'

W. R. Ullrich C. Rigano
A. Fuggi P. J. Aparicio (Eds.)

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InorganicNitrogen
in Plantsand
Microorganisms
Uptakeand Metabolism

NITRATE TRANSPORT IN CYANOBACTERTA

M -G - G uer r e r o ,
pp .

J.M.

Romero, R. Rod rig u e z

7 9 - 85

Springer-Verlag
Berlin HeidelbergNew york
London Paris Tokyo Hong Kong Barcelona
I qq o

and c.

L a ra

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,

NITRATE TRANSPORTIN CYANOBACTERIA

M.G.Guerrero,
J.M. Romero,R. Rodriguez
andC. LaraI

Introduction
Prior to iu rcduction to amrnonium and conversion of the laner into an a-amino gmup,
nitrate has to enter the cell. Transportof nitrate into photosyntheticcells remainsthe
leastunderstoodstepin the global prooessof nitrateassimilation.Actyally, very linle is
known about the biochemistry of nitrate transport in higher plants and microorgani sms .
Studieson thc entranccof nilrate have conventionallybeenconductedby following
the uptakeof the ion from the extemal medium.This is particularly true for rhosemicroalgaewhich lack stonge organelles,including the cyanobacteria(blue-greenalgae).
Such an approachdoesnot allow to distinguishexperimentally betweenthe rransport
stepand the subsequentmetabolismof nitrate,althoughit hasbeenvaluableto indicate
the participationin nitrateassimilationof transportsystemswith a high affinity for
nitrate(K, in the micromolarrange)(Syrea 1988;Ullrich 1987).With vacuolared
microalgae,suchas the diatomPhaeodactylumtricornurunt,Syren and coworken have
addressedthe study of nitrate transportby following the appearenceof rhe substrate
within the cells (seeSyrett 1988,for a review).More recently,a sensitivemethodhas
beendevelopedto,measure
low intracellularnitratelevelsand hasbeenappliedto the
studyof nitrate trarlsportin unicellular cyanobacteria(Lara et al. 1987a; Romeroet al.
1989).In parallel,ptudiesof the polypeptidecompositionof cytoplasmicmembranes
arealsoproviding dataaboutproteinsinvolved in nitratetranspon(Omata 1988;Omara
et al. 1989;Sivak et al. 1988,1989).A fast developmenrin rhe understanding
of rhe
processcan lhus be predicted.
Being the first stepin nitrate assimilation,nitrate transportrepresentsthe first possiblepoint of controlof the wholeprocess.This hasbeenoverlookedin pastyears,attention being especiallyfocused on nitrate reductaseas a regulatory enzyme
responsiblefor the regulationof nitrate assimilation(Guerreroet al. l98l). More
recenlly,however,evidencehas accumulatedin favor of nitratetransportas the first
tightly controlled rate-limiting step in nitrate utilization (Lara et al. 1987 a; Syrert
1988),reinforcingthe rclevanceof metabolicregulationat the level of substrar supply
to the cell.
In this chapter we summarizc the prcsent state of knowledge on nitrate transport in
cyanobacteria,with particularemphasison our own rcsults.
csrc, Apdo.I I r3; 4to8Gscfrtta,
spain

sic,Frcuhaddc BiologfqUnivcrsidd dc Scvillay

UU
Nltrate Transport
The study of nitrate mnsport in cyanobacteriahas been an elusive task till rccently,
mainly becauseof the unavailability of a rcliable method to measurctranspon of nitrate
independentlyof its subsequentassimilation(Guerreroand l-ara 1987).Although it is
generallyassumedthat nitrate is actively transportedinto the cells,dircct experimental
evidenceis lacking in many cases.Recently,a sensitivemethodhasbeendevelopedto
measurcintracellularnitrate.It involvescentrifugationof the cells througha layer of
high
siliconeoil into an acid solutionand the analysisof cell lysatesby ion-exchange
performance liquid chromatographyfollowed by UV detection of nitratc at 210 nm
(Laraet al. 1987a; Romeroet al. 1989).
Using this procedure,intracellularnitrateaccumulationhas beenmeasuredin the
leopoliensis 1402-l)
unicellular cyanobacteriumAnacystisnidulans (Synechococcus
(Lara et al. 1987a). The intracellularnitrate level dependson the balancebetweenthe
rate of net nitrate entranceinto the cell and the rate of its reductionby nitratereductase.
ln intact nitrate-growncells with a fulty activenitrate reductase,low but significantintracellular concentrationsofnitrate can be estimated.In order to separatenitrate transport from reduction,experimentalsystemsar most convenientin which the cells exhibit high nitratetransportactivity togetherwith negligiblenitratereductionactivity.
Such a systemis provided by tungstatetreatrnentof the cells (Lara et al.1987 a),
which under controlled conditionsproducesan inactive nitrate reductase(Herrero and
Guerrero1986),or by keepingcells in the da* (Sivak et al. 1989)wherethe lack of
photogenerated
rcductantleadsto negligiblenitratereductionin vivo.In eithersystem,
A. nidulans
intracellularnitrateaccumulates
at high levels(Fig. l ). ln tungstate-trcated
cells assayedunder illumination(Fig. I A) intracellularnitrateincreaseswith time to
reacha plateauin about 2 min. Untreatedcells assayedin the dark transportnitrate to a
similar level although4 - 5 min are requiredto rcachmaximal accumulation(Fig. lB).
Regardingthe plateauas an equilibrium situation,the changein free energyassociatedwith the build up and maintenanceof theseintracellularnitrate concenhationscan
be calculatedaccordingto the Nemst equation.For a membranepotential of -100 mV
(Paschingerl9?7; Pescheket al. 1985),an initial extemal concentrationof 50 pM
nitrate and an intemal conoenttationof 370 pM (Fig. l), the corrcspondingfrec energy
changeamountsto about+15 U permol nitrate.A. nidulanscellsare,thereforc,able
gradientof the ion. The
to accumulateintricellularnitrateagainstthe electrochemical
resultsstnonglyindicate the operationof an endergonicnitrate transpoft systemin A.
nidulans ([:ra et al. 1987 a). In this rcgard, prcliminary data show that nitrate transport in A. nidulans is sensitiveto ionophoresand dicyclohexylcarbodiimide.
The exprcssionof the nitrate transportsystemis strongly affectedby the nature
of the availablenitrogensource.High nitratetransportactivity is exhibitedby nitrategrown A. nidulans cells, whereasonly negligible activity is detectedin ammonium-

8l

./ /
/'
I

a -6-'

/
/
I

2 z4o
I

rzo

f
100

150

3Oo

.l5O

lim r ( r )
Fig. l. Timc-coursccf intraccllularnitratc accumulationin Anacystisnidulans cclls. (A), rungstarc
reatedcclls assayedin the light. (B), unrcatcdcclls assaycdin thc dark. In both cascs50 pM KNOS
wasaddedat zetotimc. Pleasenotc the diffcrcnt dme scalcon lhe X-axis.PanclA takcnfrom Laract
al. 1987,with permission
grown cells (Lara et al. 1987 a; Sivak et al. 1989). Nitrate transport activity develops
after transfer of ammonium-grown cells to media containing nitrate or no available nitrogen source, but remains absent when the cells are transferred to media containing

ammoniumor ammoniumnitrate.The rcsults.indicatethat ammonium (or a productof

ammoniumassimilation)repressesthe developmentof nitrate transponactivity, nitrate
not being requiredas an inducer(Sivak et al. 1988, 1989).Such a pauem of rcgulation
-is
analogousto that of fte nitrate reducingenzymesof A. nidulau, namely nitrate reductase(Herrero et al. 1985) and nitrite reductase(Herrero and Guerrero 1986),
suggestingthat synthesisof both the nitrate transportand he nitrate reductionsystems
is subjectto the sarnetype of nitrogencontrol.
Analysis of the polypeptidecompositionof the cytoplasmicmembranesof A. nidu/anshasrcvealedthat the level of a 47-kDapolypeptidechangessignificantlyin responseto variations in the availablenitrogen source (Sivak et al. 1988, 1989). In
nitrate-grown cells, the 47-kDa polypeptide is a major membrane cofnponent,
representingabout 25% of total cytoplasmic membraneprotein. Synthesisof the 47kDa polypeptideis also subjectto ammoniumrepressionand is not dependenton
nitrateas an inducer, indicatingthat this adaptiveprolein is involved in the nitrate
assimilationsystem.The 47-kDa polypeptidemight correspondto niuate or nitrite

82
reductasc.Ncitherenzymeactivity was detcctcd,however,in the cytoplasmic
membrane
in 0r thylakoidfraoion,nitrite
fraction.Nitratercductase
activityappearcd
andsolublefraction.Intercstingly,
a
rductase
activitybeingprcsentin bothttrylakoids
in thecytoplasmic
closeparallelism
existsbetweenthelevelof ttre47-kDapolypeptide
membraneand0remagnitudeof thenitratetransportactivityof A. nidulonscellsin all
(Sivaket al. 1989).Table I illustratesthe point,
expeiimental
situationsassayed
of boththe47-kDapolypeptideandnitrate
showingthetime-courseof thedevelopnrent
transportactivityof cells,followingtransferfromammoniumto nitratemedium.Taken
together,the dataindicatethc involvementof the 47-kDapolypeptidein nitrate
transportin A. nidulans,mostprobablyas a componentof the nitratetransporter
(Sivaket al. 1988,1989).

Table l. Incrcasc in tlrc lcvcl of nitrar ranspon and thc 47-kDa cytoplasmic mcmbrancpolypcptidc
in Anocystis nidulans following thc s'ansfcrof ammonium-growncells to a medium containing

KNOr

Time(h)
0
t.5
3.0
4. 5
6.0
24.0

Intraccllular
NO3-(rM)
<2
<2
2'l
38
76
l8l

47-kDapolypeptidc(arcaunits)

r70
t50

490
570
720
3t20

Takenfrom Sivak et al. (1989),with pcrmission

Furthersupportto this view is providedby dataobtainedin AnccysrdsR2 (SyrechococcusPCC7942)by Omataand coworkers(1988,1989).The cytoplasmic
membranes
containanadaptive45-kDapolypeptide,
whichis a
of thiscyanobacterium
majorplasmamembrane
componentin nitrate-growncellsbut is virnally absentin
ammonium-grown
cells.A mutantnamedM45, constructed
by inactivatingthegene
is specificallydevoidof thisproteinirrespcctive
of
encodingthe45-kDapolypeptide,
the growthconditions.Comparative
studiesperformedwith the wild+ype andM45
on bothgrowthandnitrate-dependent
O,
strainson theeffectof nitrateconcentration
(Omata
evolutionsuggest
thatthedefectin M45 is at thelevelof nitratetransport
et al.
r989).
Theidentityof the45-kDapolypeptide
of AnacystisR2 with the47-kDapolypepThe45 - 47 kDaproteinappears
tideof A. nidulansseemsmostprobable.
to be anessentialcomponent
of thenitratetransportsystemof Anacystis,
althoughits exactrole
in nitratetransportactivityrcmainsto bc investigated.
Otherwisethe involvementof
otherpoly-*ptidesin thenitratehansportsystemcannotbe excluded.

contrlbutlon

of Nltrate Transporl to the control of Nltrate Asslmllatlon

NitrlE utilization is efficiently rcgularcd in tlre short-term through changesin the activity of the system.Nitrate transportappearsto be the major rate-limiting stcp and the
fint operativecontrol point of the processin cyanobacteria.
ln A. nidulans and other cyanobacteria,nitrate utilization is rapidly and effectively
hampercdby the prcsenceof ammoniumin the outer medium.This inhibition is mediated by ammonium assimilation products(Florcs et al. 1980). In addition, nitrate utilizationby Arucystis exhibits a tight dependenceupon the rate of CO2fixation, which is
hampercdafter lrcatment of the cells with D,L-glyceraldehyde,a selectiveinhibitor of
CO2 fixation. The negativeeffect of ammonium and the positive effect of CO2 on nitrateutilization are solhehowrclated,sinceammoniumassimilationinhibitorsrclieve
nitrate uptake from both the ammonium inhibition and the co2 rcquircment(Floreset
al. 1983;Romeroet al. 1985;see[:ra et al. 1987b, for a review).Acually, CO2acts
as an antagonistof ammonium in the control of nitrate uptake.In cells with moderate
ammonium assimilationcapacity(after trcatmentwith S-hydroxylysine)accelerationof
CO2fixation inducedby transition from CO2-limiting to CO2-saturatingconditionsrcsuls in abolishmentof the hegativeeffect of ammonium(Romeroet al. 198?).The nitrate entrancestep seemsto be the target of the effect of ammonium assim,lationand
CO2 fixation products.As shown in Table 2, nitrate'transportactivity of Anacystis
cells is effectively inhibited by ammonium addition or by impeding CO2 fixation with
D,L-glyceraldehyde.In either case,the inhibition of nitrate transport is preventedby
blocking ammoniumassimilationwith L-methionine-D,L-sulfoximine,
indicatingthe
commonbasisof the mechanismsunderlyingthe effectsof ammoniumand CO2on nitrate transport.The sensitivity of nitrate transportactivity to thosefactors that influence
tlre overall processof nitrateulilization thus indicatesthat nitrateassimilationin this
cyanobacteriumis primarily rcgulatedat the level of substratesupply to the cell (L:ra et
al. 1987a).

Tabfe2. Inhibitionof niratc ransponin Anacyslisnidulansby ammonium

or Dt-glyceraldehydc
(DtG) andis prcvcntion
(MSX)
by L-mcthioninc-D,L-sulfoximine
Mcan intraccllular nitrate conccntration[.rM)

Nonc
MSX

NrL*
MSX and NlIa+
DLG
MSX andDlIi
Takcn from l-ara ct al.(1987) with pcrmission

208
225
0
2ll
0
196

84
The rvailabledatathusemphasize
the outstanding
role playedby thc nitrate
transporstepin the regulationof nitrateutilizationin cyanobacteria.
The situation
(Syrett1988),andits validitymustbe
seemsto be sharcdby eukaryotic
microalgae
assessed
whendrawingconclusions
on rate-limitingstepsin nitrateassimilationby
other3,reen
cells.
AcknowledgmcntJ. Rcscarchin thc authors' laboratory has bccn supportcd by granrs from Junu de
Andalucfaand DGICYT (PB8E-0019).Wc thank Dr. T. Omata for making availablc unpublished
data.

Relerenccs
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Anacystisnidulans and other cyanobactcria.Arch Microbiol 128: 137-144
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nitratcutilizationandcarbondioxide lixation in the cyanobacterium
Anacystisnidulus. Biochim
BiophysActa 725: 529-532
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Cyanobacteria.
Elsevier,Amstcrdam,pp I 63-I 86
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85
slv* Mry,Lrn c, RomcroJM,Rodrfguez
R, GucrrercMG (19g9)Rclationship
bcrwccn
a 47kDa
-Bili;-Bidhy;
cybpf$mic mgnbrylg pltylcpridc-urd
nirar ransporrii*acys* iinti'f,.
:262
RccCrrunun | 58:E7
srrcftP.l,(f988)pnakc anaurilization
of nirogcncompounds.
In: RogersLt, GallonJR(cds)Biopress,Oxford,pp 23_39
qn9qlgy _o_t_ttrc
Atgacand-Cyanobactcria.
Clar,endon
-...
Ullric.hlVR (1987)Ninatcandainnronium
upnkcin grcenalgic a"a nifficipfa"rs:Mcctanismand
nitratc.
metabolism.
Ini Ullrich-wR,AfiariciopJlsyrirt FJ,Casti.to
F (cds)
J9lalignpniq.with
Inorganic
NitrogenMctabolism,
Springcr,
BcrlinHcidclbcigNcwyort, pp 32-39