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Evidence for direct repression of nitrogenase by ammonia in the cyanobacterium Anabaena cylindrica
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 835-844
January 29, 1986
EVIDENCE FOR DIRECT REPRESSION OF NITROGENASE BY AMMONIA IN THE CYANOBACTERIUM ANABAENA CYLINDRICA
Alison H. Mackerras and Geoffrey D. Smith Department of Biochemistry, Faculty of Science, The Australian National University, G.P.O. Box 4, Canberra, A.C.T. 2601, Australia Received December 13, 1985
SUMMARY The nitrogenase a c t i v i t y of the cyanobacterium Anabaena cy-~i ca was repressed u p o n addi t i on of ammonium salts after preincubation in the presence of a concentration of L-methionine-DLsulfoximine s u f f i c i e n t to t o t a l l y i n h i b i t glutamine synthetase. Repression was also observed when urea was added to c e l l s in the presence of the glutamine synthetase i n h i b i t o r . Measurements of ammonia concentrations were made in each case and provided evidence that ammonia i t s e l f is a primary regulator of nitrogenase levels in A. c y l i n d r i c a . ® ~986A~ade~icPr.... Inc.
Nitrogenase and
heterocyst synthesis in cyanobacteria are subject
to regulation by a product of nitrogen f i x a t i o n
(I).
regulatory
and
molecule
is
uncertain.
Stewart
The i d e n t i t y of this Rowell
evidence, based on use of the glutamine synthetase i n h i b i t o r
(2)
presented
L-methionine-
DL-sulfoximine (MSOX)I, that ammonia i t s e l f was not the regulator and that glutamine or some other simple derivative of ammonia was l i k e l y to be the regulatory molecule. recently Singh et al.
More recent data have contradicted these findings and (3) and Turpin et al.
(4) have suggested that the
e a r l i e r work was indeed consistent with ammonia i t s e l f regulator
of
nitrogen
fixation
conclusion by Turpin et al.
in
cyanobacteria.
being an important
The reason for
this
(4) was the suggestion that MSOX was a potent
i n h i b i t o r of ammonium ion transport into the c e l l s in addition to i t s role as a glutamine synthetase i n h i b i t o r
(4).
Other workers have disputed the
proposition that MSOX i n h i b i t s ammonium ion transport
IAbbreviation:
(5, 6) and therefore
MSOX, L-methionine-DL-sulfoximine. 0006-291X/86 $1.50 835
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the discrepant conclusions concerning ammonia repression alternative inhibitor
explanation. concentrations
We have
reinvestigated
and conditions
which give
may require
this
problem
total
inhibition
an
using of
glutamine synthetase.
We have also used urea as an a l t e r n a t i v e method for
allowing i n t r a c e l l u l a r
ammonia accumulation, based on the observation that
c e l l s grown in the presence of nickel
ions have an active urease a c t i v i t y
and that urea is r a p i d l y transported into c e l l s and converted to ammonia. This has allowed us to reassess the action of MSOX by e f f e c t i v e l y
allowing
transport of ammonia into c e l l s by a mechanism not involving the ammonium carrier.
We have
also
made detailed
ammonium ion
concentration
measurements and present results which provide strong confirmatory evidence that ammonia i t s e l f
is a potent regulator of nitrogenase synthesis. MATERIALS AND METHODS
Cyanobacteria and t h e i r growth. Anabaena c y l i n d r i c a (ATCC 27899) was obtained Trom the American Type Culture Collection and grown as in (7) except with the growth medium at I/8 concentration of all components except phosphate and nickel which were at f u l l strength and 4 x strength, respectively. Nickel-depleted c e l l s were grown as described previously (8). Cells were grown under n i t r o g e n - f i x i n g conditions, being continuously sparged during growth and the reported experiments with 0.3% CO2 in air at approx. 170 ml/min. The l i g h t i n t e n s i t y was 150 ~Es-lm -2 (photos y n t h e t i c a l l y active r a d i a t i o n ) . Cells were collected and used in the concentration range 80 to 170 K l e t t units (9) where nitrogenase a c t i v i t y was found to be maximal on a dry weight basis. For dry weight determination c e l l s were centrifuged, washed once, and then dried overnight at 85 C. Assays. Glutamine synthetase was assayed using the biosynthetic assay -(TOT_ Cells were harvested by centrifugation and concentrated in 50 mM Hepes/KOH, pH 7.5. They were passed through a French pressure cell at 138 MPa and the debris removed by centrifugation before the assay. MSOX was obtained from the Sigma Chemical Co., St. Louis, MO, USA. Nitrogenase a c t i v i t y was measured by the acetylene reduction method as described previously ( I I ) . Acetylene reduction rates were measured over a 3 h assay period and are expressed as i n i t i a l rates. For ammonia determinations samples (I0 ml) were taken at appropriate i n t e r v a l s and centrifuged. Ammonia released into the medium was measured in duplicate aliquots of the supernatant with an Orion ammonia electrode (model no. 95-12), a f t e r adding NaOH (I0 M) to convert ammonium ions into free ammonia° Protein concentrations were measured by the method of Peterson (12). RESULTS When MSOX was added to whole c e l l s of A. c y l i n d r i c a glutamine synthetase a c t i v i t y
it
inhibited
in a time-dependent manner as shown in Fig. 836
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E 0"0~ E -6 E ._~ 0"0~
.p,o o-o~ E
.~ O ~ E '5 0 2
Fig. ].
I.
4
6
8 10 Time (h)
12
1L
16
1'8
The effect of MSOX, added to growing cells of A. cylindrica, on glutamine synthetase a c t i v i t y . The MSOXwas added to give a f i n a l concentration of I0 ~M (0) or 1 mM (1), with a control (0) to which no addition was made. At the indicated times cells were harvested, extracted and assayed as quickly as possible (within 30 min)o A c t i v i t i e s are expressed per mg of protein.
Whereas I0 ~M MSOX produced t o t a l i n h i b i t i o n only a f t e r 12-24 h, a 1 mM
concentration of the i n h i b i t o r
produced complete i n h i b i t i o n w i t h i n 2 hours,
as with A. flos-aquae
We chose
preincubation
with
1 mM MSOX, f o r
quickness of a c t i o n . of the t e s t
(4).
The f i r s t
and control
preincubation.
to
do f u r t h e r
three
reasons
work in
using
addition
a 3 h to
the
was t h a t in three hours the concentrations
cells
diverged
little
Secondly, the e x t r a c e l l u l a r
compared with
a 12-24 h
ammonia concentration measured
a f t e r 3 h in the presence of MSOX was much lower (approx. 0 . I mM) than t h a t f r e q u e n t l y observed a f t e r
a 24 h preincubation with
I0 uM MSOX (0.5-0.6
mM). T h i r d l y , nitrogenase l e v e l s of c e l l s a f t e r 24 h incubation in I0 ~M MSOX were u s u a l l y depressed to a much greater extent than a f t e r a shorter incubation with the higher i n h i b i t o r concentration
of MS0X poses
preincubation
is
clearly
concentration.
problems of
insufficient
Thus use of the lower
interpretation
to
since
ensure complete
a shorter
inhibition
of
glutamine synthetase. For the reported experiments MSOX (I mM) was added and the c e l l s incubated in the l i g h t without the i n h i b i t o r
while
sparging with
were t r e a t e d s i m i l a r l y . 837
air/0.3% C 0 2 .
Control
cells
At the end of the incubation
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100
S
8O
u
• ~
60
>
cJ
LO
c
& o
."2_ 2 O Z
0
4
8
12
16
20
24
Time (h) The effects of urea (o, o) and (NH4)2HPO~ I,FI) on the nitrogenase a c t i v i t y (measured as i n i t i a l rates) of A. c y lin d r ic a, harvested at 170 Klett units and preincubated either with (solid symbols) or without (open symbols) 1 mM MSOXfor 3 ho Control cultures with no added nitrogen source were tested s i m i l a r l y (A, A), Results are expressed as a percentage a c t i v i t y of the control to which no additions were made. The a c t i v i t y of the control culture was 370 nmol h-I mg-1 dry weight on the f i r s t day of the experiment and declined to 190 nmol h -I mg-I dry weight on the second day of the experiment due to aging. Zero time is that time at which the MSOX preincubation was commenced. The urea and ammonia were added at 3h.
c e l l s were d i v i d e d i n t o a l i q u o t s and then both t e s t incubated f u r t h e r
after
a d d i t i o n of e i t h e r
urea
and c o n t r o l c e l l s were (I
mM) or
(NH4)2HPO4 (I
mM), or w i t h o u t f u r t h e r a d d i t i o n s . Nitrogenase a c t i v i t y and again
after
overnight
was assayed immediately a f t e r these a d d i t i o n s incubation
(Fig.
2).
The c o n c e n t r a t i o n s
ammonium ions in the medium were also measured at i n t e r v a l s seen in totally
Fig.
2,
with
nickel-containing
cells,
nitrogenase
(Fig.
of
3).
As
activity
was
i n h i b i t e d w i t h i n 24 h by ammonium ions and by urea, whether or not
MSOX was present. led t o t o t a l
In the c o n t r o l c u l t u r e als o , the presence of MSOX alone
inhibition
of nitrogenase w i t h i n 24 h. 838
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2o
~E
E
c o
'6
I-0
o o 0
g 05 E E
0
Fig. 3.
In
2
4 6 Time ( h )
22
24
Ammonia concentrations in the medium of cells to which additions of 1 mM urea (e, o), 1 mM (NH4)2HPO4 (I,[]) or no additions (A, a) were made. The c e l l s were preincubated as described in Fig. 2 and the solid and open symbols, together with the time scale, have the same significance as in that figure. The arrow indicates the time at which urea and ammonia were added. On the f i r s t day of the experiment the cell concentration was 0.34 mg dry weight per ml, corresponding to 170 Klett units. After overnight incubation the concentrations were 0.53 mg/ml (0), 0.54 mg/ml ( | ) , 0.27 mg/ml ([i]) 0.30 mg/ml (m), 0.44 mg/ml (a) and 0.33 mg/ml (A).
order
to
nitrogenase a c t i v i t y nickel-depleted
determine
whether
urea
itself
was a repressor
of
the above experiments were repeated w i t h urea added to
cells
which
were
devoid
of
urease
activity.
These
experiments ( r e s u l t s not shown) revealed t h a t urea i t s e l f
caused no loss of
nitrogenase a c t i v i t y
or w i t h o u t
urea,
and t h a t the e f f e c t s
were comparable w i t h
the r e s u l t s
of MSOX, w i t h of
Fig.
2.
Interestingly,
added after
o v e r n i g h t i n c u b a t i o n of such c e l l s w i t h urea the nitrogenase a c t i v i t y
was
50-100% more a c t i v e than t h a t of c o n t r o l c e l l s to which no urea was added. The reason f o r t h i s with
i s not known.
nickel-depleted
absence of MSOX;
cells
the
The corresponding ammonia measurements
showed no ammonia formation
presence
of
this 839
reagent
from urea in the
resulted
in
a rate
of
VoI. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
ammonia formation which was the same whether urea was added or not and was comparable
with
nitrogenase,
(0)
of Fig.
prepared
ammonium chloride
3.
Furthermore,
and assayed
as
in
experiments with c e l l - f r e e
(13),
revealed
(2 mM) nor urea (I mM) i n h i b i t e d
that
neither
nitrogenase d i r e c t l y
(results not shown). Several
conclusions
which are shown in Fig.
can be made from the
3.
First,
ammonia measurements
ammonia is consumed by the c e l l s
and
consumption is t o t a l l y prevented by the preincubation with MSOX. Secondly, under the conditions
of
these experiments cultures
alone produced ammonia, apparently at i n t r a c e l l u l a r t o t a l l y repress nitrogenase a c t i v i t y ; the control
no external
incubated with levels
sufficient
to
ammonia was produced by
culture to which no additions were made.
completely converted into ammonia within 24 h.
MSOX
Thirdly,
urea was
In the presence of MSOX the
ammonia produced was all released into the medium whereas in the absence of the i n h i b i t o r only a transient concentration of ammonia was observed since assimilatory processes were s t i l l still
sufficient
This concentration was c l e a r l y
to repress nitrogenase a c t i v i t y
the nitrogenase a c t i v i t y (20-22 h).
active.
(Fig.
2).
Interestingly
reappeared at the time of depletion of the urea
These results
show that
cells
of A. c y l i n d r i c a
convert urea
into ammonia f a s t e r than i t can be incorporated. The above experiments were repeated with a 24 h preincubation of the c e l l s in I0 ~M MSOX. those reported, glutamine
The results were q u a l i t a t i v e l y
despite the
addition
to
cells
problems
was also
Glutamine (I mM) produced a t o t a l although i t s
alluded
action could well
to
studied
inhibition
above. under
consistent with The e f f e c t
these
of
conditions.
of nitrogenase within
24 h,
have been via ammonia, which accumulated
s t o i c h i o m e t r i c a l l y from the glutamine in the presence of MSOX.
DISCUSSION
Our results provide strong evidence for a role of ammonia i t s e l f in
nitrogenase
repression
in
cyanobacteria. 840
In
the
presence
of
MSOX
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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
ammonia was produced both from nitrogen f i x a t i o n containing c e l l s , r e s u l t i n g in total it
and from urea in nickel-
loss of nitrogenase within 24 h. Hence
is clear that urea transport into c e l l s
is
not i n h i b i t e d
that the ammonia which r e s u l t s from urease a c t i v i t y I t has been shown by Bone (14) that urea i t s e l f synthesis
of
nitrogenase
nickel-depleted c e l l s
in
and which
we
have
by MSOX and
represses nitrogenase.
is u n l i k e l y to repress the
demonstrated
urease a c t i v i t y
this
was absent
by
using
(results
not
shown).
Therefore, these urea results confirm the conclusion of Turpin et
al.
and others
(4)
that
ammonia plays
such a role
and contradict
the
contradiction
and
conclusion of Rowell and Stewart (2) that i t does not. There
may
be
particularly
why,
nitrogenase
repression°
concentrations
(I
in
several our
reasons
experiments, Rowell
~M) of
for
the
this
MSOX did
and
Stewart
inhibitor.
not
protect
(2)
used
As shown in
against
very
Fig. 1
a
low 3 h
preincubation with 1 mM MSOX was necessary to completely block glutamine synthetase a c t i v i t y . accumulated
in
significantly
the
One r e s u l t of this was that the ammonia levels which presence of
the
inhibitor
higher than those of Rowell
level of glutamine
synthetase a c t i v i t y
presumably quite s i g n i f i c a n t ,
our
and Stewart
within
experiments (2)
for
were
whom the
12 h of MSOX addition was
r e s u l t i n g in a depletion of the i n t r a c e l l u l a r
ammonia l e v e l , which may well be c r i t i c a l is concerned.
in
In the absence of
as far as nitrogenase repression
added f i x e d
nitrogen
there
are three
sources of ammonia, each of which may have been s i g n i f i c a n t in our c e l l s in contributing
to ammonia levels
presence of MSOX. The f i r s t
sufficient
to repress nitrogenase
of these is nitrogen f i x a t i o n
itself.
in
the
I t has
been shown previously (7, 15) that nitrogenase a c t i v i t y varies dramatically with the age of c e l l s and we were careful to harvest c e l l s when nitrogenase activity (16).
was maximal.
The second source of ammonia is
photorespiration
Although the cultures were sparged with air containing 0.3% CO2 t h i s
may not have been s u f f i c i e n t
to suppress photorespiration
The t h i r d source of ammonia is protein degradation (17). 841
entirely
(16).
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
In all our experiments the pH values were c a r e f u l l y monitored and shown to be in
the range 7 to
phosphate alone was added; experiment.
Thus the
7.4 except in
the
case where ammonium
in that case the pH dropped to 6.4 during the
pH was well
below the
pK of
ammonia and hence
comparable with the low pH experiments of Turpin et al.
(4) in which the
c e l l s would require operation of the ammonium ion c a r r i e r . (not reported)
Similar results
were obtained when the experiments were done with
resuspended in I0 mM Hepes buffer, pH 7.1, as used by Turpin et al. also obtained similar
results
using
doubled concentrations
of
cells (4). We
urea and
ammonia. That the e f f e c t
of ammonia was not d i r e c t l y
on the nitrogenase
i t s e l f was shown by experiments (results not shown) in which i t to c e l l - f r e e extracts of nitrogenase; i n h i b i t i o n of a c t i v i t y . many n i t r o g e n - f i x i n g (18).
Furthermore,
was added
neither ammonia nor urea gave any
Similar results have been obtained with ammonia in bacteria, it
is
excluding unlikely
the
that
non-sulfur the
purple
effect
of
bacteria
ammonia in
suppressing nitrogenase is due to an e f f e c t as an uncoupler on the energy or reductant generating systems since this organism is
able to grow well
with ammonia concentrations s i g n i f i c a n t l y higher than those used herein (2, 19).
Ammonia has also been postulated to i n h i b i t
nitrogenase by competing
with i t for ATP and/or reductant (20, 21) but c l e a r l y t h i s would not be the case when i t s time
incorporation
course of
nitrogenase
is i n h i b i t e d by MSOX (21). activity
loss
observed
progressive decline, more consistent with an e f f e c t than d i r e c t i n h i b i t i o n . of
nitrogenase
differentiation inhibition
at
in
2 shows a
on protein
that
some stage
to
ammonia declines
nitrogenase during
differentation.
used herein would have contained at least
It
synthesis
heterocyst
is
to not
ammonia easy
since c l e a r l y
a proportion
heterocyst c e l l s at all stages during the experiments. 842
during
becomes r e f r a c t o r y
reconcile these observations with the present results cells
Fig.
the
I t has been suggested (22, 23) that the s e n s i t i v i t y
synthesis and
Furthermore,
to the
of mature
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
The d e f i n i t i v e effectively
transportS
aspect of this
work was the
ammonia into
cells
requiring the ammonium transport system. on this urea produces ammonia. in
the
cells
confirmation
resulting that
in
under
use of
urea which
conditions
without
The subsequent action of urease
In the presence of MSOX ammonia accumulates nitrogenase
ammonia i t s e l f
is
repression.
We take
a primary regulator
of
this
as
nitrogenase
levels in A. cylindrica.
ACKNOWLEDGEMENTS
The Australian Research Grants Scheme (Grant No. D283154661) and the
Australian
financial
National
assistance
for
performing the c e l l - f r e e
University this
work.
Faculties We thank
Research Fund provided Dr D.M. Pederson for
nitrogenase assays and A. Daday for
assistance
with the glutamine synthetase assays.
REFERENCES .
2. 3. 4.
5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17.
Stewart, W.D.P. (1973). Ann. Rev. Microbiol. 27, 283-316. Stewart, W.D.P. and Rowell, P. (1975). Biochem. Biophys. Res. Commun. 65, 846-856. Singh, H.N., Rai, U.N., Rao, V.V. and Bagchi, S.N. (1983). Biochem. Biophys. Res. Commun. I I I , 180-187. Turpin, D.H., Edie, S.A. and Canvin, D.T. (1984). Plant Physiol. 74, 701-704. Boussiba, S. and Gibson, J. (1985). FEBS Lett. 180, 13-16. Rai, A.N., Rowell, P. and Stewart, W.D.P. (1984). Arch. Microbiol. 137, 241-246. Daday, A., Platz, R.A. and Smith, G.D. (1977). Appl. Environ. Microbiol. 34, 478-483. Daday, A., Mackerras, A.H. and Smith, G . D . (1985). J. Gen. Microbiol. 131, 231-238. Mallette, M.F. (1969). Methods Microbiol. l, 521-566. Dharmawardene, M.W.N., Haystead, A. and Stewart, W.D.P. (1973). Arch. Mikrobiol. 90, 281-295. Lambert, G.R. and Smith~ G.D. (1980). Arch. Biochem. Biophys. 205, 36-50. Peterson, G.L. (1977). Analyt. Biochem. 83, 346-356. Hallenbeck, P.C., Kostel, P.J. and Benemann, J.R. (1979). Eur. J. Biochem. 98, 275-284. Bone, D.H. (1972i. Arch. Mikrobiol. 86, 13-24. Weare, N.M. and Benemann, J.R. (1973). Arch. Mikrobiol. 93, 101-112. Bergman, B. (1984). Arch. Microbiol. 137, 21-25. Lambert, G.R. and Smith, G.D. (1981). Arch. Biochem. Biophys. 211, 360-367. 843
Vol. 134, No. 2, 1986 18. 19. 20. 21. 22. 23.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Gordon, J.K., Shah, V.K. and B r i l l , W.J. (1981). J. Bacteriol. 148, 884-888. Daday, A~, Lambert, G.R. and Smith, G.D. (1979). Biochem. J. 177, 139-144. Ohmori, M. and Hattori, A. (1974). Plant and Cell Physiol. 15, 131-142. Ohmori, M. and Hattori, A. (1978). Arch. Microbiol. 117, 17-20. Murry, M.A. and Benemann, J.R. (1979). Plant and Cell Physiol. 20, 1391-1401. Murry, M.A., Jensen, D.B. and Benemann, J.R. (1983). Biochim. Biophys. Acta 756, 13-19.
844
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Pages 835-844
January 29, 1986
EVIDENCE FOR DIRECT REPRESSION OF NITROGENASE BY AMMONIA IN THE CYANOBACTERIUM ANABAENA CYLINDRICA
Alison H. Mackerras and Geoffrey D. Smith Department of Biochemistry, Faculty of Science, The Australian National University, G.P.O. Box 4, Canberra, A.C.T. 2601, Australia Received December 13, 1985
SUMMARY The nitrogenase a c t i v i t y of the cyanobacterium Anabaena cy-~i ca was repressed u p o n addi t i on of ammonium salts after preincubation in the presence of a concentration of L-methionine-DLsulfoximine s u f f i c i e n t to t o t a l l y i n h i b i t glutamine synthetase. Repression was also observed when urea was added to c e l l s in the presence of the glutamine synthetase i n h i b i t o r . Measurements of ammonia concentrations were made in each case and provided evidence that ammonia i t s e l f is a primary regulator of nitrogenase levels in A. c y l i n d r i c a . ® ~986A~ade~icPr.... Inc.
Nitrogenase and
heterocyst synthesis in cyanobacteria are subject
to regulation by a product of nitrogen f i x a t i o n
(I).
regulatory
and
molecule
is
uncertain.
Stewart
The i d e n t i t y of this Rowell
evidence, based on use of the glutamine synthetase i n h i b i t o r
(2)
presented
L-methionine-
DL-sulfoximine (MSOX)I, that ammonia i t s e l f was not the regulator and that glutamine or some other simple derivative of ammonia was l i k e l y to be the regulatory molecule. recently Singh et al.
More recent data have contradicted these findings and (3) and Turpin et al.
(4) have suggested that the
e a r l i e r work was indeed consistent with ammonia i t s e l f regulator
of
nitrogen
fixation
conclusion by Turpin et al.
in
cyanobacteria.
being an important
The reason for
this
(4) was the suggestion that MSOX was a potent
i n h i b i t o r of ammonium ion transport into the c e l l s in addition to i t s role as a glutamine synthetase i n h i b i t o r
(4).
Other workers have disputed the
proposition that MSOX i n h i b i t s ammonium ion transport
IAbbreviation:
(5, 6) and therefore
MSOX, L-methionine-DL-sulfoximine. 0006-291X/86 $1.50 835
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the discrepant conclusions concerning ammonia repression alternative inhibitor
explanation. concentrations
We have
reinvestigated
and conditions
which give
may require
this
problem
total
inhibition
an
using of
glutamine synthetase.
We have also used urea as an a l t e r n a t i v e method for
allowing i n t r a c e l l u l a r
ammonia accumulation, based on the observation that
c e l l s grown in the presence of nickel
ions have an active urease a c t i v i t y
and that urea is r a p i d l y transported into c e l l s and converted to ammonia. This has allowed us to reassess the action of MSOX by e f f e c t i v e l y
allowing
transport of ammonia into c e l l s by a mechanism not involving the ammonium carrier.
We have
also
made detailed
ammonium ion
concentration
measurements and present results which provide strong confirmatory evidence that ammonia i t s e l f
is a potent regulator of nitrogenase synthesis. MATERIALS AND METHODS
Cyanobacteria and t h e i r growth. Anabaena c y l i n d r i c a (ATCC 27899) was obtained Trom the American Type Culture Collection and grown as in (7) except with the growth medium at I/8 concentration of all components except phosphate and nickel which were at f u l l strength and 4 x strength, respectively. Nickel-depleted c e l l s were grown as described previously (8). Cells were grown under n i t r o g e n - f i x i n g conditions, being continuously sparged during growth and the reported experiments with 0.3% CO2 in air at approx. 170 ml/min. The l i g h t i n t e n s i t y was 150 ~Es-lm -2 (photos y n t h e t i c a l l y active r a d i a t i o n ) . Cells were collected and used in the concentration range 80 to 170 K l e t t units (9) where nitrogenase a c t i v i t y was found to be maximal on a dry weight basis. For dry weight determination c e l l s were centrifuged, washed once, and then dried overnight at 85 C. Assays. Glutamine synthetase was assayed using the biosynthetic assay -(TOT_ Cells were harvested by centrifugation and concentrated in 50 mM Hepes/KOH, pH 7.5. They were passed through a French pressure cell at 138 MPa and the debris removed by centrifugation before the assay. MSOX was obtained from the Sigma Chemical Co., St. Louis, MO, USA. Nitrogenase a c t i v i t y was measured by the acetylene reduction method as described previously ( I I ) . Acetylene reduction rates were measured over a 3 h assay period and are expressed as i n i t i a l rates. For ammonia determinations samples (I0 ml) were taken at appropriate i n t e r v a l s and centrifuged. Ammonia released into the medium was measured in duplicate aliquots of the supernatant with an Orion ammonia electrode (model no. 95-12), a f t e r adding NaOH (I0 M) to convert ammonium ions into free ammonia° Protein concentrations were measured by the method of Peterson (12). RESULTS When MSOX was added to whole c e l l s of A. c y l i n d r i c a glutamine synthetase a c t i v i t y
it
inhibited
in a time-dependent manner as shown in Fig. 836
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E 0"0~ E -6 E ._~ 0"0~
.p,o o-o~ E
.~ O ~ E '5 0 2
Fig. ].
I.
4
6
8 10 Time (h)
12
1L
16
1'8
The effect of MSOX, added to growing cells of A. cylindrica, on glutamine synthetase a c t i v i t y . The MSOXwas added to give a f i n a l concentration of I0 ~M (0) or 1 mM (1), with a control (0) to which no addition was made. At the indicated times cells were harvested, extracted and assayed as quickly as possible (within 30 min)o A c t i v i t i e s are expressed per mg of protein.
Whereas I0 ~M MSOX produced t o t a l i n h i b i t i o n only a f t e r 12-24 h, a 1 mM
concentration of the i n h i b i t o r
produced complete i n h i b i t i o n w i t h i n 2 hours,
as with A. flos-aquae
We chose
preincubation
with
1 mM MSOX, f o r
quickness of a c t i o n . of the t e s t
(4).
The f i r s t
and control
preincubation.
to
do f u r t h e r
three
reasons
work in
using
addition
a 3 h to
the
was t h a t in three hours the concentrations
cells
diverged
little
Secondly, the e x t r a c e l l u l a r
compared with
a 12-24 h
ammonia concentration measured
a f t e r 3 h in the presence of MSOX was much lower (approx. 0 . I mM) than t h a t f r e q u e n t l y observed a f t e r
a 24 h preincubation with
I0 uM MSOX (0.5-0.6
mM). T h i r d l y , nitrogenase l e v e l s of c e l l s a f t e r 24 h incubation in I0 ~M MSOX were u s u a l l y depressed to a much greater extent than a f t e r a shorter incubation with the higher i n h i b i t o r concentration
of MS0X poses
preincubation
is
clearly
concentration.
problems of
insufficient
Thus use of the lower
interpretation
to
since
ensure complete
a shorter
inhibition
of
glutamine synthetase. For the reported experiments MSOX (I mM) was added and the c e l l s incubated in the l i g h t without the i n h i b i t o r
while
sparging with
were t r e a t e d s i m i l a r l y . 837
air/0.3% C 0 2 .
Control
cells
At the end of the incubation
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100
S
8O
u
• ~
60
>
cJ
LO
c
& o
."2_ 2 O Z
0
4
8
12
16
20
24
Time (h) The effects of urea (o, o) and (NH4)2HPO~ I,FI) on the nitrogenase a c t i v i t y (measured as i n i t i a l rates) of A. c y lin d r ic a, harvested at 170 Klett units and preincubated either with (solid symbols) or without (open symbols) 1 mM MSOXfor 3 ho Control cultures with no added nitrogen source were tested s i m i l a r l y (A, A), Results are expressed as a percentage a c t i v i t y of the control to which no additions were made. The a c t i v i t y of the control culture was 370 nmol h-I mg-1 dry weight on the f i r s t day of the experiment and declined to 190 nmol h -I mg-I dry weight on the second day of the experiment due to aging. Zero time is that time at which the MSOX preincubation was commenced. The urea and ammonia were added at 3h.
c e l l s were d i v i d e d i n t o a l i q u o t s and then both t e s t incubated f u r t h e r
after
a d d i t i o n of e i t h e r
urea
and c o n t r o l c e l l s were (I
mM) or
(NH4)2HPO4 (I
mM), or w i t h o u t f u r t h e r a d d i t i o n s . Nitrogenase a c t i v i t y and again
after
overnight
was assayed immediately a f t e r these a d d i t i o n s incubation
(Fig.
2).
The c o n c e n t r a t i o n s
ammonium ions in the medium were also measured at i n t e r v a l s seen in totally
Fig.
2,
with
nickel-containing
cells,
nitrogenase
(Fig.
of
3).
As
activity
was
i n h i b i t e d w i t h i n 24 h by ammonium ions and by urea, whether or not
MSOX was present. led t o t o t a l
In the c o n t r o l c u l t u r e als o , the presence of MSOX alone
inhibition
of nitrogenase w i t h i n 24 h. 838
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2o
~E
E
c o
'6
I-0
o o 0
g 05 E E
0
Fig. 3.
In
2
4 6 Time ( h )
22
24
Ammonia concentrations in the medium of cells to which additions of 1 mM urea (e, o), 1 mM (NH4)2HPO4 (I,[]) or no additions (A, a) were made. The c e l l s were preincubated as described in Fig. 2 and the solid and open symbols, together with the time scale, have the same significance as in that figure. The arrow indicates the time at which urea and ammonia were added. On the f i r s t day of the experiment the cell concentration was 0.34 mg dry weight per ml, corresponding to 170 Klett units. After overnight incubation the concentrations were 0.53 mg/ml (0), 0.54 mg/ml ( | ) , 0.27 mg/ml ([i]) 0.30 mg/ml (m), 0.44 mg/ml (a) and 0.33 mg/ml (A).
order
to
nitrogenase a c t i v i t y nickel-depleted
determine
whether
urea
itself
was a repressor
of
the above experiments were repeated w i t h urea added to
cells
which
were
devoid
of
urease
activity.
These
experiments ( r e s u l t s not shown) revealed t h a t urea i t s e l f
caused no loss of
nitrogenase a c t i v i t y
or w i t h o u t
urea,
and t h a t the e f f e c t s
were comparable w i t h
the r e s u l t s
of MSOX, w i t h of
Fig.
2.
Interestingly,
added after
o v e r n i g h t i n c u b a t i o n of such c e l l s w i t h urea the nitrogenase a c t i v i t y
was
50-100% more a c t i v e than t h a t of c o n t r o l c e l l s to which no urea was added. The reason f o r t h i s with
i s not known.
nickel-depleted
absence of MSOX;
cells
the
The corresponding ammonia measurements
showed no ammonia formation
presence
of
this 839
reagent
from urea in the
resulted
in
a rate
of
VoI. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
ammonia formation which was the same whether urea was added or not and was comparable
with
nitrogenase,
(0)
of Fig.
prepared
ammonium chloride
3.
Furthermore,
and assayed
as
in
experiments with c e l l - f r e e
(13),
revealed
(2 mM) nor urea (I mM) i n h i b i t e d
that
neither
nitrogenase d i r e c t l y
(results not shown). Several
conclusions
which are shown in Fig.
can be made from the
3.
First,
ammonia measurements
ammonia is consumed by the c e l l s
and
consumption is t o t a l l y prevented by the preincubation with MSOX. Secondly, under the conditions
of
these experiments cultures
alone produced ammonia, apparently at i n t r a c e l l u l a r t o t a l l y repress nitrogenase a c t i v i t y ; the control
no external
incubated with levels
sufficient
to
ammonia was produced by
culture to which no additions were made.
completely converted into ammonia within 24 h.
MSOX
Thirdly,
urea was
In the presence of MSOX the
ammonia produced was all released into the medium whereas in the absence of the i n h i b i t o r only a transient concentration of ammonia was observed since assimilatory processes were s t i l l still
sufficient
This concentration was c l e a r l y
to repress nitrogenase a c t i v i t y
the nitrogenase a c t i v i t y (20-22 h).
active.
(Fig.
2).
Interestingly
reappeared at the time of depletion of the urea
These results
show that
cells
of A. c y l i n d r i c a
convert urea
into ammonia f a s t e r than i t can be incorporated. The above experiments were repeated with a 24 h preincubation of the c e l l s in I0 ~M MSOX. those reported, glutamine
The results were q u a l i t a t i v e l y
despite the
addition
to
cells
problems
was also
Glutamine (I mM) produced a t o t a l although i t s
alluded
action could well
to
studied
inhibition
above. under
consistent with The e f f e c t
these
of
conditions.
of nitrogenase within
24 h,
have been via ammonia, which accumulated
s t o i c h i o m e t r i c a l l y from the glutamine in the presence of MSOX.
DISCUSSION
Our results provide strong evidence for a role of ammonia i t s e l f in
nitrogenase
repression
in
cyanobacteria. 840
In
the
presence
of
MSOX
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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
ammonia was produced both from nitrogen f i x a t i o n containing c e l l s , r e s u l t i n g in total it
and from urea in nickel-
loss of nitrogenase within 24 h. Hence
is clear that urea transport into c e l l s
is
not i n h i b i t e d
that the ammonia which r e s u l t s from urease a c t i v i t y I t has been shown by Bone (14) that urea i t s e l f synthesis
of
nitrogenase
nickel-depleted c e l l s
in
and which
we
have
by MSOX and
represses nitrogenase.
is u n l i k e l y to repress the
demonstrated
urease a c t i v i t y
this
was absent
by
using
(results
not
shown).
Therefore, these urea results confirm the conclusion of Turpin et
al.
and others
(4)
that
ammonia plays
such a role
and contradict
the
contradiction
and
conclusion of Rowell and Stewart (2) that i t does not. There
may
be
particularly
why,
nitrogenase
repression°
concentrations
(I
in
several our
reasons
experiments, Rowell
~M) of
for
the
this
MSOX did
and
Stewart
inhibitor.
not
protect
(2)
used
As shown in
against
very
Fig. 1
a
low 3 h
preincubation with 1 mM MSOX was necessary to completely block glutamine synthetase a c t i v i t y . accumulated
in
significantly
the
One r e s u l t of this was that the ammonia levels which presence of
the
inhibitor
higher than those of Rowell
level of glutamine
synthetase a c t i v i t y
presumably quite s i g n i f i c a n t ,
our
and Stewart
within
experiments (2)
for
were
whom the
12 h of MSOX addition was
r e s u l t i n g in a depletion of the i n t r a c e l l u l a r
ammonia l e v e l , which may well be c r i t i c a l is concerned.
in
In the absence of
as far as nitrogenase repression
added f i x e d
nitrogen
there
are three
sources of ammonia, each of which may have been s i g n i f i c a n t in our c e l l s in contributing
to ammonia levels
presence of MSOX. The f i r s t
sufficient
to repress nitrogenase
of these is nitrogen f i x a t i o n
itself.
in
the
I t has
been shown previously (7, 15) that nitrogenase a c t i v i t y varies dramatically with the age of c e l l s and we were careful to harvest c e l l s when nitrogenase activity (16).
was maximal.
The second source of ammonia is
photorespiration
Although the cultures were sparged with air containing 0.3% CO2 t h i s
may not have been s u f f i c i e n t
to suppress photorespiration
The t h i r d source of ammonia is protein degradation (17). 841
entirely
(16).
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
In all our experiments the pH values were c a r e f u l l y monitored and shown to be in
the range 7 to
phosphate alone was added; experiment.
Thus the
7.4 except in
the
case where ammonium
in that case the pH dropped to 6.4 during the
pH was well
below the
pK of
ammonia and hence
comparable with the low pH experiments of Turpin et al.
(4) in which the
c e l l s would require operation of the ammonium ion c a r r i e r . (not reported)
Similar results
were obtained when the experiments were done with
resuspended in I0 mM Hepes buffer, pH 7.1, as used by Turpin et al. also obtained similar
results
using
doubled concentrations
of
cells (4). We
urea and
ammonia. That the e f f e c t
of ammonia was not d i r e c t l y
on the nitrogenase
i t s e l f was shown by experiments (results not shown) in which i t to c e l l - f r e e extracts of nitrogenase; i n h i b i t i o n of a c t i v i t y . many n i t r o g e n - f i x i n g (18).
Furthermore,
was added
neither ammonia nor urea gave any
Similar results have been obtained with ammonia in bacteria, it
is
excluding unlikely
the
that
non-sulfur the
purple
effect
of
bacteria
ammonia in
suppressing nitrogenase is due to an e f f e c t as an uncoupler on the energy or reductant generating systems since this organism is
able to grow well
with ammonia concentrations s i g n i f i c a n t l y higher than those used herein (2, 19).
Ammonia has also been postulated to i n h i b i t
nitrogenase by competing
with i t for ATP and/or reductant (20, 21) but c l e a r l y t h i s would not be the case when i t s time
incorporation
course of
nitrogenase
is i n h i b i t e d by MSOX (21). activity
loss
observed
progressive decline, more consistent with an e f f e c t than d i r e c t i n h i b i t i o n . of
nitrogenase
differentiation inhibition
at
in
2 shows a
on protein
that
some stage
to
ammonia declines
nitrogenase during
differentation.
used herein would have contained at least
It
synthesis
heterocyst
is
to not
ammonia easy
since c l e a r l y
a proportion
heterocyst c e l l s at all stages during the experiments. 842
during
becomes r e f r a c t o r y
reconcile these observations with the present results cells
Fig.
the
I t has been suggested (22, 23) that the s e n s i t i v i t y
synthesis and
Furthermore,
to the
of mature
Vol. 134, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
The d e f i n i t i v e effectively
transportS
aspect of this
work was the
ammonia into
cells
requiring the ammonium transport system. on this urea produces ammonia. in
the
cells
confirmation
resulting that
in
under
use of
urea which
conditions
without
The subsequent action of urease
In the presence of MSOX ammonia accumulates nitrogenase
ammonia i t s e l f
is
repression.
We take
a primary regulator
of
this
as
nitrogenase
levels in A. cylindrica.
ACKNOWLEDGEMENTS
The Australian Research Grants Scheme (Grant No. D283154661) and the
Australian
financial
National
assistance
for
performing the c e l l - f r e e
University this
work.
Faculties We thank
Research Fund provided Dr D.M. Pederson for
nitrogenase assays and A. Daday for
assistance
with the glutamine synthetase assays.
REFERENCES .
2. 3. 4.
5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17.
Stewart, W.D.P. (1973). Ann. Rev. Microbiol. 27, 283-316. Stewart, W.D.P. and Rowell, P. (1975). Biochem. Biophys. Res. Commun. 65, 846-856. Singh, H.N., Rai, U.N., Rao, V.V. and Bagchi, S.N. (1983). Biochem. Biophys. Res. Commun. I I I , 180-187. Turpin, D.H., Edie, S.A. and Canvin, D.T. (1984). Plant Physiol. 74, 701-704. Boussiba, S. and Gibson, J. (1985). FEBS Lett. 180, 13-16. Rai, A.N., Rowell, P. and Stewart, W.D.P. (1984). Arch. Microbiol. 137, 241-246. Daday, A., Platz, R.A. and Smith, G.D. (1977). Appl. Environ. Microbiol. 34, 478-483. Daday, A., Mackerras, A.H. and Smith, G . D . (1985). J. Gen. Microbiol. 131, 231-238. Mallette, M.F. (1969). Methods Microbiol. l, 521-566. Dharmawardene, M.W.N., Haystead, A. and Stewart, W.D.P. (1973). Arch. Mikrobiol. 90, 281-295. Lambert, G.R. and Smith~ G.D. (1980). Arch. Biochem. Biophys. 205, 36-50. Peterson, G.L. (1977). Analyt. Biochem. 83, 346-356. Hallenbeck, P.C., Kostel, P.J. and Benemann, J.R. (1979). Eur. J. Biochem. 98, 275-284. Bone, D.H. (1972i. Arch. Mikrobiol. 86, 13-24. Weare, N.M. and Benemann, J.R. (1973). Arch. Mikrobiol. 93, 101-112. Bergman, B. (1984). Arch. Microbiol. 137, 21-25. Lambert, G.R. and Smith, G.D. (1981). Arch. Biochem. Biophys. 211, 360-367. 843
Vol. 134, No. 2, 1986 18. 19. 20. 21. 22. 23.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Gordon, J.K., Shah, V.K. and B r i l l , W.J. (1981). J. Bacteriol. 148, 884-888. Daday, A~, Lambert, G.R. and Smith, G.D. (1979). Biochem. J. 177, 139-144. Ohmori, M. and Hattori, A. (1974). Plant and Cell Physiol. 15, 131-142. Ohmori, M. and Hattori, A. (1978). Arch. Microbiol. 117, 17-20. Murry, M.A. and Benemann, J.R. (1979). Plant and Cell Physiol. 20, 1391-1401. Murry, M.A., Jensen, D.B. and Benemann, J.R. (1983). Biochim. Biophys. Acta 756, 13-19.
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