In ol emen o he C- e minal Domain o an ATP-binding Subuni
in he Regula ion o he ABC- ype Ni a e/Ni i e T anspo e o
he Cyanobac e ium Synechococcus sp. S ain PCC 7942*
(Recei ed o publica ion, Ap il 10, 1997, and July, 7, 1997)
Masaki Kobayashi‡, Rocı´o Rod ı´guez§, Ca alina La a§, and Ta suo Oma a‡¶
F om he ‡Depa men o Applied Biological Sciences, School o Ag icul u al Sciences, Nagoya Uni e si y,
Nagoya, 464-01 Japan and he §Depa amen o de Bioquı´mica Vege al y Biologı´a Molecula , Facul ad de Biologı´a,
Uni e sidad de Se illa, Apa ado 1095, 41080 Se illa, Spain
In Synechococcus sp. s ain PCC 7942, an ATP-binding
casse e anspo e encoded by he genes n A, n B,
n C, and n D media es ac i e anspo o ni a e and
ni i e, which is inhibi ed by ammonium, a p e e ed
sou ce o ni ogen o he cyanobac e ium. One o he
ATP-binding subuni s o he anspo e , N C, has a
dis inc C- e minal domain o 380 amino acid esidues. A
mu an NC2, cons uc ed by emo al o his domain us-
ing gene ic enginee ing echniques, assimila ed low
concen a ions o ni a e and ni i e and accumula ed
ni a e in acellula ly, showing ha he domain is no
essen ial o he anspo e ac i i ies. Assimila ion o
low concen a ions o ni i e was only pa ially inhib-
i ed by ammonium in NC2 bu was comple ely inhibi ed
in he wild- ype cells. Cells o NC2 and i s de i a i e
(ni a e educ ase-less s ain NC4) ca ying he un-
ca ed N C bu no he cells wi h he wild- ype N C
accumula ed ni a e in acellula ly in he p esence o
ammonium in medium. These indings indica ed ha
he C- e minal domain o N C is in ol ed in he ammo-
nium-p omo ed inhibi ion o he ni a e/ni i e ans-
po e . In he p esence o ammonium, NC2 could no
assimila e ni a e despi e i s abili y o accumula e ni-
a e in acellula ly, which sugges ed ha educ ion o
in acellula ni a e by ni a e educ ase is also subjec
o inhibi ion by ammonium.
Ni a e is a majo sou ce o ni ogen o cyanobac e ia (1). I
is anspo ed in o he cell by an ac i e anspo sys em and
educed o ammonium by he sequen ial ac ion o ni a e e-
duc ase (NR)
1
and ni i e educ ase (NiR) p io o ixa ion in o
amide ni ogen o Gln. Exp ession o he ni a e assimila ion
ac i i y is nega i ely egula ed by ammonium (1, 2). In he
unicellula non-ni ogen- ixing cyanobac e ium Synechococcus
sp. s ain PCC 7942, he genes encoding he ni a e anspo
sys em (n A, n B, n C, and n D) (3–5), NR (na B) (6, 7), and
NiR (ni A) (8, 9) o m an ni A-n ABCD-na B ope on ( he ni A
ope on), and he ansc ip ion om he ope on is inhibi ed by
he addi ion o ammonium o he cyanobac e ial cul u es (9).
Ammonium inhibi s ansc ip ion h ough i s ixa ion in o Gln,
bu Gln is no he di ec egula o o ansc ip ion (9, 10). We
ha e p oposed ha cyana e, a me aboli e o Gln ia ca -
bamoylphospha e, ac s as he me abolic signal o he ammo-
nium-p omo ed ep ession o he ni A ope on (11).
Ni a e assimila ion by cyanobac e ia is subjec also o pos -
ansla ional egula ion, being inhibi ed upon addi ion o am-
monium o he cul u es (12, 13). The majo a e-limi ing s ep o
ni a e assimila ion in cyanobac e ia is ni a e anspo in o
he cell (3, 14), which has been shown o be inhibi ed by am-
monium (14, 15). Because inhibi ion o glu amine syn he ase
by L-me hionine-DL-sul oximine abolishes he nega i e e ec o
ammonium on ni a e anspo (14), ixa ion o ammonium o
Gln is clea ly equi ed o he egula ion. Howe e , he me a-
bolic signal leading o he inhibi ion o ni a e anspo and
he molecula mechanism o he egula ion emain o be
elucida ed.
The ni a e anspo sys em o Synechococcus sp. s ain
PCC 7942 anspo s ni i e as well as ni a e (16, 17) and
hence is a ni a e/ni i e anspo e . The p oduc o he n A
gene is a 45-kDa cy oplasmic memb ane p o ein (3), which has
been shown o be a ni a e/ni i e-binding lipop o ein (18). The
deduced N B p o ein has s uc u al simila i ies o he in eg al
memb ane componen s o he ABC (ATP-binding casse e)
anspo e s, and he deduced N C and N D p o eins ha e
he sequences ypical o he ATP-binding componen s o he
ABC anspo e s (5), showing ha he cyanobac e ial ni a e/
ni i e anspo e belongs o he supe amily o ABC ans-
po e s (19) o a ic ATPases (20). N C is unique among he
ATP-binding subuni s o he ABC anspo e s in ha i con-
sis s o wo dis inc domains, one o which (amino acids 1–254)
is s ongly simila o N D and he ATP-binding subuni s o
o he ABC anspo e s, whe eas he o he (amino acids 279–
659) is 30% iden ical in amino acid sequence o N A (5). In his
wo k, we cons uc ed and cha ac e ized dele ion mu an s o
Synechococcus sp. s ain PCC 7942 lacking N C o wi h a
unca ed N C lacking he C- e minal domain. Measu emen s
o ni a e and ni i e up ake om medium and o in acellula
ni a e accumula ion and examina ion o he e ec s o ammo-
nium he eon showed ha N C is an essen ial componen o
he ni a e anspo e and ha he C- e minal domain o N C
is in ol ed in he ammonium-p omo ed inhibi ion o he
anspo e .
EXPERIMENTAL PROCEDURES
S ains and G ow h Condi ions—A de i a i e o Synechococcus sp.
s ain PCC 7942 ha is cu ed o he esiden small plasmid pUH24
(R2-SPc, Re . 6; he ea e designa ed simply as s ain PCC 7942) and
he mu an s ains de i ed he e om we e g own pho oau o ophically
unde CO
2
-su icien condi ions as desc ibed p e iously (11). The
* This wo k was suppo ed by G an s-in-aid o Scien i ic Resea ch in
P io i y A eas 09274101 and 09274103 om he Minis y o Educa ion,
Science and Cul u e, Japan, G an PB94-1191-CO2 om Di eccion
Gene al de In es igacion Cien i ica y Tecnica, Spain ( o C. L.), and
G an -in-aid o Scien i ic Resea ch 80003243 om he Minis y o
Educa ion, Science and Cul u e, Japan ( o M. K.). The cos s o publica-
ion o his a icle we e de ayed in pa by he paymen o page
cha ges. This a icle mus he e o e be he eby ma ked “ad e isemen ”
in acco dance wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac .
¶To whom co espondence should be add essed. Tel.: 81-52-789-
4106; Fax: 81-52-789-4104; E-mail: [email p o ec ed].
1
The abb e ia ions used a e: NR, ni a e educ ase; NiR, ni i e
educ ase; kbp, kilobase pai (s); Chl, chlo ophyll; HPLC, high p essu e
liquid ch oma og aphy.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 43, Issue o Oc obe 24, pp. 27197–27201, 1997
© 1997 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g 27197
This is an Open Access a icle unde he CC BY license.
g ow h empe a u e was 30 °C unless o he wise s a ed. The basal me-
dium used was a ni ogen- ee medium ob ained by modi ica ion o
BG11 medium (21) as desc ibed p e iously (11). Ammonium-, ni i e-,
and ni a e-con aining media we e p epa ed by addi ion o 3.75 mM
(NH
4
)
2
SO
4
,5mMNaNO
2
, and 15 mMKNO
3
, espec i ely, o he basal
medium unless o he wise s a ed. All media we e bu e ed wi h 20 mM
HEPES-KOH (pH 8.2). When app op ia e, kanamycin was added o he
media a 25
m
g/ml. Exp ession o he ni a e assimila ion genes was
induced by ans e o ammonium-g own cells o he ni a e- o ni i e-
con aining medium as desc ibed p e iously (9).
Dele ional Mu agenesis—Two de ined mu an s o Synechococcus,
NC2 and NC3, we e cons uc ed by dele ing he 39po ion o n C
co esponding o he C- e minal domain o N C and he en i e n C
gene, espec i ely, om he ni A-n ABCD-na B ope on by he ma ke
exchange-e ic ion mu agenesis me hod (22) using a 3.8-kbp np I-sacB
ca idge excised om pRL250 (23) as he selec ion ma ke (Fig. 1A). In
NC2, a 1179-base pai in e nal segmen o n C, co esponding o
nucleo ides 798–1976 o he 1977-nucleo ide-long coding egion (do ed
in Fig. 1A), had been dele ed om he genome, and as a consequence o
he in- ame dele ion o nucleo ides, he modi ied n C encoded a p o-
ein o 266 amino acid esidues, consis ing o he N- e minal ATP-
binding domain (amino acids 1–254) and a pa o he linke sequence
connec ing he N- e minal and C- e minal domains (amino acids 255–
266) o N C (5). The n B and n D coding egions in NC3 we e
sepa a ed by 16 nucleo ides, GGAGCCCTATGAATTC, in which he
i s 10 bases we e de i ed om he i s 10 o he 14-base-long n B-
n C in e cis onic sequence and he las 6 bases we e de i ed om he
EcoRI ecogni ion sequence ha had been c ea ed by polyme ase chain
eac ion ampli ica ion o he n B and n D agmen s du ing he con-
s uc ion o he mu an . A NR-less de i a i e o NC2 (designa ed NC4)
was cons uc ed by inac i a ing na B acco ding o a p e iously de-
sc ibed p ocedu e o cons uc ion o he a ge ed NR mu an o Syn-
echococcus sp. s ain PCC 7942 (Dna B::kan, Re . 24; he e designa ed
NR1).
Isola ion and Analysis o DNA and RNA—Ch omosomal DNAs we e
ex ac ed and pu i ied om he Synechococcus cells as desc ibed by
Williams (25). Manipula ions and analyses o DNA we e pe o med
acco ding o s anda d p o ocols (26). Fo Sou he n hyb idiza ion anal-
ysis o he genomic DNA diges s, he ollowing gene-speci ic p obes we e
used (Fig. 1A): a 0.4-kbp BamHI-XhoI agmen o n B (p obe B), a
0.6-kbp HincII agmen o he N- e minal domain o n C (p obe C1),
and a 0.8-kbp Ps I-XhoI agmen o he C- e minal domain o n C
(p obe C2). To al RNA was ex ac ed and pu i ied om Synechococcus
cells by he me hod o Aiba e al. (27). Fo No he n hyb idiza ion
analysis, a 410-base pai polyme ase chain eac ion-ampli ied n D
agmen was used as a p obe (p obe D; Fig. 1A).
Exp ession o Plasmid-encoded N C in Synechococcus—A agmen
o n C, ex ending om nucleo ides 210 o 829 wi h espec o he
ansla ion s a si e, was ampli ied by polyme ase chain eac ion and
cloned in o pT7Blue T- ec o (No agen). The 14 h base o he sense
p ime used, co esponding o he 4 h base o he coding egion, had
been changed om C in he o iginal n C sequence o G o c ea e a NcoI
ecogni ion si e a he ansla ion s a si e. A e e i ica ion o he
nucleo ide sequence, a 0.6-kbp n C agmen , co esponding o nucle-
o ides 21 o 595, was excised om he plasmid by diges ion wi h NcoI
and SalI and joined wi h a 1.6-kbp SalI-XbaI agmen o pTO1 (5)
ca ying he es o n C and he 59po ion o n D be ween he NcoI
and XbaI si es o he shu le exp ession ec o pSE1 (18). The esul ing
plasmid (pNRTC1) encoded a modi ied N C, in which he second amino
acid esidue had been changed om Se o Ala. pNRTC1 was ans-
o med in o he NC3 mu an , and exp ession o n C was induced by 1
mMisop opyl-1- hio-
b
-D-galac opy anoside.
P epa a ion o An ibody agains he N C Polypep ide and Immuno-
blo ing Analysis—A 1.1-kbp SalI-XhoI agmen o n C was excised
om pTO2 (5) and cloned in he SalI si e in he polylinke o he
exp ession ec o pQE-31 (Qiagen). The esul ing plasmid ca ied a
chime ic gene encoding a unca ed N C (amino acids 199–569) used
o an N- e minal amino acid segmen ca ying six consecu i e His
esidues. The plasmid was ans o med in o Esche ichia coli M15
[pREP4] (Qiagen), and exp ession o he chime ic gene was induced by
1mMisop opyl-1- hio-
b
-D-galac opy anoside. The his idine- agged p o-
ein was pu i ied on Ni
21
-ni ilo iace ic acid esin (28) and used o
aising an ibodies in mice. Immunoblo ing analysis o he cy oplasmic
memb ane samples p epa ed om Synechococcus cells was pe o med
as desc ibed p e iously (18), using he an ise um agains he N C
polypep ide.
Measu emen s o Ni a e and Ni i e Up ake—Up ake o ni a e and
ni i e by ni a e (20 mM)-g own Synechococcus cells was measu ed a
30 °C in he ligh by ollowing he changes in concen a ions o ni a e
and ni i e, espec i ely, in he medium as desc ibed p e iously (18),
excep ha he pH o he assay medium was 8.2 o he measu emen o
ni a e up ake; ni i e up ake was measu ed a 9.6 as in he p e ious
s udy (18) so ha passi e di usion o ni ous acid (HNO
2
) in o he cells
is negligible (29).
Measu emen s o In acellula Ni a e Concen a ion—Synechococ-
cus cells we e g own a 40 °C wi h ammonium as he ni ogen sou ce.
Ni a e anspo ac i i y was induced by ans e o he ammonium-
g own cells o ni i e-con aining medium ollowed by incuba ion unde
he g ow h condi ions o 15 h. Accumula ion o in acellula ni a e
was measu ed a 40 °C in he ligh in he p esence o 20
m
MKNO
3
and
10 mMNaHCO
3
, using he silicone oil il e ing cen i uga ion echnique
and HPLC de e mina ion o ni a e as desc ibed p e iously (16).
O he Me hods—NR and NiR ac i i ies we e de e mined a 30 °C,
using oluene-pe meabilized cells wi h di hioni e- educed me hyl iolo-
gen as he elec on dono (30, 31). Ni a e and ni i e we e de e mined
wi h a low injec ion analyze (NOX-1000W, Tokyo Chemical Indus y
Co., L d.). Ammonium was de e mined as desc ibed by Ande son and
Li le (32). Chlo ophyll and p o ein we e de e mined acco ding o
Mackinney (33) and Low y e al. (34), espec i ely.
RESULTS
Cons uc ion o n C Dele ion Mu an s—In Sou he n hyb id-
iza ion analysis o he BamHI-XbaI diges o genomic DNA
om he wild- ype s ain, he n B-speci ic p obe hyb idized
wi h a 3.0-kbp DNA agmen (Fig. 1B,lane 1), which was
ecognized also by he p obes speci ic o he 59po ion (Fig. 1B,
lane 4) and he 39po ion (Fig. 1B,lane 7)o n C, as expec ed
om he es ic ion map o he ni A ope on (Fig. 1A). Wi h he
n B-speci ic p obe, he hyb idizing band in he diges o NC2
DNA was loca ed a 1.6 kbp (Fig. 1B,lane 2), which was
ecognized by he p obe speci ic o he 59po ion o n C (Fig.
1B,lane 5) bu no by he p obe speci ic o he 39po ion o n C
(Fig. 1B,lane 8), indica ing ha he 39po ion o n C had been
dele ed om he genome o NC2. In he diges o NC3 DNA, he
band hyb idizing o he n B-speci ic p obe was loca ed a 0.7
kbp (Fig. 1B,lane 3). The 0.7-kbp band hyb idized wi h nei he
o he n C p obes (Fig. 1B,lanes 6 and 9), indica ing ha he
en i e n C gene had been dele ed om he genome o NC3.
Exp ession o he O he Ni a e Assimila ion Genes in NC2
and NC3—As desc ibed p e iously (9), ans e o ammonium-
g own wild- ype cells o ni a e-con aining medium induced
accumula ion o he ni A ope on ansc ip , which hyb idized
wi h a p obe speci ic o n D, he gene loca ed downs eam o
n C (Fig. 2, lanes 1 and 2). The size o he hyb idiza ion signal
anged om 0.25 o .7 kilobases ep esen ing apid deg ada-
ion o he ni A ope on ansc ip (9). The NC2 and NC3 mu-
an s also accumula ed he ansc ip s hyb idizing wi h he
n D-speci ic p obe when ans e ed o ni a e-con aining me-
dium (Fig. 2, lanes 3–6), showing ha n D is ansc ibed in
he mu an s unde he egula ion o he ni A ope on p omo e
as in he wild- ype s ain.
In he mu an s as well as in he wild- ype s ain, he NR and
NiR ac i i ies we e induced by ans e o he cells om am-
monium-con aining medium o ni a e-con aining medium (Ta-
ble I). Because NR and NiR a e encoded by he las (na B) and
he i s (ni A) genes o he ni A ope on, espec i ely, he
unca ion o n C o i s dele ion om he ni A ope on did no
essen ially a ec he exp ession o he o he genes in he
ope on. Howe e , he NR ac i i y in NC2 was abou 50% o he
wild- ype le el (Table I) o an unknown eason. Because he
g ow h a e o NC2 in ni a e-con aining medium was simila
o ha o he wild- ype s ain and because he a e o ni a e
u iliza ion by NC2 cells was compa able wi h ha by he wild-
ype cells (see below), i was unlikely ha he low NR ac i i y
was limi ing ni a e assimila ion in NC2.
Exp ession o T unca ed N C in NC2—In immunoblo ing
analysis, he an ibody agains he N C polypep ide eac ed
wi h h ee polypep ides ha ing appa en molecula masses o
Regula o y Domain o a Cyanobac e ial ABC T anspo e 27198
67, 55, and 45 kDa, espec i ely, in he cy oplasmic memb ane
om ni a e-g own wild- ype cells (Fig. 3, lane 2). The ain
55-kDa band was obse ed in he memb ane p epa a ions om
ammonium-g own cells as well (Fig. 3, lane 1) and was asc ibed
o nonspeci ic binding o he an ibody o a cy oplasmic mem-
b ane p o ein. O he wo bands speci ic o ni a e-g own cells,
he 45-kDa band was p obably due o N A, which is 30%
iden ical in amino acid sequence o he C- e minal po ion o
N C and is he mos abundan p o ein in he cy oplasmic
memb ane o ni a e-g own cells (3). The 67-kDa polypep ide
was iden i ied as he p oduc o he n C gene, because i s
appa en molecula mass was simila o he calcula ed molec-
ula mass o N C (72 kDa). The cy oplasmic memb ane p ep-
a a ions om ni a e-g own NC2 lacked he 67-kDa polypep-
ide co esponding o N C bu had a 34-kDa immuno eac i e
polypep ide (Fig. 3, lane 4), which was absen in he wild- ype
cells (Fig. 3, lanes 1 and 2) and ammonium-g own NC2 cells
(Fig. 3, lane 3). Because i s appa en molecula mass was
simila o he deduced molecula mass o he p o ein encoded
by he unca ed n C (29 kDa), we iden i ied he 34-kDa
polypep ide as he unca ed N C lacking he C- e minal do-
main. These indings con i med he exp ession o he N- e mi-
nal po ion o N C and i s inco po a ion in o he cy oplasmic
memb ane in NC2. In acco dance wi h i s genome s uc u e,
NC3 showed nei he he 67- no he 34-kDa immuno eac i e
polypep ide (no shown).
G ow h and Ni a e Up ake Capabili y o he Mu an s—In a
medium con aining 2 mMni a e as he ni ogen sou ce, NC2
g ew as apidly as he wild- ype s ain, whe eas NC3 g ew only
poo ly (Fig. 4A). The cell suspensions o he wild- ype s ain
and NC2 (5
m
g Chl/ml) used 120
m
Mo ni a e un il i s exhaus-
ion in 30 min, whe eas he NC3 cells could no u ilize he low
concen a ion o ni a e (Fig. 4B). These indings indica ed ha
NC2 is capable o ac i e anspo o ni a e bu NC3 is no . A
de i a i e o NC3 ca ying plasmid-bo ne n C (designa ed
NC31) u ilized ni a e un il i s exhaus ion (Fig. 4B), indica ing
ha N C i sel is essen ial o ni a e anspo . The abili y o
NC2, ha ing he unca ed N C, o anspo ni a e he e o e
indica ed ha he N- e minal ATP-binding domain o N C bu
no he C- e minal domain is essen ial o he ac i i y o he
anspo e .
Regula ion o Ni i e and Ni a e Up ake in NC2—NC2 as-
simila ed low concen a ions o ni i e as e ec i ely as he
wild- ype s ain (Fig. 5, Aand B). As p e iously epo ed in a
closely ela ed species o Synechococcus (s ain PCC 6301, o -
FIG.1.Sou he n hyb idiza ion analysis o genomic DNA om
he wild- ype s ain and he n C mu an s. A, es ic ion map o
he ni A–na B egion o he genome o he wild- ype s ain (WT) and he
NC2 and NC3 mu an s. The ba s abo e he map ep esen he p obes
used o Sou he n and No he n hyb idiza ion analyses. The illed ba
in he map ep esen he 59po ion o n C encoding he ATP-binding
domain, and he do ed ba ep esen s he 39po ion o n C encoding
an N A-like domain (5). The genome egion eplaced by a kanamycin
esis ance gene in he na B mu an s NR1 and NC4 is shown by a illed
ba below he map o he wild- ype s ain. Res ic ion endonuclease
si es a e abb e ia ed as ollows: B,BlnI; Ba,BamHI; Bg,BglII; H,
HincII; P,Ps I; S,SalI/HincII; X,XhoI; Xb,XbaI. B, Sou he n hyb id-
iza ion analysis o genomic DNA om wild ype (lanes 1,4, and 7), NC2
(lanes 2,5, and 8), and NC3 (lanes 3,6, and 9). DNA samples (2
m
g/lane)
we e diges ed wi h BamHI plus XbaI, ac iona ed on a 1% aga ose gel,
ans e ed o posi i ely cha ged nylon memb ane (Hybond N1; Ame -
sham Co p.), and hyb idized wi h he
32
P-labeled gene-speci ic p obes
as indica ed.
FIG.2.No he n blo analysis o o al RNA om he wild- ype
s ain and he n C mu an s using an n D-speci ic p obe. Am-
monium-g own cells we e ans e ed o ni a e-con aining medium,
and RNA samples we e ex ac ed om he cells be o e and 30 min a e
he ans e . The RNA samples (10
m
g/lane) om wild ype (WT,lanes
1and 2), NC2 (lanes 5 and 6), and NC3 (lanes 3 and 4) ex ac ed be o e
(lanes 1,3, and 5) and a e (lanes 2,4, and 6) he ans e we e
dena u ed by ea men wi h o mamide, ac iona ed on a 1.2% aga-
ose gel ha con ained o maldehyde, ans e ed o posi i ely cha ged
nylon memb ane (Hybond N1; Ame sham Co p.), and hyb idized wi h
he
32
P-labeled n D-speci ic p obe.
TABLE I
NR and NiR ac i i ies in ammonium- and ni a e-g own cells o he
wild- ype and mu an s ains
Ammonium-g own cells o wild- ype Synechococcus sp. s ain PCC
7942 (WT) and he mu an s NC2 and NC3 we e ans e ed o ni a e
(15 mM)-con aining medium, and he enzyme ac i i ies we e assayed
be o e and 16 h a e he ans e . The alues a e he a e ages om
h ee measu emen s, and hose in he pa en heses show he anges.
S ain NR ac i i y NiR ac i i y
NH
4
1
NO
3
2
NH
4
1
NO
3
2
m
mol mg
21
Chl h
21
WT 17 (15–20) 259 (250–268) 10 (9–11) 47 (41–58)
NC2 19 (17–23) 129 (126–131) 11 (9–14) 82 (83–87)
NC3 11 (9–13) 258 (254–263) 9 (6–11) 98 (97–99)
Regula o y Domain o a Cyanobac e ial ABC T anspo e 27199
me ly Anacys is nidulans) (29), ni i e up ake by he wild- ype
PCC 7942 cells was inhibi ed by he addi ion o ammonium o
he medium (Fig. 5A) and esumed a e consump ion o he
ammonium in he medium (da a no shown). By con as , ni-
i e u iliza ion by NC2 was only pa ially inhibi ed by ammo-
nium (Fig. 5B). Al hough he ni i e up ake a e was educed
by 60% by ammonium, he cells u ilized ni i e and ammonium
simul aneously and e en ually used up ni i e in he p esence
o ammonium (Fig. 5B). A ni i e-speci ic anspo e was e-
cen ly ound in Synechococcus sp. s ain PCC 7942 (18). How-
e e , i canno accoun o he ni i e up ake by NC2 in he
p esence o ammonium, because he anspo e is exp essed
only unde s ess o ni ogen de iciency and is subjec o am-
monium inhibi ion.
2
The indings showed ha ni i e is ans-
po ed in o NC2 cells by he ni a e/ni i e anspo e in he
p esence o ammonium.
As p e iously epo ed in o he s ains o cyanobac e ia (12,
13), ni a e u iliza ion by he wild- ype PCC 7942 s ain was
inhibi ed by ammonium and esumed a e deple ion o ammo-
nium om he medium (Fig. 5C). Unlike ni i e up ake, ni a e
up ake by NC2 was comple ely and e e sibly inhibi ed by
ammonium (Fig. 5D) as in he wild- ype s ain.
Accumula ion o In acellula Ni a e and I s Regula ion—
Up ake o ni a e, as measu ed by ollowing he ni a e con-
cen a ion in medium, includes join ly anspo and educ ion.
To de e mine speci ically ni a e anspo ac i i y, in acellu-
la ni a e accumula ion in he ligh was measu ed in he
NR-de icien mu an s NR1 (24) and NC4, which we e con-
s uc ed om he wild- ype s ain and NC2, espec i ely. As
obse ed p e iously in a mu an o s ain PCC 7942 wi h no
app eciable NR ac i i y (15), NR1 accumula ed abou 1.5 mMo
ni a e in acellula ly a 20
m
Mex e nal ni a e concen a ion
in he absence o ammonium bu did no accumula e ni a e in
he p esence o ammonium (Fig. 6A). The a ge ed NR mu an
wi h unca ed n C, NC4, accumula ed ni a e o a concen a-
ion simila o ha obse ed in NR1 in he absence o ammo-
nium and, unlike he NR1 mu an ha ing wild- ype n C, ac-
cumula ed ni a e in he p esence o ammonium as well,
al hough he ini ial a e o ni a e accumula ion and he inal
in acellula ni a e concen a ion we e educed by 60% by
ammonium (Fig. 6B). These indings showed ha ni a e is
ac i ely anspo ed in o NC4 cells in he p esence o
ammonium.
Cyanobac e ial cells wi h unc ional NR educe in acellula
ni a e in he ligh and usually accumula e only low concen-
a ion o ni a e (20–30
m
M) (14). NC2 cells, howe e , accumu-
la ed ni a e unde illumina ion o a concen a ion as high as 1
mMin he absence o ammonium (Fig. 6C), al hough he in a-
cellula ni a e was subsequen ly deple ed p esumably due o
educ ion by NR (Fig. 6C). Simila o NC4, NC2 accumula ed
2
M. Okamu a, S. Maeda, M. Kobayashi, and T. Oma a, unpublished
esul s.
FIG.3. Immunoblo ing analysis o he p oduc s o he wild-
ype and he mu an n C genes. Ammonium-g own cells we e
ans e ed o ni a e-con aining medium, and cy oplasmic memb ane
samples we e ex ac ed and pu i ied om he cells be o e and 16 h a e
he ans e . The memb ane samples (10
m
g o p o ein/lane) om wild
ype (WT,lanes 1 and 2) and NC2 (lanes 3 and 4) ex ac ed be o e (lanes
1and 3) and 16 h a e (lanes 2 and 4) he ans e we e elec opho esed
in a 10% SDS-polyac ylamide gel. A e he elec opho esis, polypep-
ides in he gel we e elec o ans e ed o poly inylidene di luo ide
memb ane o immunos aining using he an ise um agains he N C
polypep ide.
FIG.4.G ow h and ni a e up ake capabili y o he wild- ype
s ain and he n C mu an s. A, g ow h cu es in a medium con ain-
ing2m
MKNO
3
.B, changes in ni a e concen a ion in he medium
a e addi ion o ni a e o he cell suspensions con aining 5
m
go
Chl/ml. E, wild ype (WT); ‚, NC2; M, NC3; L, NC31.
FIG.5.E ec s o ammonium on he up ake o ni i e and ni-
a e by he cells o he wild- ype s ain and he NC2 mu an . A,
ni i e up ake by wild ype (WT). B, ni i e up ake by NC2. C, ni a e
up ake by wild ype. D, ni a e up ake by NC2. Ni i e o ni a e (200
m
M) was added a ime 0 o he cell suspensions con aining 5
m
go
Chl/ml, and ammonium (500
m
Min Aand B; 150
m
Min Cand D) was
added a he ime indica ed by he a ows. Changes in he ni a e/
ni i e concen a ion (ci cles) and he ammonium concen a ion ( ian-
gles) in he medium a e shown. Ni i e up ake was measu ed a pH 9.6,
whe eas ni a e up ake was measu ed a pH 8.2 (see “Expe imen al
P ocedu es”). Open ci cles, con ol; closed ci cles, plus ammonium.
Regula o y Domain o a Cyanobac e ial ABC T anspo e 27200
ni a e in he p esence o ammonium, showing no deple ion o
he in acellula ni a e (Fig. 6C). The main enance o in a-
cellula ni a e accumula ion in NC2 cells in he p esence o
ammonium bu no in i s absence sugges ed inhibi ion by am-
monium o NR ac i i y, in acco dance wi h he comple e inhi-
bi ion by ammonium o ni a e up ake in NC2 (Fig. 5D).
DISCUSSION
In NC2, he up ake o ni i e om he medium p oceeded in
he p esence o ammonium (Fig. 5B), sugges ing ha he ni-
a e/ni i e anspo e o NC2 is much less suscep ible o
ammonium han he wild- ype anspo e . This, howe e , ap-
pa en ly con adic ed wi h he comple e inhibi ion by ammo-
nium o ni a e up ake by he mu an (Fig. 5D). Di ec meas-
u emen s o in acellula ni a e concen a ion, pe o med by
HPLC de e mina ion o ni a e in acid lysa es o he cell (16),
showed ha NC2 and i s NR-less de i a i e (NC4) accumula e
high concen a ions o ni a e in acellula ly in he p esence o
ammonium (Figs. 6, Band C). These indings indica e he
unc ioning o he ni a e/ni i e anspo e wi h unca ed
N C in he p esence o ammonium and hence he in ol emen
o he C- e minal domain o N C in he ammonium-p omo ed
inhibi ion o he anspo e . The inabili y o NC2 o assimila e
ni a e in he p esence o ammonium (Fig. 5D), despi e i s
abili y o accumula e ni a e in acellula ly (Fig. 6C), sugges s
ha he educ ion o in acellula ni a e by NR is also inhib-
i ed by ammonium. Ni i e has been ecen ly ound o ac i a e
ansc ip ion o he ni a e assimila ion ope on in Synechococ-
cus sp. s ain PCC 7942 (35). The simul aneous inhibi ion by
ammonium o ni a e/ni i e anspo and ni a e educ ion
would con ibu e o nega i e egula ion o he ni a e assimi-
la ion ope on by he mechanism o induce exclusion.
The molecula mechanism o he egula ion o he ni a e/
ni i e anspo ing ac i i y by he C- e minal domain o N C
emains o be elucida ed. In Synechococcus sp. s ain PCC
6301, a plasma memb ane p o ein kinase ac i i y has been
shown o be apidly inac i a ed, whe eas a soluble phospha ase
ac i i y is ac i a ed, a e exposu e o he cells o ammonium
(36). Because he ammonium-sensi i e p o ein kinase ac i i y
has been shown o phospho yla e se e al cy oplasmic mem-
b ane p o eins including hose comig a ing wi h N A and
N D (36), he egula ion o ni a e anspo may in ol e
phospho yla ion/dephospho yla ion o he anspo e . The C-
e minal domain o N C may ac as a senso o he dephos-
pho yla ion cascade igge ed by ammonium o may in e ac
speci ically wi h he o he pa s o he dephospho yla ed ans-
po e o inhibi he anspo . On he o he hand, he simila -
i y o he C- e minal domain o N C in amino acid sequence o
N A (5), which has been shown o be a ni a e/ni i e-binding
p o ein (18), sugges s ha he C- e minal domain o N C may
be an e ec o -binding domain. Because he mal ose ans-
po e o E. coli is inhibi ed by a p o einaceous e ec o binding
o he C- e minal ex ension o i s ATP-binding subuni (37, 38),
he egula ion o he cyanobac e ial ni a e anspo e migh
also in ol e a p o einaceous e ec o . Gene ic and biochemical
s udies a e being pe o med o cla i y he molecula mecha-
nism o he egula ion o ni a e/ni i e anspo by he C-
e minal domain o he ATP-binding subuni .
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FIG.6.E ec s o ammonium on in acellula accumula ion o
ni a e by Synechococcus cells. A, NR1. B, NC4. C, NC2. Ni a e (20
m
M) was added a ime 0 o he cell suspensions con aining 33.3
m
go
Chl/ml wi h o wi hou 250
m
Mammonium. Changes in he in acellula
ni a e concen a ion a e shown. Open ci cles, con ol; closed ci cles,
plus ammonium.
Regula o y Domain o a Cyanobac e ial ABC T anspo e 27201