JOURNAL OF BACTERIOLOGY, No . 2004, p. 7337–7343 Vol. 186, No. 21
0021-9193/04/$08.00⫹0 DOI: 10.1128/JB.186.21.7337–7343.2004
Copy igh © 2004, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
The N cA-Dependen P
1
P omo e Is U ilized o glnA Exp ession in
N
2
-Fixing He e ocys s o Anabaena sp. S ain PCC 7120
Ana Vallada es, Alicia M. Mu o-Pas o , An onia He e o, and En ique Flo es*
Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones
Cien ı´ icas-Uni e sidad de Se illa, Se ille, Spain
Recei ed 15 Ap il 2004/Accep ed 19 July 2004
Exp ession o he glnA gene encoding glu amine syn he ase, a key enzyme in ni ogen me abolism, is subjec
o a a ie y o egula o y mechanisms in di e en o ganisms. In he ilamen ous, N
2
- ixing cyanobac e ium
Anabaena sp. s ain PCC 7120, glnA is exp essed om mul iple p omo e s ha gene a e se e al ansc ip s
whose abundance is in luenced by N cA, he ansc ip ion ac o exe ing global ni ogen con ol in cyanobac-
e ia. Whe eas RNA
I
o igina es om a canonical N cA-dependen p omo e (P
1
) and RNA
II
o igina es om a
␴
70
- ype p omo e (P
2
), RNA
IV
is in luenced by N cA bu he co esponding p omo e (P
3
) does no ha e he
s uc u e o N cA-ac i a ed p omo e s. Using RNA isola ed om Anabaena ilamen s g own unde di e en
ni ogen egimens, we obse ed, in addi ion o hese ansc ip s, RNA
V
, which has p e iously been de ec ed
only in in i o ansc ip ion assays and should o igina e om P
4
. Howe e , in he e ocys s, which a e
di e en ia ed cells specialized in N
2
ixa ion, RNA
I
was he almos exclusi e glnA ansc ip . Analysis o
P
glnA
::lacZ usions con aining di e en agmen s o he glnA ups eam egion con i med ha agmen s
ca ying P
1
,P
2
,o P
3
and P
4
ha e he abili y o p omo e ansc ip ion. Mu a ion o he N cA-binding si e in
P
1
elimina ed P
1
-di ec ed ansc ip ion and allowed inc eased use o P
2
. The N cA-binding si e in he P
1
p omo e and binding o N cA o his si e appea o be key ac o s in de e mining glnA gene exp ession in
ege a i e cells and he e ocys s.
Glu amine syn he ase is a key enzyme in ni ogen me abo-
lism in all li ing cells and cons i u es he ou e o inco po a-
ion o ino ganic ni ogen (in he o m o ammonium) in o
o ganic ma e ial in many mic oo ganisms and plan s. The glnA
gene encoding glu amine syn he ase is subjec o egula ion
and is exp essed a maximal le els when cells a e incuba ed
unde ni ogen-limi ing condi ions. Whe eas ac i a ion o ex-
p ession o glnA in en e ic bac e ia is media ed by he well-
cha ac e ized N B-N C wo-componen egula o y sys em
(27), in cyanobac e ia ansc ip ion o he glnA gene is unde
con ol o N cA, he global ni ogen con ol ansc ip ion ac-
o o hese o ganisms (19). The glu amine syn he ase p o ein
is abundan in cyanobac e ia (26), and glnA is a p edic ed
highly exp essed gene (30) whose ansc ip is eadily de ec ed
(15, 45).
The cyanobac e ia, which belong o he domain Bac e ia, a e
cha ac e ized by pe o ming oxygenic pho osyn hesis and a e
conside ed he e olu iona y p ecu so s o algal and highe -
plan chlo oplas s (17). The cyanobac e ial RNA polyme ase is
simila o he well-cha ac e ized en e obac e ial enzyme (43),
al hough he poC gene encoding he RNA polyme ase ␤⬘
subuni is spli in he cyanobac e ia in o wo genes, poC1 and
poC2, which encode polypep ides ␥and ␤⬘, which a e homol-
ogous o he N- e minal and C- e minal hal es o en e obac-
e ial ␤⬘, espec i ely (3, 53). Thus, he a chi ec u e o he
cyanobac e ial RNA polyme ase co e is ␣
2
␤␤⬘␥ ins ead o he
en e obac e ial ␣
2
␤␤⬘ (43). The p incipal RNA polyme ase ␴
ac o in Anabaena sp. s ain PCC 7120 ( he p oduc o he sigA
gene) is homologous o he ege a i e Esche ichia coli ␴
70
and
Bacillus sub ilis ␴
43
ac o s (5). Consis en ly, a numbe o cya-
nobac e ial gene p omo e s ha e been ound o bea a ⫺10 box
in he o m TAN
3
T (9, 22), and some o hem also ca y a
ecognizable ⫺35 box (42).
Al hough nume ous cyanobac e ial p omo e s do no con-
ain an ob ious ⫺35 box, some ha e an ex ended ⫺10 egion
(TGNTAN
3
T) simila o ha ound in a subclass o E. coli
p omo e s ha unc ion wi hou a ecognizable ⫺35 box (2,
10). This is he case o he p omo e o he bcLXS ope on
encoding ibulose-1,5-bisphospha e ca boxylase/oxygenase.
N cA-dependen p omo e s ep esen ano he class o p omo -
e s ha do no con ain a ecognizable ⫺35 box. These p o-
mo e s possess a ⫺10 box (TAN
3
T) and an N cA-binding si e
cha ac e ized by he signa u e sequence GTAN
8
TAC, which is
usually cen e ed a abou posi ion ⫺41.5 wi h espec o he
ansc ip ion s a si e (19). N cA belongs o he CAP amily o
bac e ial ansc ip ional egula o s, and he p omo e s uc u e
jus desc ibed is simila o ha o he class II CAP-dependen
p omo e s (7). N cA unc ions in esponse o he C/N balance
o he cell, and 2-oxoglu a a e has been iden i ied as a pu a i e
e ec o o N cA (35, 44, 46–48). The n cA gene appea s o be
au o egula o y, and n cA exp ession, which akes place a a
basal le el in he p esence o ammonium, inc eases in esponse
o ni ogen limi a ion (21, 22, 34, 38, 49).
Unde combined ni ogen limi a ion condi ions, ilamen ous
cyanobac e ia like Anabaena sp. s ain PCC 7120 di e en ia e
he e ocys s, which a e specialized cells ha a e he si es o N
2
ixa ion in ae obically g own ilamen s (13). He e ocys di e -
en ia ion does no ake place in n cA mu an s (15, 49) o in
mu an s wi h mu a ions in he de elopmen egula o y gene
he R (4, 6). The ex ensi e s uc u al and physiological di e -
* Co esponding au ho . Mailing add ess: Ins i u o de Bioquı´mica
Vege al y Fo osı´n esis, Cen o de In es igaciones Cien ı´ icas Isla de la
Ca uja, A da. Ame´ ico Vespucio s/n, E-41092 Se ille, Spain. Phone:
34 95 448 9523. Fax: 34 95 446 0065. E-mail: [email p o ec ed].
7337
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
ences be ween he e ocys s and ege a i e cells a e la gely he
esul o di e en ial gene exp ession (50). Thus, o ins ance,
ni HDK encoding he ni ogenase complex is exp essed only in
he e ocys s, whe eas bcLXS is exp essed only in ege a i e
cells (13). Some genes, howe e , ha e o be exp essed in bo h
ypes o cells. This is he case o glnA (13), since glu amine
syn he ase is in ol ed in ni ogen assimila ion in he ege a i e
cells and also p o ides he pa h o inco po a ion o he am-
monium p oduced in he N
2
ixa ion eac ion in he he e ocys s
(51).
The p omo e egion o he glnA gene in Anabaena sp. s ain
PCC 7120 has a complex s uc u e and gene a es se e al di -
e en RNA species co esponding o RNA
I
o RNA
V
(Fig. 1).
RNA
I
, whose 5⬘end is loca ed 93 nucleo ides ups eam o he
glnA ansla ion s a , is mo e abundan in he absence o
ammonium han in he p esence o ammonium (45), is N cA
dependen , and o igina es om a p omo e ( ha we call P
1
)
wi h he canonical s uc u e o he N cA-dependen p omo e s
(15, 19). Indeed, N cA binds wi h high e iciency o i s binding
si e in he P
1
p omo e , and his N cA-binding si e has been
oo p in ed (37). RNA
II
, whose 5⬘end is loca ed 155 (45) o
157 (15) nucleo ides ups eam o he s a codon, is de ec ed
independen o he ni ogen sou ce, al hough i s le el is some-
wha highe in he p esence o ammonium han in he absence
o ammonium. The p omo e ha o igina es RNA
II
,P
2
,is
simila o he canonical ␴
70
-dependen p omo e s and can be
used in an E. coli s ain ca ying he Anabaena glnA gene in a
plasmid (45), as well as in in i o ansc ip ion assays wi h bo h
Anabaena and E. coli RNA polyme ases (43). RNA
III
, whose
5⬘end has been loca ed ei he 244 o 247 (45) o 196 (15)
nucleo ides ups eam o he ansla ion s a , may a ise om
p ocessing o RNA
IV
a he han ep esen a ue ansc ip ion
s a si e (42). The e is some unce ain y abou he exac 5⬘end
o RNA
IV
; i is 273 (43), 275 (15), o 266 (45) nucleo ides
ups eam o he s a codon. Howe e , he egion om nucle-
o ide 282 o nucleo ide 277 ups eam o he ansla ion s a
has a sequence ha ep esen s an accep able ␴
70
- ype ⫺10 box
(TATTAA). In e es ingly, conside ing a ew ups eam nucle-
o ides, a sequence (TGNTATTAA) ha could ep esen an
ex ended ⫺10 box is e iden . The p omo e ha gene a es
RNA
IV
,P
3
, has been desc ibed as N cA dependen (15), al-
hough i has no e iden N cA-binding si e, and i can be used
in in i o ansc ip ion assays wi h bo h Anabaena and E. coli
RNA polyme ases (43). Finally, RNA
V
, whose 5⬘end is lo-
ca ed 319 nucleo ides ups eam o he ansla ion s a , co e-
sponds o a ansc ip ha has been de ec ed only in in i o
ansc ip ion assays (43), al hough he pu a i e p omo e o
his ansc ip ion s a poin has a ecognizable ⫺10 box (42).
The complex pa e n o exp ession summa ized abo e has
been in e p e ed in e ms o exp ession o he glnA gene
mainly om a ␴
70
- ype p omo e (P
2
) in ammonium-g own
Anabaena cells and om a ni -like p omo e (P
1
, which was
la e iden i ied as an N cA- ype p omo e [15]) unde N
2
- ixing
condi ions (45). In his wo k, we show ha P
1
is he p omo e
used in he e ocys s.
MATERIALS AND METHODS
Bac e ial s ains and g ow h condi ions. Anabaena sp. s ain PCC 7120 was
g own axenically in BG11 medium (39), which con ains 17.6 mM NaNO
3
,in
BG11
0
(ni ogen- ee) medium, o in BG11
0
medium supplemen ed wi h 2 mM
NH
4
Cl and 4 mM N- is(hyd oxyme hyl)me hyl-2-aminoe hanesul onic acid
(TES)–NaOH bu e (pH 7.5). Fo pla es, he media we e solidi ied wi h sepa-
a ely au ocla ed 1% aga (Di co). Cul u es we e g own a 30°C in he ligh (75
mic oeins eins 䡠m
⫺2
䡠s
⫺1
) wi h shaking (80 o 90 pm) o liquid cul u es.
Anabaena sp. s ain PCC 7120 de i a i es ca ying a Km
Nm
gene casse e (12)
we e ou inely g own in medium supplemen ed wi h 25 ␮g o neomycin 䡠ml
⫺1
,
and s ains ca ying Sm
Sp
gene casse e C.S3 (12, 36) we e g own in medium
supplemen ed wi h 2 o 5 ␮g o spec inomycin 䡠ml
⫺1
and2 o5␮go
s ep omycin 䡠ml
⫺1
. The chlo ophyll acon en s o he cul u es we e de e mined
by using me hanolic ex ac s o cells (23).
Fo RNA isola ion, cells g owing exponen ially in BG11
0
C medium (BG11
0
medium plus 10 mM NaHCO
3
) o BG11
0
C medium supplemen ed wi h 5 o 8
mM NH
4
Cl (plus a double concen a ion o TES-NaOH bu e [pH 7.5]) and
bubbled wi h a mix u e o CO
2
and ai (1:99, ol/ ol) we e used. Fo induc ion
expe imen s, cells g own exponen ially in BG11
0
C medium supplemen ed wi h 5
o8mMNH
4
Cl we e ha es ed a oom empe a u e, washed wi h and esus-
pended in BG11
0
C medium, and hen incuba ed unde cul u e condi ions o he
numbe s o hou s indica ed below. Cul u es used o isola ion o RNA om
he e ocys s we e g own in BG11
0
C medium supplemen ed wi h 5 o 8 mM
NH
4
Cl un il he chlo ophyll aconcen a ion eached 3 o 5 ␮g䡠ml
⫺1
. Cells we e
hen washed wi h and esuspended in BG11
0
C medium and incuba ed un il
ma u e he e ocys s we e obse ed (19 h). He e ocys s we e isola ed as desc ibed
p e iously (18).
E. coli s ain DH5␣was g own in Lu ia-Be ani medium con aining, when
necessa y, 50 ␮g o ampicillin 䡠ml
⫺1
,50␮g o kanamycin 䡠ml
⫺1
,30␮go
chlo amphenicol 䡠ml
⫺1
,25␮g o s ep omycin 䡠ml
⫺1
,o 100␮go
spec inomycin 䡠ml
⫺1
.
␤-Galac osidase ac i i y. ␤-Galac osidase ac i i y was de e mined as p e i-
ously desc ibed (48) by using chlo o o m- ea ed ilamen s om shaken cul u es
FIG. 1. Sequence o he Anabaena glnA genomic egion om nucleo ide 391 ups eam o he ansla ion s a o nucleo ide 10 downs eam o
he ansla ion s a . RNA 5⬘ends iden i ied in his wo k (which do no always exac ly coincide wi h hose p e iously epo ed) a e indica ed, along
wi h he ollowing possible p omo e ea u es: ⫺10 boxes o RNA
I
, RNA
II
, and RNA
V
, ex ended ⫺10 box [⫺10 (E)] o RNA
IV
,⫺35 box o
RNA
II
, and he P
1
N cA-binding si e. The N cA- oo p in ed egion (37), including he N cA-binding si e sequence signa u e (GTAN
8
TAC), he
glnA ATG s a codon (double unde lined), and he loca ion o oligonucleo ides GA7 and GA13 used o p ime ex ension analysis a e also
indica ed. The limi s o he di e en DNA p omo e agmen s analyzed in his wo k we e as ollows: agmen C, posi ions 381 and 246; agmen
CB, posi ions 381 and 139; agmen CBA, posi ions 381 and 70; agmen B, posi ions 237 and 139; agmen BA, posi ions 237 and 70; and
agmen A, posi ions 159 and 70 (posi ions indica e nucleo ides ups eam om he glnA ansla ion s a ).
7338 VALLADARES ET AL. J. BACTERIOL.
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
ha we e g own in BG11
0
medium con aining NH
4
⫹
and incuba ed o 3 days in
BG11
0
medium wi h ai le els o CO
2
. One uni o ␤-galac osidase ac i i y
co esponded o p oduc ion o 1 ␮mol o o-ni ophenol 䡠min
⫺1
. The da a p e-
sen ed below a e he means and s anda d de ia ions o esul s ob ained wi h
h ee o six independen cul u es. The p o ein concen a ion was de e mined by
a modi ied Low y p ocedu e (24) by using 0.2-ml aliquo s o he cul u es.
Mu agenesis o he N cA-binding si e in he glnA P
1
p omo e . The deoxyoli-
gonucleo ide p ime s used in his wo k a e shown in Table 1. Si e-di ec ed
mu agenesis o he P
1
p omo e N cA-binding si e was ca ied ou by PCR as
desc ibed p e iously (1). The mu agenic oligonucleo ides used we e GA9 and
GA10, and he lanking oligonucleo ides we e GA1 and GA7; pAN503, which
con ains he ups eam egion and coding sequence o he glnA gene (14), was
used as he empla e. A e cloning o he PCR-gene a ed agmen s, he inse
o he plasmid gene a ed, pCSAV117, con aining he mu a ed e sion (CBA*) o
he glnA ups eam egion, was sequenced by using oligonucleo ide GA6 in o de
o check ha i con ained only he desi ed change (CAT ins ead o GTA).
Cons uc ion o Anabaena s ains wi h ansc ip ional usions. Di e en ag-
men s om he glnA gene ups eam egion we e ampli ied by PCR by using
plasmid pAN503 as he empla e and oligonucleo ides GA1 plus GA2, GA1 plus
GA4, GA1 plus GA6, GA3 plus GA4, GA3 plus GA6, and GA5 plus GA6, which
esul ed in agmen s C, CB, CBA, B, BA, and A, espec i ely (see Fig. 3). The
ampli ied p oduc s we e cloned in ec o pGEM-T (P omega). SphI/SalI ag-
men s om he esul ing plasmids (con aining he cloned agmen ) and om
plasmid pCSAV117 (which bea s agmen CBA*) we e inse ed be ween SphI
and SalI si es in ec o pIC20R (25). A 2-kb Sp
Sm
gene casse e wi h HindIII
ends was inse ed in o he unique HindIII si e o pIC20R. A BamHI agmen
om pPE20 con aining he p omo e less lacZ gene (20) was cloned in o a
BamHI si e jus a e he glnA p omo e agmen . A plasmid wi h no p omo e
was also cons uc ed by using he same s eps and was used as a nega i e con ol.
In o de o in eg a e hese cons uc s bea ing ansc ip ional usions in o he
cyanobac e ial genome, hey we e ans e ed o plasmid pCSAV81. This plas-
mid consis s o pCSAM28, which con ains a agmen o he nucA egion om
Anabaena sp. s ain PCC 7120 cloned be ween he EcoRI and EcoRV si es o
pBR322 (31), in which he nucA gene was mu a ed by diges ion wi h HindIII and
illing in wi h he Klenow enzyme ( his inac i a ion was necessa y because he
inc ease in nucA exp ession di ec ed om he Km
casse e inse ed ups eam o
his gene could be le hal o he E. coli hos ). BglII agmen s con aining he Sm
Sp
casse e, he p omo e agmen , and he lacZ gene we e liga ed o BglII-
diges ed pCSAV81, gene a ing plasmids pCSAV44, pCSAV45, pCSAV46,
pCSAV47, pCSAV48, pCSAV49, and pCSAV127 con aining p omo e ag-
men s C, CB, CBA, B, BA, A, and CBA*, espec i ely, and pCSAV50 con aining
no p omo e agmen .
In i o-gene a ed cons uc s ca ying ansc ip ional usions be ween di e en
agmen s o he glnA ups eam egion and he lacZ gene we e ans e ed by
conjuga ion (52) o Anabaena sp. s ain PCC 7120 and, as indica ed below, also
o s ain CSE2 (n cA [15]) o gene a e s ains bea ing hese ansc ip ional
usions in he nucA genomic egion. Fo gene a ion o hese s ains, E. coli
HB101 con aining plasmid pCSAV44, pCSAV45, pCSAV46, pCSAV47,
pCSAV48, pCSAV49, pCSAV50, o pCSAV127 and helpe plasmids pRL528
and pRL591-W45 (11) was mixed wi h E. coli ED8654 ca ying he conjuga i e
plasmid pRL443 and hen wi h Anabaena sp. Exconjugan s we e isola ed and
iden i ied as clones esis an o neomycin, s ep omycin, and spec inomycin, and
hei ch omosome s uc u e in he nucA egion was con i med by Sou he n
analysis.
DNA and RNA isola ion and analysis. To al DNA (8) and RNA (34) om
Anabaena sp. s ain PCC 7120 and i s de i a i es we e isola ed as p e iously
desc ibed. Sequencing was ca ied ou by he dideoxy chain e mina ion me hod
by using a T
7
Sequencing ki (Ame sham Biosciences) and [␣-
35
S] hio-dATP.
DNA agmen s we e pu i ied om aga ose gels wi h a Geneclean II ki (Bio
101). Plasmid isola ion om E. coli, ans o ma ion o E. coli, diges ion o DNA
wi h es ic ion endonucleases, liga ion wi h T4 ligase, and PCR we e pe o med
by s anda d p ocedu es (1, 41).
Sou he n analysis was ca ied ou by s anda d me hods by using Hybond-N
⫹
memb anes (Ame sham Biosciences). Fo No he n analysis, 70 ␮g o RNA was
loaded pe lane and elec opho esed in 1% aga ose dena u ing o maldehyde
gels. T ans e and ixa ion o Hybond-N
⫹
memb anes (Ame sham Biosciences)
we e ca ied ou by using 0.1 M NaOH. Hyb idiza ion was pe o med a 65°C
acco ding o he ecommenda ions o he manu ac u e s o he memb anes. The
ni H and bcL p obes we e in e nal agmen s o hese genes ampli ied by PCR.
All p obes we e
32
P labeled wi h a Ready o Go DNA labeling ki (Ame sham
Biosciences) by using [␣-
32
P]dCTP.
P ime ex ension analysis was ca ied ou as desc ibed p e iously (33). The
oligonucleo ides used o analysis o he glnA ansc ip we e GA7 and GA13.
Plasmid pAN503 was used o gene a e dideoxy sequencing ladde s by using he
same p ime s. Fo de e mina ion o ansc ip ion s a poin s o P
glnA
::lacZ
usions, oligonucleo ide LZ3 (complemen a y o a sequence loca ed ups eam o
he lacZ gene) was used as he p ime , and plasmid pCSAV127 was used o
gene a e dideoxy sequencing ladde s by using he same p ime . Images o a-
dioac i e il e s we e ob ained and quan i ied by using a Cyclone s o age phos-
pho sys em and he Op iQuan image analysis so wa e (Packa d). P ime ex-
ension analyses we e ca ied ou wi h i e (p ime ex ensions wi h he GA7 and
GA13 p ime s) o ou (p ime ex ensions wi h he lacZ p ime ) independen
RNA p epa a ions o wi h wo independen p epa a ions in he case o he e o-
cys RNA, and a ep esen a i e example is shown in each case below.
Band shi assays and DNase I oo p in ing. DNA agmen s o be used in
elec opho e ic mobili y shi assays we e ob ained by PCR ampli ica ion. Oli-
gonucleo ides GA1, GA2, GA3, GA4, GA5, and GA6 (Table 1; see Fig. 3A) and
plasmids pAN503 and pCSAV117 we e used o PCR ampli ica ion o he glnA
ups eam egion (wild- ype and mu a ed e sions, espec i ely). Oligonucleo-
ides (pUC/M13 o wa d and e e se p ime s) and plasmid pBluesc ip SK(⫹)
we e used o ampli ica ion o a DNA agmen ha was used as a nega i e
con ol. Binding assays we e ca ied ou as desc ibed p e iously (28) by using as
a sou ce o N cA a cell ex ac (1.4 ␮g o p o ein) o E. coli s ain
BL21(pCSAM70, pREP4), which o e p oduces he Anabaena sp. s ain PCC
7120 N cA p o ein (33), and, as a con ol, a cell ex ac o E. coli s ain
BL21(pQE9, pREP4).
The DNase I p o ec ion assay was ca ied ou as desc ibed p e iously (16). The
DNA used was he inse o pCSAV26 (con aining agmen CBA in he pIC20R
ec o ) limi ed by BamHI-EcoRV o HindIII-SacI si es and 3⬘end labeled wi h
he DNA polyme ase Klenow agmen and [␣-
32
P]dCTP (3,000 Ci 䡠mmol
⫺1
). A
cell ex ac o he Anabaena N cA-o e exp essing E. coli s ain BL21(pCSAM70,
pREP4) was used as a sou ce o N cA (33).
RESULTS
T ansc ip ion s a poin s. To in es iga e he pu a i e an-
sc ip ion s a poin (s) used o he glnA gene in he e ocys s o
Anabaena sp. s ain PCC 7120, o al RNA was ex ac ed om
he e ocys s isola ed om N
2
- ixing ilamen s. This RNA p ep-
a a ion did no show any de ec able hyb idiza ion wi h an bcL
p obe, whe eas i exhibi ed s ong hyb idiza ion wi h a ni H
p obe, indica ing ha i consis ed o highly speci ic he e ocys
RNA (da a no shown). P ime ex ension analysis wi h he
GA7 (da a no shown) o GA13 (Fig. 2A) p ime was pe -
o med wi h he he e ocys RNA p epa a ion and, as con ols,
wi h RNA p epa a ions om whole ilamen s g own wi h am-
monium o wi h N
2
as he ni ogen sou ce. Consis en wi h
p e iously epo ed da a (15, 45), we obse ed se e al 5⬘ends
o glnA ansc ip s when RNA isola ed om whole ilamen s
was used. T ansc ip ion s a poin s loca ed 93 (co esponding
o RNA
I
) and 274 (RNA
IV
) nucleo ides ups eam o he glnA
s a codon we e used a somewha highe le els in dia-
zo ophically g own ilamen s han in ammonium-g own ila-
men s, whe eas he ansc ip ion s a poin loca ed 157 nucle-
TABLE 1. Deoxyoligonucleo ide p ime s used in his wo k
P ime Sequence (5⬘-3⬘)
GA1 ...................................AAGCCTGTTACTGCATCGCGCATTCC
GA2 ...................................CAGATAGATTGTTTTTGTGCC
GA3 ...................................GGATTTTATGTCAAAGTTGACCCC
GA4 ...................................TACAGAACGTCTGGATTACAGG
GA5 ...................................CGAAAGAAAGGTTAATATTACCTG
GA6 ...................................CGAAACAAAGTTGATGAC
GA7 ...................................CTTTTCAAGACTTCTTGTGGGGG
GA9 ...................................CCAGACGTTCTCATACAAAGACTAC
GA10 .................................GTAGTCTTTGTATGAGAACGTCTGG
GA13 .................................CTCCTTCTCTGCCAATTTC
LZ3 ....................................CAATCACTGCTCAATGCCC
VOL. 186, 2004 ANABAENA glnA PROMOTER 7339
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
o ides ups eam o he s a codon (co esponding o RNA
II
)
was used a simila le els in ammonium- and N
2
-g own cells
(Fig. 2A). The p e iously desc ibed RNA
III
, conside ed o be
a deg ada ion p oduc o RNA
IV
, was obse ed only in some
expe imen s and is no e iden in Fig. 2. Howe e , we obse ed
a ansc ip ion s a poin 318 nucleo ides ups eam o he
ansla ion s a ha co esponded o RNA
V
, a ansc ip p e-
iously de ec ed only in in i o ansc ip ion assays (43).
RNA
V
showed a egula o y pa e n simila o ha o RNA
IV
.
In con as o hese esul s ob ained wi h RNA isola ed om
whole ilamen s, he he e ocys RNA p epa a ion p oduced a
s ong signal ha co esponded o RNA
I
and only a ain signal
co esponding o RNA
IV
.
To es he e ec o a he R mu a ion on he use o he glnA
ansc ip ion s a poin s, RNA isola ed om ilamen s g own
wi h ammonium o g own wi h ammonium and subjec ed o
ni ogen dep i a ion o se e al hou s was used in p ime ex-
ension assays wi h he GA7 (da a no shown) o GA13 (Fig.
2B) p ime . RNA p epa a ions om Anabaena sp. s ain
DR884a (he R [4]) and, as con ols, om s ain CSE2 (n cA
[15]) and wild- ype s ain PCC 7120 we e used. Whe eas in he
n cA mu an RNA
I
was no de ec able and RNA
IV
was in-
duced a a low le el (15), bo h RNAs we e exp essed in he
he R mu an a le els simila o hose ound in he wild ype
(Fig. 2B). RNA
V
was also N cA dependen bu independen o
He R (Fig. 2B).
T ansc ip ional usions. To u he analyze he p omo e o
he glnA gene, di e en DNA agmen s o he glnA ups eam
egion we e cloned in on o a p omo e less lacZ gene, and
he esul ing cons uc s we e ans e ed o Anabaena sp. s ain
PCC 7120. A schema ic ep esen a ion o he DNA agmen s
used is shown in Fig. 3A. Each agmen was ampli ied by PCR
and cloned jux aposed o he C.S3 gene casse e, which bea s
ansc ip ion e mina o s, and he lacZ gene (Fig. 3B), and
clones wi h he p omo e agmen o ien ed so ha i di ec ed
exp ession o lacZ we e chosen (see Ma e ials and Me hods
o de ails). These cons uc s we e inco po a ed in o he nucA
egion o he Anabaena genome, which is loca ed in he ␣
megaplasmid (32). This egion was chosen because he nucA
gene encodes a nuclease ha is dispensable o g ow h o his
cyanobac e ium (31). The usions we e inco po a ed in o
Anabaena sp. s ain PCC 7120 as desc ibed in Ma e ials and
Me hods, which p oduced he genomic s uc u e shown in Fig.
3B. To ensu e ha a co ec clone, ca ying a P
glnA
::lacZ u-
sion, was chosen o u he cha ac e iza ion, he s uc u e o
he nucA egion o wo exconjugan s o each cons uc was
con i med by Sou he n analysis (da a no shown). Like he
pa en al s ain PCC 7120, hese s ains we e able o de elop
he e ocys s and o g ow on N
2
(da a no shown).
Analysis o he lacZ-ca ying s ains showed ha only a low
ac i i y o ␤-galac osidase, 0.5 ⫾0.02 mU 䡠mg o p o ein
⫺1
,
was p oduced in he absence o a p omo e agmen (s ain
bea ing he con ol cons uc om plasmid pCSAV50). Inco -
po a ion o a glnA ups eam agmen , agmen C, B, o A
(Fig. 3), esul ed in ␤-galac osidase ac i i ies o 5.1 ⫾1.1
mU 䡠mg o p o ein
⫺1
( agmen C), 1.7 ⫾0.46 mU 䡠mg o
FIG. 2. T ansc ip ion s a poin s o he Anabaena glnA gene.
P ime ex ension analysis was ca ied ou wi h p ime GA13, and a
sequencing ladde was gene a ed wi h he glnA-con aining plasmid
pAN503 and he same p ime . (A) RNA isola ed om ammonium-
g own ilamen s (lane 1), om N
2
-g own ilamen s (lane 2), and om
isola ed he e ocys s (lane 3) o Anabaena sp. s ain PCC 7120.
(B) RNA isola ed om ilamen s o wild- ype s ain PCC 7120 (WT),
n cA s ain CSE2 (n cA), and he R s ain DR884a (he R) g own wi h
ammonium (lanes 0) o g own wi h ammonium and incuba ed o 9 h
(lanes 9) o 24 h (lanes 24) wi hou combined ni ogen. The a ow-
heads indica e he iden i ied RNA 5⬘ends.
FIG. 3. S uc u e o P
glnA
::lacZ usions. (A) Schema ic ep esen a-
ion o he egion om nucleo ide 381 o nucleo ide 70 ups eam o he
Anabaena glnA gene. The loca ions o he ansc ip ion s a poin s
gene a ing RNA
I
, RNA
II
, RNA
IV
, and RNA
V
, he si e de ining
RNA
III
, and he oligonucleo ides used o PCR ampli ica ion o he
agmen s shown below a e indica ed. (B) S uc u e o he nucA
genomic egion in he s ains ca ying P
glnA
::lacZ usions. The di e en
p omo e agmen s shown in panel A ( ep esen ed by a solid a ow)
we e cloned jux aposed o C.S3 and lacZ o di ec exp ession o lacZ.
The app oxima e loca ion o he LZ3 oligonucleo ide p ime is indi-
ca ed. No e ha an app oxima ely 2.2-kb agmen o he nucA egion
is duplica ed in hese s ains.
7340 VALLADARES ET AL. J. BACTERIOL.
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
p o ein
⫺1
( agmen B), and 3.8 ⫾0.7 mU 䡠mg o p o ein
⫺1
( agmen A). These esul s indica ed ha he e was p omo e
ac i i y o he DNA agmen s es ed, bu he ␤-galac osidase
ac i i ies exhibi ed by he di e en s ains should no be com-
pa ed o each o he , since he p omo e agmen s gene a ed
ansc ip s wi h di e en 5⬘ egions ha may ha e had di e en
s abili ies and/o ansla ion e iciencies. On he o he hand,
␤-galac osidase ac i i ies d i en by agmen s A, BA, and CBA
o by agmen s B and CB can be compa ed. F agmen BA
d o e a ␤-galac osidase ac i i y o 12.2 ⫾1.9 mU 䡠mg o p o-
ein
⫺1
, which is highe han he ac i i y d i en by agmen A.
In con as , agmen s CB (1.5 ⫾0.56 mU 䡠mg o p o ein
⫺1
)
and CBA (10.6 ⫾2.3 mU 䡠mg o p o ein
⫺1
) di ec ed ac i i ies
ha we e simila o hose p oduced by agmen s B and BA,
espec i ely.
Mu a ion o he N cA-binding si e. As shown abo e, use o
he P
1
p omo e ha gene a es RNA
I
is dependen on N cA
(15). This p omo e ca ies an N cA-binding si e (GTAN
8
TAC) ha is cen e ed a posi ion ⫺41.5 wi h espec o he
ansc ip ion s a poin . To cha ac e ize he ole o his N cA-
binding si e in ansc ip ion om he glnA p omo e , we gen-
e a ed a mu a ed CBA agmen (CBA*) in which he GTA
iple o he N cA-binding si e was eplaced by a CAT iple
(see Ma e ials and Me hods o de ails). This mu a ed ag-
men was inco po a ed in o he lacZ epo e sys em and ans-
e ed o Anabaena sp. s ain PCC 7120, whe e i di ec ed a
␤-galac osidase ac i i y o 154.8 ⫾6.7 mU 䡠mg o p o ein
⫺1
,
which is abou 15- old highe han he ac i i y obse ed wi h
he o iginal CBA agmen .
To es he e ec o mu a ion o he N cA-binding si e on he
use o he glnA p omo e (s), p ime ex ension assays we e
pe o med wi h a p ime om he lacZ ups eam egion and
RNA isola ed om ammonium-g own ilamen s incuba ed o
9 h in he absence o combined ni ogen. A e ans e o he
CBA::lacZ and CBA*::lacZ cons uc s o s ain CSE2 (n cA),
hese expe imen s we e ca ied ou in he wild- ype and n cA
gene ic backg ounds. The CBA agmen p oduced a se o
p ime ex ension p oduc s ha included he same RNA species
ha we e obse ed wi h he na i e glnA p omo e (Fig. 4A,
lane 1) (RNA
V
was no shown in his expe imen ). Howe e ,
RNA
I
was no obse ed in an n cA mu an backg ound (Fig.
4A, lane 2). These esul s con i med he p ope ope a ion as a
p omo e o agmen CBA cloned in on o lacZ. When he
mu a ed CBA agmen ( agmen CBA*) was analyzed, an-
sc ip ion was obse ed o o igina e mainly om a ansc ip ion
s a poin co esponding o RNA
II
, and RNA
I
was de ec ed
nei he in he wild ype (Fig. 4B, lane 2) no in he n cA
backg ound (Fig. 4B, lane 3).
Binding o N cA. The glnA P
1
p omo e bea s a e y e icien
N cA-binding si e (37). To es o he possible N cA in e ac-
ions wi h he Anabaena glnA ups eam egion used in his
wo k, binding o N cA o he di e en p omo e agmen s was
es ed by a band shi assay. Binding was obse ed as long as
he sequences co esponding o P
1
we e p esen in he DNA
agmen es ed bu did no ake place when he N cA-binding
si e in P
1
was mu a ed (Fig. 5). DNase I oo p in ing o a DNA
agmen co e ing he whole glnA ups eam egion ha we
in es iga ed ( agmen CBA) did no show any N cA-sensi i e
egion in addi ion o he egion p e iously desc ibed by Ra-
masub amanian e al. (37), which co esponds o he N cA-
binding si e in he P
1
p omo e ( esul s no shown).
DISCUSSION
The glnA gene o Anabaena sp. s ain PCC 7120 appea s o
be ansc ibed om ou independen ansc ip ion s a
poin s, which gi e ise o he RNA species known as RNA
I
,
RNA
II
, RNA
IV
, and RNA
V
(Fig. 1). These ou RNA species
a e obse ed in ammonium-g own Anabaena ilamen s; how-
e e , whe eas RNA
II
appea s o be cons i u i e, RNA
I
,
RNA
IV
, and RNA
V
a e induced a e ni ogen dep i a ion (15;
his s udy). DNA agmen s ca ying sequences ups eam om
RNA
I
( agmen A), RNA
II
( agmen B), and RNA
IV
( ag-
men C) d i e ansc ip ional ac i i y when hey a e used o a
lacZ epo e gene, demons a ing ha hese RNA species
de ine ue gene p omo e s (P
1
,P
2
, and P
3
, espec i ely). F ag-
men C also co e s RNA
V
, and he e o e an addi ional p o-
mo e , P
4
, may be p esen in his DNA agmen . F agmen BA
p oduces highe ␤-galac osidase ac i i y han agmen A,
which, consis en wi h de ec ion o RNA
II
and RNA
I
in sam-
ples o RNA isola ed om whole ilamen s, sugges s ha he e
FIG. 4. T ansc ip ion s a poin s o he CBA and CBA*
P
glnA
::lacZ usions in Anabaena sp. s ains PCC 7120 (wild ype) and
CSE2 (n cA). P ime ex ension analysis was ca ied ou wi h p ime
LZ3 (Fig. 3B), and a sequencing ladde was gene a ed wi h plasmid
pCSAV127 and he same p ime . (A) RNA isola ed om ilamen s o
s ains PCC 7120 (lane 1) and CSE2 (lane 2) ca ying he CBA::lacZ
usion g own wi h ammonium and incuba ed o 9 h wi h no sou ce o
combined ni ogen. (B) RNA isola ed om ilamen s o s ain PCC
7120 ca ying CBA::lacZ (lane 1) o CBA*::lacZ (lane 2) o o s ain
CSE2 ca ying CBA*::lacZ (lane 3) g own wi h ammonium and incu-
ba ed o 9 h wi h no sou ce o combined ni ogen.
VOL. 186, 2004 ANABAENA glnA PROMOTER 7341
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om

is simul aneous ope a ion o he P
2
and P
1
p omo e s. In con-
as , al hough agmen C i sel d i es a subs an ial ␤-galac-
osidase ac i i y and RNA
IV
and RNA
V
ha e been obse ed in
di e en RNA samples, agmen s CB and CBA do no di ec
highe le els o ac i i y han agmen s B and BA, espec i ely.
The eason o his lack o s imula ion o ␤-galac osidase p o-
duc ion by agmen C is unknown, bu he p esence in ag-
men B o he pu a i e RNA p ocessing si e ha gene a es
RNA
III
(Fig. 1 and 3), which could in e e e wi h ansc ip ion
d i en by p omo e s P
3
and P
4
, should be no ed.
In he e ocys s, exp ession akes place essen ially as RNA
I
,
indica ing ha P
1
ep esen s he glnA p omo e in his cell ype
(Fig. 2A). A ansc ip co esponding o RNA
I
has also been
shown o ep esen he main glnA ansc ip du ing he e ocys
di e en ia ion (49) o in cells s a ed o ni ogen unde an-
ae obic condi ions (45). Howe e , P
1
is no a p omo e ha is
used exclusi ely in he he e ocys s since i is also u ilized in
ege a i e cells (i.e., in cells o ilamen s g own wi h ammo-
nium) (Fig. 2A) and in a he R mu an (a s ain ha does no
de elop he e ocys s) (Fig. 2B). P
1
ep esen s a canonical
N cA- ype p omo e whose use is s ic ly dependen on N cA
(Fig. 2B and 4A) (15). Mu a ion o he N cA-binding si e in P
1
h ough eplacemen o he GTA iple by CAT elimina es
N cA binding in i o (Fig. 5) and he use o P
1
in i o (Fig.
4B), indica ing he impo ance o his N cA-binding si e o
N cA-dependen ansc ip ion ac i a ion. The amoun o N cA
p esen in ege a i e cells (34, 38) appea s o de e mine le els
o binding o N cA o he e icien N cA-binding si e in P
1
ha
a e su icien o he use o P
1
in his ype o cells, while he
high le els o ac i e N cA likely p esen in he he e ocys s (34,
37, 49) should con ibu e o s ong use o P
1
in hese di e en-
ia ed cells.
P
2
ep esen s a canonical ␴
70
- ype p omo e (Fig. 1) ha is
used in ege a i e cells bu no in he e ocys s (Fig. 2A). The
p esence o he N cA-binding si e in P
1
appea s o a ec he
ope a ion o P
2
. Thus, mu a ion o he N cA-binding si e in P
1
esul s in a d ama ic inc ease in ␤-galac osidase ac i i y and in
inc eased ansc ip ion om P
2
(Fig. 4B). Howe e , when e-
sul s wi h he CBA and CBA* agmen s a e compa ed, he
inc ease in RNA
II
le els de ec ed by p ime ex ension analysis
is no quan i a i ely compa able o he obse ed inc ease in
␤-galac osidase ac i i y, sugges ing ha , in addi ion o an-
sc ip ional ac i i y, ansc ip s abili y o ansla ion e iciency
may a ec he p oduc ion o ␤-galac osidase. None heless, be-
cause he N cA-binding si e in P
1
( oo p in ed egion in Fig. 1)
is 9 bp downs eam o he RNA
II
ansc ip ion s a poin ,
N cA bound o his si e could ep ess P
2
ei he by occluding
RNA polyme ase binding o by inhibi ing p omo e escape (29,
40). Rep ession by N cA migh he e o e con ibu e o he lack
o use o P
2
in he e ocys s. Howe e , inc eased ansc ip ion
om P
2
is ha dly obse ed when he n cA gene, a he han he
N cA-binding si e, is inac i a ed (Fig. 2B and 4A). Apa om
he possible ep esso ole o N cA, i is possible ha he
N cA-binding sequence i sel in e e es wi h he use o P
2
.
P omo e s P
3
and P
4
a e posi i ely in luenced by N cA,
al hough sequence sc u iny does no pe mi iden i ying any o
hese p omo e s as an N cA- ype p omo e . N cA-dependen
p omo e s ha canno be ecognized as N cA- ype p omo e s
ha e also been desc ibed o some o he Anabaena genes,
sugges ing ha he e is indi ec ope a ion o N cA. This is he
case o he he R gene, which in Anabaena sp. s ain PCC 7120
is ansc ibed om ou p omo e s, wo o which a e N cA
dependen bu do no con ain sequences ma ching he consen-
sus sequence o N cA-ac i a ed p omo e s (34). Lack o bind-
ing o N cA o agmen C (Fig. 5) is consis en wi h indi ec
egula ion by N cA o he glnA P
3
and P
4
p omo e s.
To summa ize, exp ession o he glnA gene, which encodes
he key ni ogen assimila ion enzyme glu amine syn he ase,
akes place in Anabaena sp. s ain PCC 7120 om a se o
p omo e s whose u iliza ion is in luenced by he global N-
con ol ansc ip ion ac o N cA. Whe eas he u iliza ion o
P
1
is s ic ly dependen on he binding o N cA o his p o-
mo e , ope a ion o p omo e s P
3
and P
4
appea s o be indi-
ec ly dependen on N cA. In con as , he N cA-binding si e in
P
1
in e e es wi h ansc ip ion om he P
2
p omo e .
ACKNOWLEDGMENTS
We hank J. Casadesu´s, R. Dixon, and E. San e o o use ul discus-
sions.
This wo k was suppo ed by g an s BMC2002-03902 and BMC2001-
0509 om Minis e io de Ciencia y Tecnologı´a, Spain.
REFERENCES
1. Ausubel, F. M., R. B en , R. E. Kings on, D. D. Moo e, J. G. Seidman, J. A.
Smi h, and K. S uhl (ed.). 2003. Cu en p o ocols in molecula biology.
G eene/Wiley-In e science, New Yo k, N.Y.
2. Ba ne, K. A., J. A. Bown, S. J. W. Busby, and S. D. Minchin. 1997. Region
2.5 o he Esche ichia coli RNA polyme ase ␴
70
subuni is esponsible o he
ecogni ion o he “ex ended ⫺10”mo i a p omo e s. EMBO J. 13:4034–
4040.
FIG. 5. Band shi assays o he binding o N cA o he Anabaena
glnA ups eam egion. The DNA agmen s indica ed a he op we e
ob ained by PCR, and 75 o 100 ng o each o hem was used in binding
assays wi h ex ac s o E. coli ca ying he N cA-o e p oducing plasmid
pCSAM70 (lanes ⫹) o , as a con ol, he pQE9 ec o (lanes ⫺). The
a owheads indica e e a ded agmen s, which we e obse ed only
wi h he A, BA, and CBA agmen s and he N cA-con aining ex ac .
pBS, DNA agmen om pBluesc ip SK(⫹) used as a nega i e con-
ol.
7342 VALLADARES ET AL. J. BACTERIOL.
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om
3. Be gsland, K. J., and R. Haselko n. 1991. E olu iona y ela ionships among
eubac e ia, cyanobac e ia, and chlo oplas s: e idence om he poC1 gene o
Anabaena sp. s ain PCC 7120. J. Bac e iol. 173:3446–3455.
4. Black, T. A., Y. Cai, Y., and C. P. Wolk. 1993. Spa ial exp ession and
au o egula ion o he R, a gene in ol ed in he con ol o he e ocys de el-
opmen in Anabaena. Mol. Mic obiol. 9:77–84.
5. B ahamsha, B., and R. Haselko n. 1991. Isola ion and cha ac e iza ion o
he gene encoding he p incipal sigma ac o o he ege a i e cell RNA
polyme ase om he cyanobac e ium Anabaena sp. s ain PCC 7120. J.
Bac e iol. 173:2442–2450.
6. Buikema, W. J., and R. Haselko n. 1991. Cha ac e iza ion o a gene con-
olling he e ocys di e en ia ion in he cyanobac e ium Anabaena 7120.
Genes De . 5:321–330.
7. Busby, S., and R. H. Eb igh . 1997. T ansc ip ion ac i a ion a class II
CAP-dependen p omo e s. Mol. Mic obiol. 23:853–859.
8. Cai, Y., and C. P. Wolk. 1990. Use o a condi ionally le hal gene in Anabaena
sp. s ain PCC 7120 o selec o double ecombinan s and o en ap inse ion
sequences. J. Bac e iol. 172:3138–3145.
9. Cu is, S. E., and J. A. Ma in. 1994. The ansc ip ion appa a us and he
egula ion o ansc ip ion ini ia ion, p. 613–639. In D. A. B yan (ed.), The
molecula biology o cyanobac e ia. Kluwe Academic Publishe s, Do d e-
ch , The Ne he lands.
10. deHase h, P. L., M. L. Zupancic, and M. T. Reco d, J . 1998. RNA poly-
me ase-p omo e in e ac ions: he comings and goings o RNA polyme ase.
J. Bac e iol. 180:3019–3025.
11. Elhai, J., Y. Cai, and C. P. Wolk. 1994. Conduc ion o pEC22, a plasmid
coding o MR.EcoT22I, media ed by a esiden Tn3-like ansposon,
Tn5396. J. Bac e iol. 176:5059–5067.
12. Elhai, J., and C. P. Wolk. 1988. A e sa ile class o posi i e-selec ion ec o s
based on he non iabili y o palind ome-con aining plasmids ha allows
cloning in o long polylinke s. Gene 68:119–138.
13. Elhai, J., and C. P. Wolk. 1990. De elopmen al egula ion and spa ial pa -
e n o exp ession o he s uc u al genes o ni ogenase in he cyanobac-
e ium Anabaena. EMBO J. 9:3379–3388.
14. Fishe , R., R. Tuli, and R. Haselko n. 1981. A cloned cyanobac e ial gene o
glu amine syn he ase unc ions in Esche ichia coli, bu he enzyme is no
adenylyla ed. P oc. Na l. Acad. Sci. USA 78:3393–3397.
15. F ı´as, J. E., E. Flo es, and A. He e o. 1994. Requi emen o he egula o y
p o ein N cA o he exp ession o ni ogen assimila ion and he e ocys
de elopmen genes in he cyanobac e ium Anabaena sp. s ain PCC 7120.
Mol. Mic obiol. 14:823–832.
16. F ı´as, J. E., E. Flo es, and A. He e o. 2000. Ac i a ion o he Anabaena ni
ope on p omo e equi es bo h N cA (CAP amily) and N cB (LysR amily)
ansc ip ion ac o s. Mol. Mic obiol. 38:613–625.
17. Gio annoni, S. J., S. Tu ne , G. J. Olsen, S. Ba ns, D. J. Lane, and N. R.
Pace. 1988. E olu iona y ela ionships among cyanobac e ia and g een chlo-
oplas s. J. Bac e iol. 170:3584–3592.
18. Golden, J. W., L. L. Who , and D. R. Wies . 1991. Independen egula ion
o ni HDK ope on ansc ip ion and DNA ea angemen du ing he e ocys
di e en ia ion in he cyanobac e ium Anabaena sp. s ain PCC 7120. J.
Bac e iol. 173:7098–7105.
19. He e o, A., A. M. Mu o-Pas o , and E. Flo es. 2001. Ni ogen con ol in
cyanobac e ia. J. Bac e iol. 183:411–425.
20. Koko ek, W., and W. Lo z. 1989. Cons uc ion o a lacZ-kanamycin- esis-
ance casse e, use ul o si e-di ec ed mu agenesis and as a p omo e p obe.
Gene 84:467–471.
21. Lindell, D., E. Padan, and A. F. Pos . 1998. Regula ion o n cA exp ession
and ni i e up ake in he ma ine Synechococcus sp. s ain WH 7803. J.
Bac e iol. 180:1878–1886.
22. Luque, I., E. Flo es, and A. He e o. 1994. Molecula mechanism o he
ope a ion o ni ogen con ol in cyanobac e ia. EMBO J. 13:2862–2869.
23. Mackinney, G. 1941. Abso p ion o ligh by chlo ophyll solu ions. J. Biol.
Chem. 140:315–322.
24. Ma kwell, M. A. K., S. M. Hass, L. L. Biebe , and N. E. Tolbe . 1978. A
modi ica ion o he Low y p ocedu e o simpli y p o ein de e mina ion in
memb ane and lipop o ein samples. Anal. Biochem. 87:206–210.
25. Ma sh, J. L., M. E le, and E. J. Wykes. 1984. The pIC plasmid and phage
ec o s wi h e sa ile cloning si es o ecombinan selec ion by inse ional
inac i a ion. Gene 32:481–485.
26. Me´ ida, A., L. Leu en op, P. Candau, and F. J. Flo encio. 1990. Pu i ica ion
and p ope ies o glu amine syn he ases om he cyanobac e ia Synechocys-
is sp. s ain PCC 6803 and Calo h ix sp. s ain PCC 7601. J. Bac e iol.
172:4732–4735.
27. Me ick, M. J., and R. A. Edwa ds. 1995. Ni ogen con ol in bac e ia.
Mic obiol. Re . 59:604–622.
28. Mon esinos, M. L., A. M. Mu o-Pas o , A. He e o, and E. Flo es. 1998.
Ammonium/me hylammonium pe meases o a cyanobac e ium. Iden i ica-
ion and analysis o h ee ni ogen- egula ed am genes in Synechocys is sp.
PCC 6803. J. Biol. Chem. 273:31463–31470.
29. Mooney, R. A., I. A simo i ch, and R. Landick. 1998. In o ma ion p ocess-
ing by RNA polyme ase: ecogni ion o egula o y signals du ing RNA chain
elonga ion. J. Bac e iol. 180:3265–3275.
30. M a´zek, J., D. Bhaya, A. R. G ossman, and S. Ka lin. 2001. Highly exp essed
and alien genes o he Synechocys is genome. Nucleic Acids Res. 29:1590–
1601.
31. Mu o-Pas o , A. M., E. Flo es, A. He e o, and C. P. Wolk. 1992. Iden i i-
ca ion, gene ic analysis and cha ac e iza ion o a suga -non-speci ic nuclease
om he cyanobac e ium Anabaena sp. s ain PCC 7120. Mol. Mic obiol.
6:3021–3030.
32. Mu o-Pas o , A. M., T. Ku i z, E. Flo es, A. He e o, and C. P. Wolk. 1994.
T ans e o a gene ic ma ke om a megaplasmid o Anabaena sp. s ain
PCC 7120 o a megaplasmid o a di e en Anabaena s ain. J. Bac e iol.
176:1093–1098.
33. Mu o-Pas o , A. M., A. Vallada es, E. Flo es, and A. He e o. 1999. The
he C gene is a di ec a ge o he N cA ansc ip ional egula o in cya-
nobac e ial he e ocys de elopmen . J. Bac e iol. 181:6664–6669.
34. Mu o-Pas o , A. M., A. Vallada es, E. Flo es, and A. He e o. 2002. Mu ual
dependence o he exp ession o he cell di e en ia ion egula o y p o ein
He R and he global ni ogen egula o N cA du ing he e ocys de elop-
men . Mol. Mic obiol. 44:1377–1385.
35. Mu o-Pas o , M. I., J. C. Reyes, and F. J. Flo encio. 2001. Cyanobac e ia
pe cei e ni ogen s a us by sensing in acellula 2-oxoglu a a e le els. J. Biol.
Chem. 276:38320–38328.
36. P en ki, P., and H. M. K isch. 1984. In i o inse ional mu agenesis wi h a
selec able DNA agmen . Gene 29:303–313.
37. Ramasub amanian, T. S., T.-F. Wei, and J. W. Golden. 1994. Two Anabaena
sp. s ain PCC 7120 DNA-binding ac o s in e ac wi h ege a i e cell-and
he e ocys -speci ic genes. J. Bac e iol. 176:1214–1223.
38. Ramasub amanian, T. S., T.-F. Wei, A. K. Oldham, and J. W. Golden. 1996.
T ansc ip ion o he Anabaena sp. s ain PCC 7120 n cA gene: mul iple
ansc ip s and N cA binding. J. Bac e iol. 178:922–926.
39. Rippka, R., J. De uelles, J. B. Wa e bu y, M. He dman, and R. Y. S anie .
1979. Gene ic assignmen s, s ain s o ies and p ope ies o pu e cul u es o
cyanobac e ia. J. Gen. Mic obiol. 111:1–61.
40. Rojo, F. 1999. Rep ession o ansc ip ion ini ia ion in bac e ia. J. Bac e iol.
181:2987–2991.
41. Samb ook, J., E. F. F i sch, and T. Mania is. 1989. Molecula cloning: a
labo a o y manual, 2nd ed. Cold Sp ing ha bo Labo a o y, Cold Sp ing
Ha bo , N.Y.
42. Schneide , G. J., J. D. Lang, and R. Haselko n. 1991. P omo e ecogni ion
by he RNA polyme ase om ege a i e cells o he cyanobac e ium
Anabaena 7120. Gene 105:51–60.
43. Schneide , G. J., N. E. Tume , C. Richaud, G. Bo bely, and R. Haselko n.
1987. Pu i ica ion and cha ac e iza ion o RNA polyme ase om he cya-
nobac e ium Anabaena 7120. J. Biol. Chem. 262:14633–14639.
44. Tanigawa, R., M. Shi okane, S. Maeda, T. Oma a, K. Tanaka, and H.
Takahashi. 2002. T ansc ip ional ac i a ion o N cA-dependen p omo e s
o Synechococcus sp. PCC 7942 by 2-oxoglu a a e in i o. P oc. Na l. Acad.
Sci. USA 99:4251–4255.
45. Tume , N. E., S. J. Robinson, and R. Haselko n. 1983. Di e en p omo e s
o he Anabaena glu amine syn he ase gene du ing g ow h using molecula
o ixed ni ogen. Na u e 306:1–6.
46. Va´zquez-Be mu´dez, M. F., A. He e o, and E. Flo es. 2002. 2-Oxoglu a a e
inc eases he binding a ini y o he N cA (ni ogen con ol) ansc ip ion
ac o o he Synechococcus glnA p omo e . FEBS Le . 512:71–74.
47. Va´zquez-Be mu´dez, M. F., A. He e o, and E. Flo es. 2003. Ca bon supply
and 2-oxoglu a a e e ec s on exp ession o ni a e educ ase and ni ogen-
egula ed genes in Synechococcus sp. s ain PCC 7942. FEMS Mic obiol.
Le . 221:155–159.
48. Va´zquez-Be mu´dez, M. F., J. Paz-Yepes, A. He e o, and E. Flo es. 2002.
The N cA-ac i a ed am 1 gene encodes a pe mease equi ed o up ake o
low concen a ions o ammonium in he cyanobac e ium Synechococcus sp.
PCC 7942. Mic obiology 148:861–869.
49. Wei, T.-F., T. S. Ramasub amanian, and J. W. Golden. 1994. Anabaena sp.
s ain PCC 7120 n cA gene equi ed o g ow h on ni a e and he e ocys
de elopmen . J. Bac e iol. 176:4473–4482.
50. Wolk, C. P., A. E ns , and J. Elhai. 1994. He e ocys me abolism and de el-
opmen , p. 769–823. In D. A. B yan (ed.), The molecula biology o cya-
nobac e ia. Kluwe Academic Publishe s, Do d ech , The Ne he lands.
51. Wolk, C. P., J. Thomas, P. W. Sha e , S. M. Aus in, and A. Galonsky. 1976.
Pa hway o ni ogen me abolism a e ixa ion o
13
N-labeled ni ogen gas by
he cyanobac e ium, Anabaena cylind ica. J. Biol. Chem. 251:5027–5034.
52. Wolk, C. P., A. Vonshak, P. Kehoe, and J. Elhai. 1984. Cons uc ion o
shu le ec o s capable o conjuga i e ans e om Esche ichia coli o ni-
ogen- ixing ilamen ous cyanobac e ia. P oc. Na l. Acad. Sci. USA 81:
1561–1565.
53. Xie, W. Q., K. Ja¨ge , and M. Po s. 1989. Cyanobac e ial RNA polyme ase
genes poC1 and poC2 co espond o poC o Esche ichia coli. J. Bac e iol.
171:1967–1973.
VOL. 186, 2004 ANABAENA glnA PROMOTER 7343
on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om