
lip@rum to K. pneumoniae (an organism normally devoid of these genes) and demonstrate
that they could function. The expressed draT
and drag genes allowed K. pneumoniae to respond to NH 4 with a reversible regulation of
nitrogenase activity that was correlated with the
reversible ADP-ribosylation of dinitrogenase reductase. One can conclude that DRAT and
D R A G in K. pneumoniae are sufficient to support NH 4+-switch-off-on,
"
and that ADP-ribosylation can serve as a regulatory mechanism for this
organism.
Fu et al. (1990) cloned the draTG genes from
A. lipoferum and demonstrated that they are
located next to nifH (structural gene for dinitrogenase reductase) as in R. rubrum. The
similarity in organization strongly suggests that
these genes have been conserved in these two
organisms during evolution. This is the first identification of this regional homology outside the
photosynthetic bacteria.
Herbaspirillum seropedicae is an organism that
has an unusually low optimal pO 2 for nitrogen
fixation as measured by reduction of acetylene.
As indicated in Figure 2, the optimal acetylene
reduction occurred at about 0.1 kPa. A. brasilense had optimal acetylene reduction at about
three times this pO 2.
Acetobacter diazotrophicus (formerly Acetobacter nitrocaptans) is a nitrogen-fixing organism
with intriguing properties. This organism was
Burris et al.
132
300
800
!
x 600
...200
-
g
oJ
,..~ 400
"6
E=
~_t00
200
o
0
i
i
t
2
4
6
kPa Oxygen
Fig. 2. Influence of pO 2 on acetylene reduction by Acetobacter diazotrophicus (-[3-) in comparison with AzospirilIurn
brasilense Sp7 (-[]-) and Herbaspirillurn seropedicae Z176
(-Ii,-). Samples (4 mL) of N2-fixingcultures were transferred
to an O2-electrode chamber, C2H2 was added, and samples
were removed for measurement of C2H 2 reduction at different dissolved O 2 levels. The OD580 of the culture was about
1.0 and contained about 500/xg protein mL-:.
isolated f r o m sugar cane and described by Boddey and D 6 b e r e i n e r (1988) and D 6 b e r e i n e r
(1989). They have summarized recent studies on
associative nitrogen fixation. Most free-living
and associative nitrogen fixers achieve limited N 2
fixation because of the dearth of energy-yielding
substrates available to them. H o w e v e r , A.
diazotrophicus by growing in association with
sugar cane has chosen a location rich in carbohydrate. It takes advantage of the available sucrose, grows actively in or on the cane stem and
roots, and fixes an abundance of N 2. The physiology of the organism is particularly r e m a r k a b l e in
two aspects: (a) it grows in high concentrations
of sucrose (we grow it routinely in 10% sucrose
and D 6 b e r e i n e r , 1989, reports that it grows in
30% sucrose), and (b) it fixes N 2 at a remarkably
low p H (it grows and reduces acetylene below
p H 3.0). T h e r e is no clear definition of the
internal p H of the organism, but it grows on a
nitrogen-free m e d i u m at p H 3.0 and lower.
T h e r e are indications that A. diazotrophicus and
other organisms that m a y be associated with it
m a y fix over 200 kg N per hectare in a year.
The data in Figure 3 are from an experiment
in which amino acids and azaserine were added
0
0
10
20
M1nutes
30
40
50
Fig. 3. Effect of amino acids and azaserine on nitrogenase
activity of Acetobacter diazotrophicus PAL-5. At 15 min,
2mM azaserine (-[3-), 5mM asparagine (-~-), 5mM
glutamic acid (-<>-) or water as control (-[]-) was added.
Reactions were run in an O2-electrode chamber at 0.10.2 kPa 02.
to Ne-fixing cultures of A. diazotrophicus. A t 15
min 2 m M azaserine, 5 m M glutamic acid and
5 m M asparagine were added to the chamber.
Glutamic acid and asparagine inhibited about
25% and azaserine about 50%.
As discussed, it has been shown that N 2 fixation can be turned off by N H 4 and that the
mechanism in R. rubrum, A. lipoferurn and A.
brasilense is by ADP-ribosylation of arginine 101
of one of the protein subunits of dinitrogenase
reductase. W h e n this occurs, the two subunits
can be separated by gel electrophoresis, because
one has had its properties altered by adding the
A D P - r i b o s e at arginine 101. We found no evidence for such a switch-off-on process in A.
diazotrophicus, so we examined the electrophoretic pattern of dinitrogenase reductase
from this organism. Figure 4 indicates that A.
diazotrophicus is not subject to control by covalent modification of its dinitrogenase reductase. The left lane shows the separation of two
c o m p o n e n t s f r o m A. brasilense that had been
treated with N H 2 ; this is an organism that undergoes switch-off by ADP-ribosylation of its
dinitrogenase reductase. The normal subunit and
the modified subunit from its dinitrogenase reductase are clearly separated. In contrast, only
one band appears in extracts from A. diazo-
Control of nitrogenase in Azospirillum
133
trophicus. Lane 2 is before and lane 3 after a
30 min treatment with 10 mM NH,Cl. Cells for
lane 3 were held under 6 kPa 0, for 30 min and
those for lane 5 were anaeorobic for 30min.
Under no conditions was the dinitrogenase reductase altered by covalent modification as it is
in A. brasilense. Figure 5 supports these conclusions and indicates that A. diazotrophicus lacks
draT and draG genes.
Fig. 4. Immunoblots of crude extracts from Acetobacfer
diazotrophicus
PAL-5 separated by SDS-PAGE with antiserum against dinitrogenase reductase of Azotobacter
vinelandii.
Lane 1, crude extract of Azospirillum
brasilense
treated with 5 mM NH,Cl for 30 min. Lanes 2, 3, 4, 5 from
crude extracts of A. diazotrophicus
PAL-5 before (lane 2)
and after 30 min treatment with 10mM NH,Cl (lane 3).
Lane 4, 30min at 6.0 kPa 0,; and lane 5, anaerobic for
30min. The crude extracts were prepared by sonication
(lanes 1 and 2) or by quick extraction (lanes 3, 4, 5).
Fig. 5. Southern
hybridization
of genomic DNA
of
Acetobacter
diazotrophicus
PAL-5 with a draTG probe from
Azospirillum
lipoferum.
Total DNAs from A. lipoferum
(lane 1) and A. diazobophicus
(lane 2) were digested with
EcoRI and hybridized to a “P-labeled draTG probe from A.
lipoferum.
(A)Ethidium bromide-stained DNA on the agar-
ose gel, (B) autoradiogram. Bacteriophage A-DNA cut with
Hind III was used as a size marker and control (lane 3).
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