JOURNAL OF BACTERIOLOGY, No . 2008, p. 7584–7590 Vol. 190, No. 22
0021-9193/08/$08.00⫹0 doi:10.1128/JB.00856-08
Copy igh © 2008, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Role o Two N cA-Binding Si es in he Complex n cA Gene P omo e o
he He e ocys -Fo ming Cyanobac e ium Anabaena sp. S ain PCC 7120
䌤
†
El i a Olmedo-Ve d, Ana Vallada es, En ique Flo es, An onia He e o, and Alicia M. Mu o-Pas o *
Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones Cien í icas and Uni e sidad de
Se illa, E-41092 Se ille, Spain
Recei ed 23 June 2008/Accep ed 8 Sep embe 2008
Anabaena sp. s ain PCC 7120 is a ilamen ous cyanobac e ium ha ixes N
2
in specialized cells called
he e ocys s, which di e en ia e om ege a i e cells in a p ocess ha equi es he ni ogen con ol ansc ip-
ion ac o N cA. 2-Oxoglu a a e-s imula ed binding o pu i ied N cA o wild- ype and modi ied e sions o he
n cA gene p omo e om Anabaena sp. was analyzed by mobili y shi and DNase I oo p in ing assays, and he
ole o N cA-binding si es in he exp ession o he n cA gene du ing he e ocys di e en ia ion was s udied in
i o by using an n cA-g p ansla ional usion and p ime ex ension analysis. Mu a ion o nei he o he wo
iden i ied N cA-binding si es elimina ed localized exp ession o n cA in p ohe e ocys s, bu mu a ion o bo h
si es led o e y low, nonlocalized exp ession.
Ni ogen ixa ion in many cyanobac e ia, including Anabaena
sp. s ain PCC 7120, equi es di e en ia ion o a speci ic cell
ype, called a he e ocys , in which he machine y o ni ogen
ixa ion is con ined (6, 27). The di e en ia ion o he e ocys s
begins sho ly a e ni ogen dep i a ion and, unde ou expe -
imen al condi ions, equi es abou 24 h o comple e. He e o-
cys di e en ia ion depends on he N cA p o ein, a ansc ip-
ional egula o belonging o he cyclic AMP ecep o p o ein
(CAP [o CRP]) amily (5, 8, 26). In he absence o combined
ni ogen, N cA ac i a es he exp ession o genes equi ed o
he e ocys di e en ia ion and ni ogen ixa ion (7). In o de o
ac i a e gene exp ession, N cA binds ups eam om he an-
sc ip ional s a s (7), and a consensus sequence o N cA bind-
ing (GTAN
8
TAC) has been de ined (7, 11). Binding o N cA o
DNA and N cA-media ed ansc ip ional ac i a ion a e in lu-
enced by he C- o-N balance o he cell, and 2-oxoglu a a e has
been iden i ied as a posi i e e ec o (10, 21, 22, 24, 25). He -
e ocys di e en ia ion in Anabaena sp. s ain PCC 7120 also
equi es He R (2, 3). The he R gene encodes a posi i e-ac ing
ac o ha exhibi s au op o ease (28) and DNA-binding (9)
ac i i ies in i o. Induc ion o exp ession o n cA and he R is
mu ually dependen in he con ex o he e ocys di e en ia ion
(13), and induc ion o bo h genes is mos ly localized o cells
de eloping in o he e ocys s (2, 16).
The n cA gene is ansc ibed om a complex p omo e ha
p oduces h ee ansc ip ional s a poin s (TSPs) (13, 16, 18)
(see he sequence o he p omo e egion in Fig. 3B). P omo -
e s P
1
,P
2
, and P
3
p oduce TSPs a posi ions ⫺49, ⫺136, and
⫺180, espec i ely, wi h espec o he ansla ional s a o
n cA.P
2
unc ions independen ly o he ni ogen sou ce,
whe eas P
1
unc ions in he p esence o combined ni ogen,
bu i s use ansien ly inc eases a e 6 o 12 h o ni ogen
dep i a ion in an N cA- and He R-dependen manne (13).
Finally, P
3
unc ions only in he absence o combined ni ogen
and i s use is ansien ly induced a e 6 o 12 h o ni ogen
dep i a ion in an N cA- and He R-dependen manne (13).
The p omo e o he n cA gene con ains wo pu a i e N cA-
binding si es sepa a ed by 40 nucleo ides and cen e ed a po-
si ions ⫺143.5 (GTAN
8
AAC) and ⫺103.5 (GTAN
8
TAC) wi h
espec o he ansla ional s a o n cA. (We will e e o hem
as “dis al” and “p oximal,” espec i ely, acco ding o hei
posi ion wi h espec o he n cA open eading ame.) The
dis al si e o e laps he ⫺10 hexame o he P
2
p omo e (13).
In i o binding o N cA o he dis al si e, bu no o he
p oximal si e, has been demons a ed by DNase I p o ec ion
oo p in ing assays (18). Howe e , wo e a ded bands ha e
been obse ed in elec opho e ic mobili y shi assays (EMSAs)
wi h N cA and a agmen om he n cA p omo e (10). In his
s udy, we used a combina ion o in i o and in i o app oaches
in o de o unde s and he ope a ion o he complex Anabaena
n cA p omo e egion, which is unc ional in he wo di e en
cell ypes, ege a i e cells and he e ocys s, o a ni ogen- ixing
ilamen .
Me hods. Anabaena sp. (also known as Nos oc sp.) s ain
PCC 7120 and i s de i a i es (see Table S1 in he supplemen al
ma e ial) we e g own pho oau o ophically a 30°C in BG11
0
C
medium (BG11 medium [19] wi hou NaNO
3
and supple-
men ed wi h 10 mM NaHCO
3
) supplemen ed wi h 6 mM
NH
4
Cl plus 12 mM N-T is (hyd oxyme hyl) me hyl-2-amino-
e hanesul onic acid (TES)-NaOH bu e (pH 7.5) and bubbled
wi h a mix u e o CO
2
and ai (1% [ ol/ ol]). Media we e
supplemen ed wi h 2 ␮gml
⫺1
o s ep omycin and 2 ␮gml
⫺1
o spec inomycin in he case o s ains bea ing he P
n cA
-g p
usions. Fo de e mina ion o g een luo escen p o ein (GFP)
luo escence and o RNA isola ion, cells g owing exponen-
ially (abou 3 o 5 ␮g chlo ophyll aml
⫺1
) in BG11
0
C medium
supplemen ed wi h NH
4
Cl we e ha es ed by il a ion a oom
empe a u e and ei he used di ec ly o washed wi h BG11
0
C
(ni ogen- ee) medium, esuspended in BG11
0
C medium, and
u he incuba ed unde cul u e condi ions o he numbe o
* Co esponding au ho . Mailing add ess: Ins i u o de Bioquímica
Vege al y Fo osín esis, Cen o de In es igaciones Cien í icas Isla de la
Ca uja, A da. Ame´ ico Vespucio 49, E-41092 Se ille, Spain. Phone:
34 95 448 9521. Fax: 34 95 446 0065. E-mail: [email p o ec ed].
† Supplemen al ma e ial o his a icle may be ound a h p://jb
.asm.o g/.
䌤
Published ahead o p in on 19 Sep embe 2008.
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hou s indica ed in each expe imen . To al DNA (4) and RNA
(13) 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. S anda d molecula bi-
ology me hods we e used (1). The plasmids used in his s udy
a e lis ed in Table S1 in he supplemen al ma e ial. O e ex-
p ession o N cA in Esche ichia coli cells bea ing plasmid
pCSAM61 (13) and pu i ica ion o N cA using HiT ap hepa in
HP columns (Pha macia Bio ech) we e ca ied ou as de-
sc ibed p e iously (22). Images o adioac i e gels we e ob-
ained and analyzed by using a Cyclone s o age phospho sys-
em and Op iquan image analysis so wa e (Packa d). The
accumula ion o GFP epo e was analyzed by lase con ocal
mic oscopy as p e iously desc ibed (16) by collec ion o emis-
sions in a window o 500 o 570 nm. All images we e collec ed
using he same se ings, so ha he in ensi ies can be com-
pa ed. (see also he supplemen al ma e ial.)
Binding o N cA o wild- ype and mu a ed e sions o he
n cA p omo e . As a i s s ep o unde s and he ole o he wo
pu a i e N cA-binding si es p esen in he p omo e egion o
he n cA gene, we p epa ed modi ied e sions o his egion
bea ing mu a ions in one o bo h si es, and ca ied ou EMSA
wi h pu i ied N cA p o ein ollowing p ocedu es p e iously
desc ibed (11). Th ee mu a ed e sions o he n cA ups eam
egion (posi ions ⫺603 o ⫹18 wi h espec o he ansla ional
s a o n cA) we e gene a ed, wi h changes in he sequence o
he p oximal (GTAN
8
TAC3CATN
8
TAC), dis al (GTAN
8
A
AC3CATN
8
AAC), o bo h N cA-binding si es. As p e iously
obse ed (10), binding o N cA o a wild- ype agmen p o-
duced wo e a ded bands ha likely co espond o binding o
N cA o one (lowe e a ded band) o bo h (uppe e a ded
band) binding si es (Fig. 1A). Mu a ion o he p oximal si e
esul ed, as expec ed, in he absence o he uppe e a ded
band ha likely co esponds o binding o N cA o wo si es
(Fig. 1B). Howe e , mu a ion o he dis al si e did no abolish
binding o N cA o his si e, since he uppe e a ded band was
s ill obse ed, al hough a highe N cA concen a ions han in
he case o he wild- ype agmen (compa e Fig. 1A and C).
Consis en ly, N cA was s ill able o bind o he agmen con-
FIG. 1. Binding o pu i ied N cA o wild- ype and mu a ed e sions o he n cA p omo e egion. EMSA was ca ied ou wi h a wild- ype
agmen (A) o wi h agmen s bea ing mu a ions in he p oximal (B), dis al (C), o bo h (D) N cA-binding si es. A scheme o he agmen used
in each case, including he posi ions o TSPs ⫺180, ⫺136, and ⫺49, oge he wi h he sequences o wild- ype and mu a ed N cA-binding si es
p esen in each agmen , is shown in panels A h ough D. Mu a ions in oduced in each agmen a e shown in g ay unde he wild- ype sequence.
The posi ions o ee DNA agmen s (open iangles), e a ded bands (black iangles), and ain bands appea ing when a high concen a ion o
N cA was used (as e isks), as e e ed o in he ex , a e indica ed. Assay mix u es con ained 0.1 mol o labeled DNA agmen .
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aining mu a ions in bo h N cA-binding si es (Fig. 1D). The
p esence o ain bands appea ing when a high concen a ion
o N cA was used (Fig. 1A, B, and C) sugges s low-a ini y
binding o N cA o addi ional posi ions. In ac , impe ec
N cA-binding si es a e p esen in he DNA agmen used, jus
ups eam om posi ions ⫺180 (GTAN
8
TAT) and ⫺49
(GTAN
8
TAA) (Fig. 3B). Howe e , because mu a ion o he
dis al si e did no abolish N cA binding o ha si e (see abo e),
he e a ded band shown in Fig. 1D ( agmen wi h bo h si es
mu a ed) could esul om binding o N cA o he mu a ed
dis al si e.
In he case o agmen s con aining a mu a ed p oximal si e
(Fig. 1B), he e a ded band p esumably co esponding o
binding o N cA o only one si e was al eady obse ed a he
lowes N cA concen a ion used, sugges ing ha N cA exhibi s
high a ini y o he dis al si e. In con as , binding o N cA o
he p oximal si e equi ed la ge amoun s o N cA (lowe e-
a ded band in Fig. 1C), indica ing lowe a ini y o N cA o
his si e. Thus, he lowe e a ded band obse ed wi h he
wild- ype agmen (Fig. 1A) should co espond mos ly o
binding o N cA o he dis al si e. Binding o N cA o all ou
agmen s could be compe ed by addi ion o an unlabeled
agmen co esponding o he glnA p omo e , which con ains
a high-a ini y N cA-binding si e, con i ming ha he obse ed
shi s we e in ac due o speci ic N cA binding (no shown).
Pa allel mobili y shi assays we e ca ied ou in he absence
o p esence o 0.6 mM 2-oxoglu a a e in o de o es he
esponsi eness o N cA binding o his e ec o (24) (see Fig.
S1 in he supplemen al ma e ial). The amoun o agmen in
e a ded complexes co esponding o binding o only one
N cA-binding si e was quan i ied in assays ca ied ou wi h
agmen s mu a ed in he p oximal (see Fig. S1B in he sup-
plemen al ma e ial) o dis al (see Fig. S1C in he supplemen al
ma e ial) N cA-binding si e. The esul s shown in Fig. 2 indi-
ca e ha binding o bo h si es was posi i ely in luenced by
2-oxoglu a a e. Al hough N cA alone showed highe a ini y
o he dis al han o he p oximal si e, a ini y appea ed o be
sligh ly highe o he p oximal han o he dis al si e in he
p esence o 2-oxoglu a a e. I is wo h no ing ha he dis al
si e (GTAN
8
AAC) does no exac ly ma ch he consensus o
N cA-binding si es (GTAN
8
TAC). Howe e , he p esence o
nucleo ides C and A in he second and hi d posi ions o he
8-nucleo ide spacing be ween iple s (unde lined in Fig. 1)
mos likely con ibu es o N cA binding o his si e (7, 23).
The esponsi eness o he me abolic signal e ec o makes
he p oximal si e a good candida e o an N cA ac i a o si e o
P
1
in esponse o he cellula condi ion o ni ogen dep i a ion,
which in cyanobac e ia esul s in high le els o 2-oxoglu a a e
(14). The high a ini y o N cA o he dis al si e in he absence
o 2-oxoglu a a e sugges s ha his si e would be he one p e -
e ably occupied unde cellula condi ions in which le els o
N cA a e low. Howe e , because binding o N cA o he dis al
si e would mos likely be incompa ible wi h binding o RNA
polyme ase o, and ansc ip ion om, P
2
, and because no
ep ession o ansc ip ion om P
2
is obse ed in cells o e ex-
p essing N cA (15), binding o N cA o his si e in i o could be
ela i ely low wi h espec o RNA polyme ase binding o P
2
.
To de e mine he DNA sequences in e ac ing wi h N cA in
wild- ype and mu an e sions o he n cA p omo e , binding o
N cA in he p esence o 2-oxoglu a a e was also analyzed by
DNase I p o ec ion oo p in ing assays ca ied ou as desc ibed
p e iously (22) (Fig. 3 and see Fig. S2 in he supplemen al
ma e ial). The DNA agmen s used we e PCR ampli ied wi h
oligonucleo ides NA8 and NA14 and plasmid pCSEL18 (wild-
ype p omo e ), pCSEL35 (p oximal si e mu a ed), pCSEL44
(dis al si e mu a ed), o pCSEL47 (bo h si es mu a ed) as a
empla e. One o he wo oligonucleo ides used o he PCRs
was end labeled wi h [␥-
32
P]dATP and polynucleo ide kinase.
Oligonucleo ide NA8 was labeled in he expe imen shown in
Fig. 3A, whe eas oligonucleo ide NA14 was labeled in he
expe imen shown in Fig. S2 in he supplemen al ma e ial. Two
egions ( a -le panel in Fig. 3A), each co esponding o one
N cA-binding si e, we e p o ec ed in he wild- ype agmen in
he p esence o e e y N cA concen a ion es ed. The a ea
p o ec ed by N cA a ound he dis al si e included he ⫺10
de e minan o he P
2
p omo e , as well as he posi ion co -
esponding o TSP
2
(⫺136) (Fig. 3B). Mu a ion o he p oxi-
mal si e elimina ed p o ec ion o he co esponding sequences
(second and a igh panels in Fig. 3A). Mu a ion o he dis al
si e did no comple ely elimina e p o ec ion o sequences co -
esponding o ha si e ( hi d and a igh panels in Fig. 3A),
hus indica ing ha N cA could s ill bind o hose posi ions,
al hough a la ge amoun o N cA was equi ed in o de o see
a window o p o ec ion. This obse a ion is consis en wi h he
ac ha e a ded bands ha migh be due o N cA binding o
he dis al si e we e obse ed in EMSA wi h agmen s bea ing
mu a ions in such a si e (Fig. 1).
Binding o N cA in i o o ei he si e did no seem o ely on
binding o he o he si e, al hough coope a i e e ec s canno
be excluded in i o. In e es ingly, binding o N cA modi ied
he pa e n o DNase I diges ion in he a ea co esponding o
he p oximal binding si e, e en when ha si e was mu a ed
(ei he alone o in combina ion wi h a mu a ion o he dis al
FIG. 2. E ec o 2-oxoglu a a e on binding o N cA o DNA ag-
men s con aining he n cA p omo e egion bea ing mu a ions in he
p oximal (A) o dis al (B) N cA-binding si es. The pe cen age o ag-
men e a ded (one band in panel A and wo bands in panel B) in
pa allel assays ca ied ou in he absence (open ci cles) o p esence
(closed ci cles) o 2-oxoglu a a e (see Fig. S1 in he supplemen al
ma e ial) is ep esen ed wi h espec o he N cA concen a ion in he
assays.
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si e) (Fig. 3A and see Fig. S2 in he supplemen al ma e ial).
This modi ica ion could be due o con o ma ional changes
induced by binding o N cA o o he posi ions, including he
dis al si e. Binding o N cA o he dis al (Fig. 3A) o he
p oximal (see Fig. S2 in he supplemen al ma e ial) si e p o-
duced a band hype sensi i e o DNase I diges ion, co espond-
ing o he T o he GTA iple (Fig. 3A and see Fig. S2 in he
supplemen al ma e ial). Such hype sensi i i y, also obse ed in
he case o N cA binding o he glnA and bcL p omo e s (17),
indica es ha N cA binding, simila o CRP binding (20),
FIG. 3. Sequences o he n cA p omo e egion p o ec ed by N cA in he p esence o 2-oxoglu a a e. A ows labeled ⫺49, ⫺136, and ⫺180
indica e he posi ion and di ec ion o TSPs co esponding o P
1
,P
2
, and P
3
, espec i ely. (A) DNase I p o ec ion oo p in assays ca ied ou wi h
pu i ied N cA and wild- ype and mu a ed e sions o he n cA p omo e egion. The agmen s we e ampli ied by PCR using oligonucleo ides NA8
(
32
P labeled) and NA14 (unlabeled). Assay mix u es con ained 2.5 mol o labeled agmen and 0.068, 0.137, 0.274, o 0.412 ␮M o pu i ied N cA
in he p esence o 0.6 mM 2-oxoglu a a e. Windows o p o ec ion due o N cA binding ( e ical lines), posi ions p o ec ed in all ou agmen s
(solid iangles), and posi ions al e ed in he p esence o N cA in agmen s con aining a mu a ed p oximal si e (open iangles) a e indica ed.
Thick e ical lines indica e egions p o ec ed in he p esence o e e y N cA concen a ion es ed. Thin e ical lines indica e ha mu a ion o he
dis al si e did no comple ely elimina e p o ec ion o he sequence co esponding o ha si e. The posi ions o N cA-binding si es (s iped boxes)
a e also indica ed on he le . As e isks deno e a posi ion hype sensi i e o DNase I diges ion as e e ed o in he ex . (B) Sequence o he n cA
p omo e , om posi ion ⫺253 o he ansla ional s a o he n cA gene (ATG). T iple s co esponding o he N cA-binding si es a e indica ed
in bold ace. The posi ions a ound he N cA-binding si es p o ec ed in bo h s ands o he wild- ype agmen a e indica ed wi h ho izon al lines
abo e o below he sequence. Pu a i e ⫺10 and ⫺35 de e minan s a e indica ed wi h g ay boxes. Solid iangles poin o posi ions p o ec ed in all
ou agmen s. The posi ions o TSPs o P
1
,P
2
, and P
3
a e unde lined.
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migh p oduce con o ma ional changes in he DNA backbone.
Addi ional sub le changes ha would co espond o pa ial
p o ec ion o sequences loca ed ups eam om he dis al
N cA-binding si e (a ound TSP
3
) (Fig. 3A) and downs eam
om he p oximal N cA binding si e (ups eam o TSP
1
) (see
Fig. S2 in he supplemen al ma e ial) we e obse ed wi h all
ou DNA agmen s used in oo p in ing expe imen s. Se-
quences p o ec ed by N cA binding in bo h s ands a e sum-
ma ized in Fig. 3B.
In i o ope a ion o mu a ed e sions o he n cA p omo e .
We ha e p e iously analyzed he exp ession o an n cA-g p
ansla ional usion om he wild ype and sho ened e sions
o he n cA p omo e (16). Ou p e ious esul s indica e ha
(i) he e is a ce ain le el o ope a ion o P
1
in he absence o
N cA in all cells o he ilamen (13); (ii) in he absence o P
3
,
he agmen co e ing posi ions ⫺143 o ⫺182 exe s a nega-
i e e ec on exp ession o P
1
(16); and (iii) P
1
alone appea s
capable o d i ing localized induc ion o n cA in di e en ia ing
cells in he absence o he agmen om posi ions ⫺143 o
⫺182, bu in i s p esence, localized exp ession o P
1
in p ohe -
e ocys s equi es P
3
(s ain CSEL7) (16). We ha e now p e-
pa ed cons uc s in which he n cA-g p ansla ional usion is
exp essed om modi ied e sions o he n cA p omo e (P
n cA
-
g p). In o de o p ese e any au o egula o y e ec o N cA, all
o he P
n cA
-g p cons uc s we e in eg a ed, h ough homolo-
gous ecombina ion, in he nucA-nuiA egion o he ␣
megaplasmid o Anabaena sp. s ain PCC 7120, so ha exp es-
sion o he n cA gene in he ch omosome emained unal e ed.
Cons uc s bea ing ansla ional usions o he mu a ed e -
sions o he n cA p omo e wi h he g p gene (GFP-mu 2) we e
p epa ed as desc ibed by Olmedo-Ve d e al. (16; see he
supplemen al ma e ial o de ails). S ains CSEL10, CSEL11,
and CSEL12 con ained he g p gene used o mu a ed e sions
o he n cA p omo e , including mu a ions in he p oximal,
dis al, o bo h N cA-binding si es, espec i ely. S ain CSEL6
bea ing he wild- ype e sion o he n cA p omo e ansla ion-
ally used o g p (16) was used as a con ol.
Ammonium-g own ilamen s we e subjec ed o ni ogen de-
p i a ion, and exp ession o GFP was analyzed by means o
luo escence imaging and p ime ex ension. GFP luo escence
in s ains ca ying di e en e sions o he P
n cA
-g p usion is
shown in Fig. 4. Mu a ions elimina ing N cA binding o he
p oximal si e (s ain CSEL10) o educing N cA binding o he
dis al si e (s ain CSEL11) did no abolish localized inc ease o
GFP luo escence. Howe e , mu a ion o bo h N cA-binding
si es esul ed in low, nonlocalized exp ession o he usion
(s ain CSEL12).
In o de o de e mine which p omo e s we e ac i e in each
FIG. 4. GFP luo escence o s ains ca ying he P
n cA
-g p usions in eg a ed in he nucA-nuiA genomic egion o he Anabaena ␣megaplasmid.
GFP luo escence mic og aphs o ammonium-g own ilamen s (panels labeled 0 h) o ammonium-g own ilamen s incuba ed o 6, 10 o 24 h in
a medium lacking combined ni ogen a e shown o he indica ed CSEL s ains. The N cA-binding si e o si es mu a ed in each s ain a e indica ed.
7588 NOTES J. BACTERIOL.
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s ain, p ime ex ension assays o n cA-g p ansc ip s we e
ca ied ou as desc ibed p e iously (12) using oligonucleo ide
GFP4 (5⬘CAAGAATTGGGACAACTCC3⬘; complemen a y
o posi ions ⫹46 o ⫹28 wi h espec o he ansla ional s a
o he g p gene). Resul s om one ep esen a i e expe imen
a e shown in Fig. 5. When he p oduc s o p ime ex ension
we e compa ed o hose ob ained in he con ol s ain bea ing
a wild- ype P
n cA
-g p usion (s ain CSEL6), induc ion o P
1
could no be obse ed in s ain CSEL10, indica ing ha N cA
binding o he p oximal si e is equi ed o P
1
induc ion. In
con as , exp ession om P
1
was unal e ed (o sligh ly in-
c eased) in s ain CSEL11, sugges ing ha binding o N cA o
he p oximal si e is su icien o P
1
ac i a ion. This is consis-
en wi h p e ious esul s showing high P
1
exp ession om a
agmen bea ing he p oximal N cA binding si e and only hal
o he dis al si e (s ain CSEL8 [16]). The loca ion o he
p oximal binding si e wi h espec o TSP
1
(cen e ed a posi-
ion ⫺54.5) does no , howe e , co espond o ha obse ed in
class II (a ound ⫺42) o class I (nea ⫺93, ⫺83, ⫺72, o ⫺62)
bac e ial ac i a ed p omo e s. As expec ed, because mu a ion
o he dis al N cA-binding si e a ec s he ⫺10 de e minan o
P
2
, no ansc ip co esponding o TSP
2
was obse ed in s ain
CSEL11. U iliza ion o P
3
was unal e ed in s ains CSEL10
and CSEL11. Finally, mu a ion o bo h N cA-binding si es
(s ain CSEL12) led o a basal le el o exp ession om P
1
(as
obse ed in s ain CSEL10), no exp ession om P
2
(as ob-
se ed in s ain CSEL11), and ela i ely low exp ession om
P
3
. The basis o he obse ed e ec o mu a ions o bo h
N cA-binding si es on he u iliza ion o P
3
exp ession is cu -
en ly unknown. I is wo h men ioning ha all P
n cA
-g p con-
s uc s analyzed a e in eg a ed in he ␣megaplasmid, so ha
exp ession o he n cA gene and le els o he N cA p o ein
emain unal e ed wi h espec o hose o he wild- ype s ain.
Concluding ema ks. Taking in o accoun p esen and p e-
ious esul s (13, 16), a model o he ope a ion o he n cA
complex p omo e egion can be sugges ed as ollows. Basal
ope a ion o he n cA p omo e (i.e., in he p esence o com-
bined ni ogen) would include exp ession om P
2
and basal
exp ession om P
1
and would no equi e N cA o He R (13).
When he cells a e subjec ed o ni ogen de iciency, which
esul s in an inc ease in he le el o 2-oxoglu a a e, binding o
low, p eexis ing amoun s o N cA p o ein o he p oximal
N cA-binding si e would lead o inc eased ansc ip ion om
P
1
. This may esul in inc eased ansc ip ion in all cells o he
ilamen . Then he p esence o He R and/o o he He R-de-
penden elemen s in speci ic cells o he ilamen ha a e
becoming he e ocys s would p omo e ansien exp ession
om P
3
, which is equi ed o ansien localized induc ion o
P
1
in he comple e p omo e agmen (s ain CSEL7 [16]).
T ansien induc ion o P
1
and P
3
exhibi s simila iming and
dependence on N cA and He R (Fig. 5; see also e e ence 13)
and migh hus be somewha coupled. Exp ession om P
1
and
P
3
likely accoun s o he app oxima ely i e old ansien in-
c ease in he amoun o n cA ansc ip s p e iously shown o
ake place in whole ilamen s o he wild- ype s ain, bu no in
he he R mu an , du ing ni ogen s ep down (13). Acco dingly,
we ha e obse ed a simila i e old inc ease in he amoun o
g p ansc ip s in s ain CSEL6 by means o No he n hyb id-
iza ion (no shown). Occupancy o he p oximal N cA-binding
si e appea s o play a key ole in de e mining he p omo e (s)
o be used and he o al a e o ansc ip ion o n cA unde
di e en ni ogen egimens and in he wo cell ypes o he
ni ogen- ixing ilamen .
We hank Jose´ En ique F ías and Ignacio Luque o c i ically ead-
ing he manusc ip and Alicia O ea o help wi h con ocal mic oscopy.
This wo k was suppo ed by g an s BFU2004-00872 and BFU2007-
60457 om he Minis e io de Educacio´n y Ciencia, Spain.
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