FEMS MicrobiologyReviewsgg (1991) 1-14

© 1991 Federation of European MicrobiologicalSocieties0168-6445/91/$03.50

Published by Elsevier
ADONIS 016~6~5910t~0517

FEMSRE 00212

Ammonia translocation in cyanobactcria

Sammy Boussiba ~ and Jane Gibson 2

Micro-Algal Biotechnology Laboratory, The Biaustein Institute for Desert Research, Sede Boqer, Israel
and 2 Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Corndl University. Ithaca,
New York, U.S.A.

Received26 January 1990

Accepted 19 March 1990

Key words: Ammonium/methylammonium transport; Ammonia uptake; Cyanobacteria

1. SUMMARY through its inhibition on ghtamine synthetase,

Ammonia is the inorganic nitrogen source pref-
erentially used by most cyanobacteria. Moreover,
even in nitrate utilizers or N2-fixers, ammonia is
an obligate intraeellular intermediate in nitrogen
assimilation. It also affects the synthesis and the
activation of several key enzymes in nitrogen
metabolism such as nitrogenase, ghtamine syn-
thetase and nitrate reductase. The mechanism by
which NH3/NH~ enters different cyanobacteria
(N2-fixers, non.fixers, neutrophilic) was thus
studied. Using 14CH3NH~, the convenient radio-
active analog of ammonium, we have shown that
the neutrophilic A. nidulans R-2 possibly pos-
sesses an active transport system for this cation.
The conditions leading to repression and depres-
sion of this transport system have been studied; it
appears that de novo protein synthesis is required
for the acquisition of the transport ability. We
have also provided evidence that methionine
sulfoximine affects ammonium uptake only
Correspondence to: S. Boussiba, Micro-Algal Biotcchnology
Laboratory, The Blaustein Institut© for Desert Research, Scde
BoqerM990,Israel.
and found no support for the possible interaction
of this inhibitor with the ammonium transporter.
In the alkalophilic cyanobacterium Spirulina
platensis, an active mechanism to translocate am-
monium is probably not needed. Our data suggest
that net uptake of ammonia to support optimal
growth could be explained by a pH driven diffu-
sion process.
In all the different strains tested net ammonia
uptake was observed only when conditions permit
continuous amidation through the activity glut-
amine synthetase and inhibition of this enzyme by
methionine sulfoximine caused the excretion of
ammonia to the external medium. The weight of
experimentation suggests that ammonia leaks from
cells because of the inherent permeability of NH 3,
and that no specific carrier is involved in its
release.
2. INTRODUCTION
Ammonia occupies an unique biochemical posi-
tion, being the only inorganic form of nitrogen
that is directly incorporated into organic linkage,
and thus an obligate intermediate in the utilization
of other inorganic nitrogen sources. It tnight be
anticipated that maintenance of the intracellular
pool of ammonia within fairly narrow limits would
be important to ensure an appropriate substrate
supply for the enzymes involved in the synthesis
of amino groups. The problems to be overcome by
a free living cell in order to maintain constant
pools will depend on a number of factors, includ-
ing the nitrogen source being utilized. Cells grow-
ing in nitrate or dinitrogen will generate ammonia
internally, but this compound may be totally ab-
sent extracellularly, so that there will be a tend-
ency for loss to the environment, to an extent
defined by the permeability of a cell's limiting
membrane to the unionized and ionized forms of
ammonia as well as the properties of specific
membrane channels. In contrast, if ammonia is
present in significant concentrations in the sur-
rounding solution, diffusion alone might suffice to
supply growth needs, but the internal pool in this
case would probably be almost unregulated, and
subject to marked changes if the pH of the en-
vironment was altered. Specific transport systems
have evolved to deal with such problems, and over
the last years, systems for concentrative uptake of
ammonium ions have been defined in many free.
living microorganisms (see [1,2] for reviews).
Free-living cyanobacteria inhabit environments
that contain in the main low dissolved concentra-
tions of the nutrients required in largest quantity
for growth, such as carbon dioxide and nitrogen
sources. Most filamentous forms capable of di-
nitrogen fixation carry out this process in special-
ized cells termed heterocysts, although a few
nitrogen-fvdng forms in which no such differentia-
tion occurs are also known. It is rarely possible to
detect extracellular ammonia in nitrate- or di-
nitrogen-using cultures, although the intracellular
pool of ammonia in all cases where it has been
measured is of the order of 1 mM [3-6]. In sym-
biotic forms, in contrast, upwards of 60% of the
total products of 13N2 reduction formed in a 5-rain
incubation of freshly isolated Azolla symbionts
appeared as 13NH~ in the surrounding medium
[7], indicating that the limiting membrane of these
cyanobacteria must be permeated by at least one
of the two possible forms of ammonia, NH 3 or
NH~'. As discussed further below, the extensive
production of NH4~ from both exogenous and
endogenous sources by other cyanobacteria in-
cubated under special conditions suggests that this
is a general phenomenon. It has generally been
assumed that specialized channels or carriers
would be required to allow significant movements
of the ionic form NH~ across any membrane, but
that NH 3, a small, gaseous molecule, should be
quite freely permeable. However, in very few cases
has this assumption been supported by measure-
ments. The only determination of NH3 permeabil-
ity in a cyanobacterium appears to be for gyn-
echococcus R-2 [6]; the values for P of approxi-
mately 6 am s"" are very similar to values ob-
tained for the charophyte Chara corallina [8].
Since the cells' limiting membrane is evidently
moderately permeable to NH 3, the internal con-
centration of total ammonia (NH 3 + NH~') will
be influenced not only by the total external con-
centration (very often no more than/~molar), but
also by the pH gradient between the inside and
outside of the cell; at steady state, total intracellu-
lar ammonia will exceed the external to an extent
defined by the Henderson-Hasselbalch equation
at external pH values above 7.6, the average in-
tracellular pH of illuminated cells [9,10]. It is
perhaps significant in terms of cellular ammonia
economy that cyanobacteria are generally very
tolerant to high pH, and many can thrive in
environments where the pH is 11 or above [11].
Some, but by no means all, become sensitive to
raised ammonia concentrations at high pH [12,13].
However, acidophilic strains are almost unknown
[14], and one possible explanation is that low
external pH would tend to drain the cells of
ammonia through diffusion, followed by trappir:g
in the protonated form in the outside compart-
ment. Clearly, the presence of specific transport
systems might materially affect the ability of cells
to continue growth under conditions otherwise
unfavorable for maintaining internal pools.
Internal pools of total ammonia in cyanobac-
teria, as well as in other prokaryotes, generally
amount to 0.6-1.5 mM [3,15,16]. The cyano-
bacterial cell is, however, divided into distinct
intrathylakoid and cytoplasmic compartments, and
a pH gradient across the thylakoid membrane in
excess of 2 pH units has been estimated [17]. If, as
estimated in that study, the intrathylakcid com-
partment is 10~ of the internal volume of the cell,
and if the permeability of the thylakoids to NH 3
is similar to that of ~.hecytoplasmic membrane, a
significant proportion of the total ammonia pool
extracted by acid would presumably be seques-
tered in the thylakoids while cells are illuminated.
More detailed work on the ammonium/methyl-
ammonium transport system of Escherichia coli
that has appeared since the comprehensive reviews
of Kleiner [1,2] has demonstrated that this amt
system is under the control of the general nitrogen
regulatory system ntr [18]. The physiological ef-
fects of transposon insertions strongly suggest that
a single system is responsible for ammonium
transiocation in E. coli [19]; this conclusion was
also drawn from investigations with a mutant of
Klebsiellapl~eumoniae [20]. However, reliance upon
a single system for ammonium transport does not
appear to be a general rule among micro-
organisms. In addition to the case in c.yano-
bacteria discussed later in this review, more than
one system for NH~" translocation appears to be
present in Rhodobacter sphaeroides [16] and in R.
capsulatus [21]. Multiple systems, generally de-
freed genetically, have been found in many
eukaryotic microorganisms, including Aspergillus
nidulans [22], Saccharomyces cerevisae [23], Neu-
rospora crassa [24] and Chlamydomonas reinhardtii
[25],
Three aspects of ammonia translocation specifi-
cally in cyanobacteria will be discussed in this
review: (1) Uptake of NH~/NH3; (2) transport of
| +
the radioactive analogue CH3NH 3 ; and (3) am-
monia excretion. As is now usual, the term am-
monia is used to cover both the protonated and
unprotonated forms of this compound, while NH 3
or NH~" is used when either form is meant specifi-
cally.
3. GENERAL METHODOLOGY
Ammonium uptake determined only by the rate
of removal of NH~" from the medium is almost
certain to underestimate the actual rate of trans-
port, and reflect closely the net rate of assimila-
tion into organic molecules within the cell. Incom-
ing ions will be added to an existing pool, which is
drawn on by the amination and amidation reac-
tions and replenished by turnover of proteins and
amino acids. The intracellular pool of ammonia in
unicellular strains [3,26] and Anabaena [5,27]
quickly returns to 'normal' levels even when the
cells are deprived of an external nitrogen source,
and can be maintained under these conditions, so
that the only source of this ammonia has to be
endogenous. The extent to which protein and
amino acid turnover continues in growing cells is
not known. Additional factors contribute to un-
derestimation of transport rate: first, transport
systems are inherently reversible, as demonstrated
in the case of a cyanobacterium by exchange-dif-
fusion experiments [28], so that it is not possible
to measure accurately the rate at which individual
ions move in and out of the cell using a natural
substrate, initially present on both sides of the
membrane. Secondly, at the intracellular pH of
about 7.6 maintained by illuminated cells [6,9],
approximately 1~ of the internal pool of ammonia
is unprotonated and more freely permeable
through the membrane than the charged ion, lead-
ing to a second mode of loss from the internal
compartment, l~topic methods, which permit de-
termination of initia~ rates, can minimize these
~3roblems, but the inconveniently short half-life of
N, and the relatively large samples needed for
tSN/14N ratio measurements, has restricted their
use in such investigations. Instead, very extensive
use has been made of 14C-labelled methylamine,
which, unlike the next higher homologue ethyl-
amine, appears to be a reliable analogue for stud-
ies of ammonia transport as judged by the criteria
discussed by a number of workers [e.g., 2,27,29].
Two caveats concerning its use require greater
emphasis than is usually given, however; tint, the
common assumption is made that the unproton-
ated forms of both the natural substrate and its
analogue have similar permeabilities through the
cytoplasmic membranes. Few direct measurements
have been published, for there are inherent diffi-
culties in accurate determinations of permeabili-
ties across the cytoplasmic barrier of small cells,
such as the very short time needed to reach equi-
librium and the tendency for overestimation of
surface area when using microscopic methods [6].
However, both absolute measurements, and an
indirect procedure based on concentration of
amine needed to collapse pH gradients, indicate
that methylamine is close to 10 times more per-
meable than is ammonia (P for NH 3, 6-8 ~tm s-l;
for CH3NH 2 about 60-100 ~/m s -!) not only in a
unicellular cyanobacterium but also in two other
eubacteria [6,30]. The higher permeability of the
unprotonated form of the analogue as compared
with the natural substrate will affect both the
apparent rate of uptake into cells and the final
steady-state concentration achieved. The overall
effect of the higher permeability will, however, be
substantially offset by the smaller ratio of unpro-
tonated to total amine in the internal environment
consequent on its higher pK a (10.65 at 25°C as
compared with 9.25 for NH3). A second and more
controversial problem in the use of methylam-
monium as an analogue for ammonium in trans-
port studies is that, although there have been no
reports that methyl~mmoniumcan be utilized as a
nitrogen source by cyanobacteria, the intracellular
formation of T-methyl 81utamine by both uni-
cellular and filamentous species has been amply
demonstrated [4,27,28]. TI~s requires that a dis-
tinction be made between free and chemic~dly
modified radioactive analogue associated with the
cells in all experiments; this point is brought out
further in ~he discussion of investigations of meth-
ylammonium uptake below.
4. UPTAKE OF NH~'/NH 3
4.1. Ammonia as the preferred nitrogen source for
growth
Cyanobacteria can utilize a number of nitrogen
yources to support growth. All can use nitrate or
ammonia when available, and some are also able
to fix atmospheric nitrogen. When I/ixed nitrogen
is available, this is almost always used in prefer-
ence to dinitrogen. Nitrate or dinitrogen must first
be reduced to ammonia before assimilation, and
the preferential use of ammonia could thus be
accounted for in terms of cell energy balance. The
high energetic cost of dinitrogen reduction has
been extensivelydiscussed, and reduction of nitrate
must also involve a significant diversion of re-
ductant from carbon dio:tide fixation. We have
recently determined that Anabaena siamensis, a
nitrogen-fixing cyanobacterium isolated from rice
paddies in Thailand, grew faster under limiting
illumination when using ~,.nunonia than with
nitrate or under nitrogen-fixit~gconditions, with
doubling times of 3.7 h and 5.~ h, respectively
(Boussiba and Thomas, unpublished).
4.2. Ammonium repression of nitrate assimilation
When suppled with both ammonia and nitrate
as potential nitrogen sources in the growth
medium, cyanobacteria will first completely de-
plete the medium of ammonia and only then utilize
nitrate [31]. It may be inferred that ammonia
regulates key enzymes involved in nitrate assimila-
tion. A direct effect on the activity or synthesis of
nitrate reductase has be.en ruled out, since reduc-
tion of nitrate uptake by whole cells occurred
almost immediately on adding ammonia, and the
activity of nitrate reductase measured in per-
meabilized ceils that had been exposed to am-
monia for short periods was barely affected. An
alternative explanation for inhibition of nitrate
uptake through a direct effect of ammonia on
nitrate transport has been suggested [31-33]. The
inhibition of nitrate uptake by ammonia is
eliminated if $1utamine synthetase, which in
cyanobacteria appears to constitute the main, if
not the only, route of assimilation into organic
linkage [34,35], is blocked by the specific inhibitor
methionine sulfoximine (MSX) [36,37]. Thus am-
monia inhibition of nitrate uptake requires con-
tinued assimilation of internal ammonia by
8tutamine synthetase. This situation is comparable
to the effect of MSX on nitrogenase in the pres-
ence of ammonia: the addition of ammonia to an
uninhibited nitrogen-fixing culture represses
nitrogenase [37], but nitrogenase activity is main-
tained if MSX is also added. Based on such find-
ings, it has been suggested that glutamine, or a
closely related metabolite derived from that com-
pound, mediates inhibition of both nitrate and
dinitrogen utilization.
4.3. Toxic effects of ammonia at high pH
The preponderance of NH 3 over NH + at pH
values greater than about 9.2, the pK,, for am-
monia at 20-30°C, and the relatively high per-
meability of the unionized form of ammonia, po-
ses a potential hazard for photosynthetic micro-
organisms in which pH gradients play a key role
in energy conservation. Inhibition of photophos-
phorylation, associated with elevated intrathyl-
akoid pH and decreased transmembrane pH
gradients, has been thoroughly documented in iso-
lated chloroplast thylakoids.
Ammonia has also been reported to uncouple
cyanobactcrial photosynthesis at high pH [12],
since 5 mM ammonia severely inhibited t4CO2
photoassimilation at pH 9 in a number of strains.
The degree of inhibition appears, however, to be
much reduced in strains that are restricted to
growth in alkaline environments, and so may be
considered alkalophilic. Figure 1 shows that while
5 mM ammonia inhibited light-dependent 02
evolution by 90% in a neutrophilic Anabaena
species, only 30~ inhibition was observed in the
alkalophilic Spirulina platensis at the same pH.
These observations may be due to the different
pH gradients maintained by the two organisms;
the much greater gradient in Anabaena, which
maintains an average internal pH of about 7.5, as
compared with 8.5 in gpirulina (see below) might
be expected to result in a larger influx of NH 3 in
the neutrophile and hence greater disruption of
energy conservation.
IO0~N~
o 8o
} .
o 5 z'~ z5 ~'6
Ammonia :
Fig. l. Effect of ammoniaon oxygenevolutionof ~,.platerais
and Ana~aena 7120, at pH 10. 100~ activitywere: 6.36 and
4.52/,mol oxygenmg-i chl rain-i. Open symbols, Spirulina;
dosed symbols,Anabaena.
4.4. Mechanism of uptake
One of the chezacteristic features of active
transport is accumulation of the solute against its
concentration gradient. When the solute is an ion,
then the magnitude and sign of the membrane
potential must also be considered. We have shown
that the unicellular Anacystis nidulans (now re-
named Synechococcus) can accumulate mnn~nia
against a concentration gradient of close to 1000,
leading to an intern~,:pool of 1-1.5 raM, while the
external concentration was reduced to below the
detection limit of approximately 2 pM [3]. The
membratte potential maintained by the cells under
comparable conditions was approximately -120
mV, implying significantly higher internal pools
than could be accounted for by electrical equi-
libration (Ritchie and Gibson, unpublished). The
pool was slightly reduced, but not lost, when cells
were maintained in darkness for over 30 rain, and
the membrane potential was also maintained al-
most unchanged when cells were no longer il-
luminated. Ammonia was taken up at the same
rate from solutions at pH 6.6-8.7 (Table 1), with
no detectable change in affinity; an apparent K m
of less than 1/tM was inferred from these experi-
ments. It is reasonable to conclude that the am-
monia species transported must be NH~, since its
relative concentration is changed by less than 50%
by proton dissociation over the pH range studied,
while NH 3 would constitute only about 5 × 10-3
of the total ammonia present at pH 6.6. The pH
insensitivity of ammonia uptake found in our ex-
periments [3,38] contrasts sharply with the find-
ings on what are thought to be ammonium/meth-
ylammonium systems studied with the analogue in
other microorganisms [2].
The effects of inhibitors on ammonia uptake
(Table 2) suggest that amidation is critical in
regulating net uptake. Compounds such as iono*
phores and dicyclohexylcarbodlimlde (DCCD) af-
f~t energy conservation primarily, but indirectly
will also reduce energy available for membrane
transport as well as COa fixation and the supply
of acceptor for ammonia assimilation. Methionine
sulfoximine, by inhibiting ghtamine synthetase,
also prevents further net ammonia uptake. It is
not possible in such experiments to distinguish
between inhibition of the transport system itself
Table 1 (the shortest time measured), followed by a slower
Lack of effect of pH on ammonia uptake rate in Anacyst/s phase (Fi 8. 2). The fast phase was unaffected
nidulans R-2 when 81utamine synthetase was completely in-
Cellsfrom an exponentiallygrowingculture in BOll +NO~" hibited by MSX. The slow phase, in contrast, was
were washed and resuspended in N-free BGll containin 8 20 eliminated by MSX. Preliminary results (Boussiba
mM K phosphate. The pH was adjusted by small additions of and Belkin~ unpublished data) indicate that at an
0o$ M HCI or KOH, and did not change during the uptake
measurements.
external pH of 10.0, the cytoplasmic pH is ap-
proximately 8.5, as has been found also for other
pH Uptake rate alkalophilic bacteria [39]. The ApH is thus large
(nmol nfin-I rag-i protein)
enough and of the correct sign to lead to an
6.2 26.7 intracellular accumulation of ammonia, following
7.0 34.1
diffusion of NH 3 [40,41]. Indeed when cells were
8.1 29.7
83 31.5 treated with toluene to eliminate any pH 8radient,
the fast influx of ammonia was abolished. Follow-
in8 influx, the continuous activity of glutamine
synthetase supports further net entry by withdraw-
and an indirect effect exerted through glutamine
ing ammonia from the internal pool.
synthetase, while flux through the transporter con-
tinues unabated.
These considerations lead to the conclusion that
the major form of ammonia crossing the cell mem.
brahe of neutrophilic cyanobacteria wowing on
this nitrogen source in the pH range 7-8 is prim. 340
arily NH~, and that some form of active transport
must be involved in its accumulation. In al.
kalophilic strains such as Spirulina platensis and
Cyanospira rippkae, which grow optimally at pH
over 10 using ammonia as nitrogen source, a sim-
ple mechanism of NH3 diffusion followed by trap-
ping through protonation in the more acidic inter-
hal compartment may be sufficient. Recent data
obtained using S. platensis [13] support this
suggestion. At pH 10, uptake of ammonia con-
sisted of two phases: an initial rapid disap- 260

pearance of added ammonia, lasting less than 5 s

Table 2
Effect of inhibitorson ammonia uptake rates in A. nidulans 2~I0 i i I i I ~ i I
R-2 0 S 15 45 GO

Addition Finalconcentration Uptakerate

(/~M) (%of control)" Time : see
Fi E. 2. Effect of MSX on short.term ammonia uptake by S.
IX:CD 5 0
CCCP 5 l0 platensis at pH 10. gpirullna cells taken from cultures growing
SF 6847 5 4
at steady state were washed in fresh medium, resuspended in
MSX 50 0b
the appropriate buffer and concentrated up to 1 ms protein
nd-1. Amraoniaat timezerowas 350 pM. MSX (100 tiM)was
a Average uptake rate 28.5 nmo| rain -1 m8 - I protein, added $ rain before ammonia uptake assay started, by which
b Suspensions (50 Fg protein ml - t ) were incubated 10 rain time GS acti~ty wascomplete|yinhibited,o, withoutMSX'e,
~ t h MSX before uptake measurements. with MSX.
5. METHYLAMMONIUM UPTAKE
As h~s been the case for studies of ammonium
uptake, the major interest in the investigations
that have made use of the analogue methylam-
monium is in connection with broader aspects of
nitrogen metabolism and its regulation. Much em-
phasis has therefore been placed on correlating
methylammonium uptake with nitrogen fixation
and with intracellular amidation carried out by
81utamine synthetasc.
5.1. Effect of growth conditions
Cyanobacteria grown either with nitrate or with
dinitrogen as nitrogen source have regularly been
found to take up CH3NH~ from suspending
media buffered at approximately neutral pH. Ob-
served uptake rates are up to one tenth of the rate
at which NH~" can be removed from the medium.
However, growth with ammonia represses methyl-
amine uptake to less than 105 of the rate mea-
sured in cells grown on other nitrogen sources,
although such cells remain able to remove am.
monia to below measurable concentrations. This
response of CH3NH~ uptake to growth environ-
ment in itself provides a strong argument for
existence of a specific transport system.
5.2. Assay conditions
The pH of the suspending medium governs the
extent to which an amine is protonated, and there-
fore the proportion of the total molecules avail-
able for transport as the ion or through passive
diffusion. Many investigators have therefore cho-
sen to work at external pH values of 7.0-7.5,
where only 0.t~ of the total methylamine added is
uncharged, or it~ the range of 9 ~0, where around
50~ is in this form. In other respects, the assay
conditions used have differed widely', for example,
most investigations witi~ Anabaena species from
Stewart's (e.g. [4,27A2]) and Singh's [29,,t3]
laboratories used cells from nitrogen~fixing cul-
tures washed and suspended in buffe~ only, while
growth conditions were used in work with A.
flos.aquae [44], and buffers supplemented with
bicarbonate have been used for Synechoc6xus
[28,45], The differences in carbon supply io cells
may influence uptake kinetics, as is further dis-
cussed below.
Both equilibration and uptake measurements
have commonly been carried out under illumina-
tion, and although some uptake may occur in
darkness, there is general agreement that signifi-
cant uptake of methylammonium at neutral, as
opposed to alkaline, pH requires energy, and is
driven by the membrane potential (B~) compo-
nent of protonmotive force. In gynechococcus, for
example, CH3NH ~" uptake was as rapid at pH 7.0
as at 7.5, where external and intracel!ular pH are
equal, while transient reduction of B~/' by adding
high concentrations of K +, or more permanent
collapse by nigericin, is strongly inhibitory [28].
In Synechococcus, CH3NH ~" uptake requires
the presence of Na + in the external medium (Fig.
3), but the praise role played by this ion in
transport has not been defined. In common with
most other types of cells, b/a + is extruded from
energized cyanohatteria [46], and the very rapid
flux in illuminated cells establishes a sodium mo-
tive force (dpbla +) that exceeds dpH + by 15-20
mV (Ritchie and Gibson, unpublished). Coupled
movements of CH3NH + and reentering Na + in
w
k- 6
rr
o
...w 4
r'-
E
cL 7
e-
[ 2
*-'~.
~m
0 -- J
I0
,,,!
20
I
30
I,,,
40 SO
I
NaCI: mM
Fi8. 3. Effect of Na + on methylammonium uptake rate in ,4.
t,idulans R-2. Exponentially 8rowing cells were wazhed twice in
29 mM potassium phosphate buffer and resuspended to 135/Ag
of protein ml-t in 20 mM potassium phosphate-10 mM
KHCO3(pH 7.1)•Suspension~werepreincubatedfor 15 rainat
30°C with illumination from a 100-W lamp at a distance of 30
cm. NaCI ~ddifions were made just before the assay was
started by t.he addition of 9 am 14CH3NH3CI(specific activity,
56 mCi nmol-I). Samples were taken at 5 and 60 s and
a,~sayedfor retain~ activityas describedin the text•
response to the membrane potential is a possible
explanation. Published experiments with fila-
mentous cyanobacteria have been carried out in
sodium-containing media, so that a specific re-
quirement for this ion if it is indeed common to all
cyanobacteria would not have been observed. A
further difference between the uptakes of
CH3NH ~ and of NH~ is that a requirement for
Na + cannot be shown for the natural substrate.
5.3. Kinetics
With a small number of exceptions, noted be-
low, uptake of methylammonium by both fila-
mentous and unicellular cyanobacteria is biphasic,
an initial rapid phase which lasts about 1 rain
being followed by a slower, sustained uptake which
may continue linearly for at least I h [4,28,29,44].
The rapid phase shows saturation kinetics, with
apparent K m values usually around 10/tM (Fig.
4), while the slow phase appears little affected by
concentration at neutral pH in Synechococcus, al-
though it is increased at pH 9 or above [27,44].
When excess NH~ or a higher concentration of
t2CH3NH~ is added to the suspension at the end
24~
~. 20o
f by addin8 NH~ to the suspension, so that the
~ 12C o,4
T O.3
q_ S,-~ .
~-~, :,,, T , - , - ,4
0 I 2 3
TIME:n~in
Fig. 4. Kinetics of methylammonium transport A. nidulans
R-2. Exponentially growing cells were washed and resuspended
in 20 mM potassiumphosphate-10mM NaHCO3(pH 7.1)as
describedin the legendof Fig. 3. After 15 min of preincuba-
tion at ~O°C,s~l~:nsionsweretransferredto 15°C and kept
at this temperaturefor 5 rainbeforeadditionof 14CH3NH3Ci
and samplingfor radioactivitytaken up. Shownin the main
figureis the time-courseof uptake;|4CH3NH3C!9 ItM;specific
activity,56 mCi mmoI-t. Symbols:o, fight; e, dark. Inset:
Double reciprocal plot of data (15°C) using 2-20 /~M
t'CH3NH~ (specificactivity,56 mCi nunol-t) and 40 pM
14CH3NHsC!(specificactivity,3.6 mCi retool-l). S, micro-
molar;V, nanomol/min/mgof protein).
of the rapid phase, all the radioactivity is released
from the cells (Fig. 5). However, after the slow
phase has continued for some time, only a quan-
tity of radioactivity approximately equal to that
taken up in the rapid phase is released. In Syn-
echococcus [28] and in A. variabilis [4,26,27] the
proportion of radioactivity retained corresponds
accurately with the accumulation of T-methyl
glutamine within the cells. Since this accumulation
is prevented in cells that have been exposed to
methionine sulfoximine, the slow phase in these
organisms appears clearly associated with the
$1utamine synthetase activity, and the ability of
this enzyme to use the analosue in place of am-
monia. Different conclusions have been reached as
a result of experiments with A. flos-aquae [44] and
A. doliolum and A. cycadae [29,43,47], in part on
the basis of experiments with MSX and its effect
on the slow phase of uptake. An interestin8
81utamine auxotroph of A. cycadae [29] isolated
on the basis of resistance to MSX had no detect-
able $1utamine synthetase activity as measured by
the transferase assay, yet showed a slow phase of
uptake comparable to that seen in the parent.
Only a portion of the total radioactivity?ssociated
with the cells which had accumulated 14CHsNH~.
for an extended time was chased out of the cells
possibility that the analogue had been metabolized
by the mutant, perhaps through some pathway
independent of glutamine syntbetase, has not been
eliminated. Much of the discussion revolves around
the effect and specific action of MSX which may
have other targets in addition to glutamine syn-
thetase, although it has been shown to be highly
specific for this enzyme, with which it forms a
covalent adduct [36]. Both Canvin's and Singh's
groups have su~ested a direct effect on a second
specific transport system responsible for the slow
phase of uptake. The interpretation of these ex-
periments "is complicated by the fact that MSX
added to intact cells has itself to be transported
into the cell to reach glutamine synthetase, and
appears to use a transport system which also func-
tions with glutamine [48]. Hence the onset of
enzyme inhibition is slow relative to CH3NH~"
uptake (see [28,44]). Since ammonia evolution sets
in with a comparable time course, it has also been
 8t 1 ity of glutamate as an acceptor in the glutamine
? may vary in different strains, or even be depen-
Z ~"
w.a
-rE
1- E
t3t-
L~ I .... [ cyanobacterial glutamine synthetase is not regu-
I 2 5
TIME(mln)
Fig. 5. Effectof NH~ additionon t4CH3NH~ accumulatedin
the presence of methionine sulfoximine 4. nidulans R-2.
Time-courseof 14CHzNH~"accumulationis shown.Exponen-
tiallygrowingcellswerewashedand resuspendedin potassium
phosphate-lOmM NaHCO3 (pH 7.1)and equilibratedin light
at 30oC for 10 rain; 100 pM methioninesulfoximinewas then
added and incubationwas continuedfor 10 rain.14CH3NH3Ct
(9 ~tM;specificactivity,56 mCi mmol-t) was added, followed
1 min later (at arrow) by the addition of 5 pM NH4CI.
Samplesweretakenat the timesindicated.
suggested that the considerably delayed inhibition
of CH3NH ~" uptake by MSX in Synechococcus,
which affects the rapid phase of uptake also, is a
result of net internal NH~" generation when
glutamine synthetase is inactivated [28].
Two uptake phases are not always observed.
The rate of the second phase of uptake varies in
different strains or species, and Kerby et al. [26,27]
noted that it was much slower in A. cylindrica
than in A. variabilis. Although free-living strains
of A. cycadae clearly show both rapid and slow
phases of uptake, cyanobionts harvested freshly
from host roots took up CH3NH ~ rapidly at first,
but no slow phase was observed; all accumulated
radioactivity was released by added NH~ [47], so
that, although glutamine synthetase was active in
broken cells, and was able to react with CH3NH ~
in place of NH~', this reaction was not occurring
in the cells. Again, some different interpretations
of the experiments are possible: the cells used in
both of these investigations were incubated in
buffers without bicarbonate, so that the availabil-
synthetase reaction may have been rate-limiting. A
second suggestion is that the relative affinities of
glutami~e synthetases for CH~NH~ and NH~
dent on growth conditions; some preliminary sup-
port for the former suggestion has been obtained
[27], and the observation that the cyanobionts
were unable to take up NH~ could also be the
result of regulatory effects on glutamine syn-
thetase within the cell, that were relieved after
extraction. It has been shown repeatedly that
lated by the adenylylation/deadenytylation sys-
tem found in enteric bacteria; however, activation
of the enzyme by light-reduced thioredoxin has
been demonstrated [49]. Preliminary experiments
with antiserum prepared against Synechococcus
strain R-2 glutamine synthetase have shown at
least two electrophoretically separable forms of
the native enzyme, whose ratio is affected by the
nitrogen source used for ~owth (Brake and Gib-
son, unpublished), and it is evident that the regu-
lation of the enzyme in vivo is not yet fully
understood.
5.4. Regulation
It has already been noted that growth of all the
strains of cyanobacteria used in these investiga-
tions with ammonia as nitrogen source yields cells
that fail to take up CH3NH ~ rapidly at neutral
pH. Nitrate-grown, and therefore uptake-com-
petent, Synechococcus cells lose this activity when
incubated in growth medium either with NH~" or
CH3NH ~ , although in the latter the cells become
chlorotic and the increase in cell mass is limited.
In contrast, the rate of transport of CH3NH ~ is
maintained and even enhanced if the medium
contains glutamine, which in this strain supports
only slow growth, presumably because of trans-
port limitation [50]. The interpretation of dere-
pression kinetics in unicellular forms, as is the
case with diazotrophic strains, is complicated by
the accompanying nitrogen deficiency and need
for synthesis of enzymes for utilizing the new
nitrogen source, whether this be nitrate or di-
nitrogen. However, a rise in CH3NH~" uptake
becomes apparent within 1-2 h, and is dependent
on new protein synthesis [50,51]. These experi.
ments indicate a coordinated response between
transF.)rt and the utilization of nitrogen sources
other than ammonia, but details of the regulatory
system remain to be established. However, two
recent publications bear on the genetic control of
CH3NH ~" transport. Mutants of A. variabilis,
selected for resistance to concentrations of
CH3NH~ toxic to the parent at pH 7, had greatly
reduced fast and slow phases of uptake at pH 7,
although diazotrophic growth was unimpaired [42].
This mutant therefore appears to lack the trans-
port system responsible for rapid uptake, but not
to suffer any ill effects from this lesion in terms of
its ability to reduce and incorporate dinitrogen.
These observations appear inconsistent with the
suggested role of the transport system in recaptur-
ing internally generated NH4+ [2,28]. It must be
remembered, however, that the primary product of
nitrogen fixatio~ is assimilated into organic com-
pounds within the heterocysts, rather than being
transferred as such into the vegetative cells which
are presumably the main site of CH3NH~" trans-
port. Considerable differences in detailed function
between differentiated diazotrophs and unicellular
cyanobacteria using nitrate, and experimental def-
inition of CH3NH~ transport in filamentous
cyanobacteria grown with nitrate does not appear
to have been attempted. An unexpected finding
with the mutant was that growth in the presence
of CH3NH ~ appeared to result in expression of a
further transport system for this compound al-
though, in common with all cyanobacteria,
CH3NH ~ will not support growth of the mutant
and was metabolized no further than to y-methyl
$1utamine. A mutant of lVostoc muscorum, re-
sistant to the herbicide glyphosate, had lost the
ability to differentiate heterocysts, to fix dinitro-
gen, and also to accumulate CH3NH ~" [52]. Some
general regulatory phenomenon appears therefore
to be involved in coordinating responses to changes
in nitrogen supply, perhaps analogous to the ntr
system of enteric bacteria [18].
6. AMMONIUM EXCRETION
Cyanobacteria can take up ammonia from their
environments at a rate that appears to be limited
less by the activity of their transport systems for
NH4+ at neutral pH or by diffusion of NH3 at
alkaline pH than it is by the net rate at which the
internal pool of ammonia is converted into amino
groups. The internal pools appear relatively con-
stant [e.g. 3,26], suggesting that factors leading to
internal ammonia production will also result in
net release of this compound. Substantial produc-
tion of ammonia by cyanobacteria can indeed be
observed under special conditions, and the bio-
technological implications for tropical agriculture
have received considerable attention.
6.1. Conditions favoring ammonium excretion
Ammonia formation is generally observed when
assimilation is blocked by pretreatment of cells
with MSX to covalently inhibit glutamine syn-
thetase. Nitrate supplied to such cells is reduced
to anmionia and released [53-55], and cells re-
main active for long periods. Substantial ammonia
production from dinitrogen has also been ob-
served in the presence of MSX [56-62]. Lower
rates of MSX-induced ammonia production result-
ing from protein breakdown in strains incapable
of nitrogen fixation have also been reported
[26,63]. A number of mutants of nitrogen-fixing
strains that release ammonia continuously have
been isolated. The most common features of these
mutants are reduced 81utamine synthetase activity
together with increased nitrogenase [26,64-67].
6.2. The mechanism of ammonium excretion
A common feature of these experimental sys-
tems is that 81utamine synthetase is impaired while
energy supply and alternate nitrogen sources con-
tinue to be available. One may ask whether am-
monia is lost from the cells as NH~', possibly
through reversal of the transport system, or
through diffusion of NH 3, which, as discussed
above, is able to diffuse quite rapidly through the
cell membrane. Indirect evidence obtained re-
cently suggests that ammonia is lost from the cell
as NH 3 in a process that is not energy-dependent.
In Spirulina platensis the loss of ammonia was pH
dependent, the rate increasing when the external
pH was more acid (Fig. 6). Ammonia release
brought on by MSX inhibition was unaffected by
pretreatment of the cells with N-ethylmaleimide,

4O
.=..,
3o
+~ 10
• I t I t I . I
30 60 90 120
Time: min
Fig. 6. Ammonium excretion from S, platensis treated with
methionine sulfoximine.Cells treated as in Fig. 2. MSX was
added to a final concentration of 100 itM. Sample were taken
up every 30 rain, and ammonia was determined in the filtrate.
D, pH 7.0; It pH 10.
which inhibited ammonia uptake without affecting
energy balance over a period of at least 1 h; this
may indicate that uptake and release are indepen-
dent processes, and that excretion is not mediated
by channels involved in NH~ uptake, in contrast
to the mutants of enteric bacteria that lack the
a m t system [19,20] and release ammonia, a re-
cently isolated mutant from a nitrogen-fixing
cyanobacterium (Boussiba nnd Thomas, unpub-
lished) released ammonia continuously while fix-
ing nitrogen, but still possesses an ammonium
uptake ~stem as evidenced by the ability to accu-
mulate ! CH3NH~," Taken together, the weight of
experimentation suggests that ammonia leaks from
cells because of the inherent permeability of NH 3,
and that no specific carrier is involved in its
release.
7. CONCLUSION
It has become obvious over the last five years
that cyanobacteria, whether symbiotic or free-liv-
ing, diazotrophic or requiting fixed nitrogen
sources for growth, possess transport systems
which can function in the accumulation and reten-
tion of ammonia, and of its analogue methyl-
amine, from neutral .environments. The effective
competition between ammonium and methylum-
monium ions in uptake experiments carried out at
pH values around neutrality leads to the conclu-
sion that the ions share a common u p ~ e path-
way. However, cells in which methylammonium
transport is repressed arc unimpaired in their abil-
ity to take up ammonium, and differences be-
tween ionic requirements for uptake of the two
ions have also been found. The suggestion that
multiple systems exist for transporting NH~, and
that only one of these also transports CH3NH~,
has therefore been put forward~ but requires sub-
stantiation for exampJc by genetic evidence. There
are strong similarities among different species be-
tween the relative uptake affinities for true sub-
strafe and analogue, and in some features of
CH3NH ~ uptake regulation. As in other bacteria,
these systems are driven by membrane potential,
but there is suggestive evidence for Na + involve-
ment in the transport system responsible for
CH3NH ~ uptake, as yet in only one species.
The major uncertainties at present concern the
number and relative substrate affinities of such
systems in any one strain of cyanobacterium, the
membrane structures involved in mediating trans-
port, and the detailed molecular components in-
volved in their regulation. General lines of experi-
mentation and inference suggest that more than
one system for moving NH~/CH3NH ~" against
concentration gradients exist, and a parallel with
the multiple transport systems for critical ions
such as K* and ghosphate in enteric bacteria can
be drawn. Clearly, molecular approaches will be
invaluable in answeting the questior ~ outstanding,
and in establishing the physiological significance
of the transport systems as well as in attempts to
exploit the potential of ¢yanobacteria for increas-
ing soil fertility. A number of interesting mtd
relevant mutants have been described recently,
and the tremendous advances made in the last few
years in molecular analysis of cyanobacteria opens
the possibility for better characterization of the
organization and control of these systems. Further
support for the existence of one or more types of
ammonium carrier in ¢yanobaeteria could be ob-
tained with active cytoplasmic membrane vesicles,
which should help greatly to resolve some of the
apparent differences between the translocation of
the natural substrate NH4+ and its convenient
analogue 14CH3NH+.
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