Co E om
Myxococcus xan hus
Is a Coppe -Dependen
RNA Polyme ase Sigma Fac o
Nu ia Go
´mez-San os, Juana Pe
´ ez, Ma ı
´a Celes ina Sa
´nchez-Su il, Au elio Mo aleda-Mun
˜oz, Jose
´Mun
˜oz-
Do ado*
Depa amen o de Mic obiologı
´a, Facul ad de Ciencias, Uni e sidad de G anada, G anada, Spain
Abs ac
The dual oxici y/essen iali y o coppe o ces cells o main ain a igh ly egula ed homeos asis o his me al in all li ing
o ganisms, om bac e ia o humans. Consequen ly, many genes ha e p e iously been epo ed o pa icipa e in coppe
de oxi ica ion in bac e ia. Myxococcus xan hus, a p oka yo e, encodes many p o eins in ol ed in coppe homeos asis ha
a e di e en ially egula ed by his me al. A s ac o o he ECF (ex acy oplasmic unc ion) amily, Co E, has been ound o
egula e he exp ession o he mul icoppe oxidase cuoB, he P1B- ype ATPases copA and copB, and a gene encoding a
p o ein wi h a hea y-me al-associa ed domain. Cha ac e iza ion o Co E has e ealed ha i equi es coppe o bind DNA in
i o. Genes egula ed by Co E exhibi a cha ac e is ic exp ession p o ile, wi h a peak a 2 h a e coppe addi ion. Exp ession
apidly dec eases he ea e o basal le els, al hough he me al is s ill p esen in he medium, indica ing ha he ac i i y o
Co E is modula ed by a p ocess o ac i a ion and inac i a ion. The use o mono alen and di alen me als o mimic Cu(I)
and Cu(II), espec i ely, and o addi i es ha a o he o ma ion o he wo edox s a es o his me al, has e ealed ha
Co E is ac i a ed by Cu(II) and inac i a ed by Cu(I). The ac i a ion/inac i a ion p ope ies o Co E eside in a Cys- ich domain
loca ed a he C e minus o he p o ein. Poin mu a ions a hese esidues ha e allowed he iden i ica ion o se e al Cys
in ol ed in he ac i a ion and inac i a ion o Co E. Based on hese da a, along wi h compa a i e genomic s udies, a new
g oup o ECF s ac o s is p oposed, which no only clea ly di e s mechanis ically om he o he s ac o s so a
cha ac e ized, bu also om o he me al egula o s.
Ci a ion: Go
´mez-San os N, Pe
´ ez J, Sa
´nchez-Su il MC, Mo aleda-Mun
˜oz A, Mun
˜oz-Do ado J (2011) Co E om Myxococcus xan hus Is a Coppe -Dependen RNA
Polyme ase Sigma Fac o . PLoS Gene 7(6): e1002106. doi:10.1371/jou nal.pgen.1002106
Edi o : Josep Casadesu
´s, Uni e sidad de Se illa, Spain
Recei ed Janua y 19, 2011; Accep ed Ap il 11, 2011; Published June 2, 2011
Copy igh : ß2011 Go
´mez-San os e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This wo k has been suppo ed by g an s om ‘‘Minis e io de Ciencia e Inno acio
´n,’’ Spain (BFU2006-00972/BMC, 70% unded by FEDER; and he
p og am CONSOLIDER-INGENIO 2010, e . CSD2009-00006) and Jun a de Andalucı
´a (CVI-1377). AM-M has been unded by a pos doc o al ellowship om he
‘‘Plan P opio de la Uni e sidad de G anada.’’ The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he
manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]
In oduc ion
Myxococcus xan hus is a soil-dwelling d-p o eobac e ium o he
g oup o myxobac e ia used as a model o s udy mul icellula
beha io and di e en ia ion, because i exhibi s a complex
de elopmen al cycle igge ed by s a a ion [1]. Howe e , M.
xan hus cells no only ha e o adap hei me abolism and beha io
o changing nu i ional concen a ions, bu also o o he
pa ame e s, such as me als.
Coppe is a ansi ion me al ha unc ions as an ideal biological
co ac o due o i s abili y o al e na e be ween he edox s a es
Cu(I) and Cu(II). Howe e , coppe also gene a es eac i e oxygen
species ha cause cell damage [2]. This duali y o ces o ganisms o
main ain a s ic homeos asis o his me al. The mos ep esen-
a i e examples o he e ec o dis u bances in coppe homeos asis
a e wo inhe i ed human diso de s, Wilson disease and Menkes
synd ome, which a e di ec ly linked o o e load and de iciency o
his me al, espec i ely [3].
Coppe is equi ed by p oka yo es in ace amoun s because i is
used as a co ac o by a ew p o eins. Hence, mos bac e ial
homeos a ic mechanisms a e de o ed o con e ing esis ance o his
me al. The mos common mechanisms a e coppe - anspo ing
P1B- ype ATPases, coppe chape ones, mul icoppe oxidases
(MCOs), and Cus sys ems [4]. In bac e ia such as Esche ichia coli,
one o each o hese elemen s is encoded in he genome [4]. In o he
bac e ia, he homeos a ic mechanism is e en simple , consis ing o
wo P1B- ype ATPases and one chape one (Synechocys is PCC6803,
En e ococcus hi ae, and Lac ococcus lac is), o one ATPase and one
chape one (Bacillus sub ilis) [4,5]. In con as , he la ge M. xan hus
genome encodes a la ge numbe o pa alogous genes o con e
coppe ole ance: h ee MCOs, a leas wo Cus sys ems, and h ee
P1B- ype ATPases, as well as he genes equi ed o he biosyn hesis
o ca o enoids [6–9]. This gene edundancy indica es ha coppe
homeos asis in his myxobac e ium is mo e complex han in o he
p oka yo es. All o hese genes ha e been shown o be di e en ially
egula ed [6–9], sugges ing ha his sophis ica ed ne wo k mus be
inely egula ed by speci ic me al senso s.
One o he signal ansduc ion mechanisms used by bac e ia o
di ec gene exp ession a he ansc ip ional le el in esponse o s ess
signalsis ep esen edbyal e na i es ac o s [10]. The la ges g oup
o al e na i e s ac o s is he ECF (ex acy oplasmic unc ion) amily,
which co esponds o g oup 4 o he s
70
p o eins [11]. ECF s ac o s
a e small p o eins, qui e di e gen in sequence, ha con ain only wo
egions (s
2
and s
4
) equi ed o in e ac ion wi h he RNA
polyme ase co e enzyme and ecogni ion o he p omo e [12].
Thei abili y o p omo e ansc ip ion elies on a p o ein ha is
PLoS Gene ics | www.plosgene ics.o g 1 June 2011 | Volume 7 | Issue 6 | e1002106
no mally co ansc ibed wi h he s ac o , named an i-s ac o . In he
absence o ex e nal signals, ECF s ac o s a e seques e ed by hei
cogna e an i-s. A e de ec ing he speci ic s imulus, he an i-s ac o
eleases he ssubuni , which can hen p omo e gene exp ession a e
ec ui men o he co e RNA polyme ase [13–16].
In his epo , we iden i y a no el me al senso in ol ed in
coppe homeos asis in M. xan hus named Co E ( o coppe -
egula ed ECF s ac o ). We demons a e ha Co E equi es
coppe in o de o bind o DNA and ha i s ac i i y is modula ed
by he edox s a e o his me al. Acco ding o hese da a, we
p opose a new g oup o ECF s ac o s, de ined by a Cys- ich
domain (CRD) loca ed a he C e minus o he p o ein, which is
essen ial o ac i a ion and inac i a ion o he p o ein.
Resul s/Discussion
Co E is an ECF s ac o in ol ed in coppe homeos asis
Mos M. xan hus genes in ol ed in coppe homeos asis a e
loca ed in he genome in wo clus e s [8]. In coppe egion 2, and
nex o he MCO cuoB, a gene encoding a p o ein wi h high
simila i y o ECF s ac o s was ound (MXAN_3426), sugges ing
ha i could egula e he exp ession o genes in ol ed in con e ing
coppe esis ance. This s ac o has been designa ed as Co E. The
analysis o he Co E sequence has e ealed a domain a chi ec u e
wi h he conse ed egions s
2
(sigma70_ 2, PF04542; E- alue o
3.2e-14) and s
4
(sigma70_ 4_2, PF08281; E- alue o 4.3e-06)
ypical o his ype o s ac o s [11,12].
To de e mine he ole o Co E in coppe homeos asis, a s ain
ha bo ing a co E-lacZ usion was cons uc ed, and he analysis o
his s ain e ealed ha co E was up- egula ed by coppe
(Figu e 1A). Addi ionally, an in- ame dele ion mu an (Dco E)
was also gene a ed, and he pheno ypic analysis o his s ain
con i med ha his egula o con e ed coppe ole ance
(Figu e 1B).
Genes egula ed by Co E
To iden i y genes egula ed by Co E, plasmids con aining
usions be ween he genes ha ha e so a been in ol ed in coppe
and/o o he me al homeos asis in M. xan hus and lacZ we e
elec opo a ed in o he Dco E mu an . When he exp ession
p o iles o hese genes in he mu an we e compa ed wi h hose
exhibi ed in he wild- ype (WT) s ain, i was obse ed ha only
he MCO cuoB and he P1B- ype ATPase copB emained
unde ec able in he Dco E backg ound in he p esence o coppe
(Figu e 2A and Figu e S1), indica ing ha hey a e egula ed by
his s ac o . In e es ingly, hese wo M. xan hus genes exhibi a
cha ac e is ic exp ession p o ile, wi h a peak a 2 h a e he
addi ion o exogenous coppe .
As ECF s ac o s a e usually au o egula ed, co E exp ession was
analyzed in he Dco E mu an . The esul s ob ained showed ha
his s ac o is only pa ially esponsible o i s own up- egula ion
by coppe , especially in he ea ly s ages a e me al addi ion.
Howe e , some up- egula ion by he me al s ill emains in he
mu an (Figu e 1A, ed lines), indica ing ha al hough cuoB and
co E a e e y close in he genome (Figu e S2), hei egula ion
exhibi s some di e ences.
The compa ison and analysis o he ups eam egions o cuoB
and copB has allowed he iden i ica ion o wo simila sequences
ha could unc ion as he p omo e elemen s ecognized by Co E
(Figu e S3), one loca ed ups eam o copB, and he o he ups eam
o a hi d gene gene ically linked o cuoB and co E which encodes
an ou e memb ane e lux p o ein (MXAN_3424). A manual
sea ch o homologous sequences o his pu a i e Co E-binding
si e in he M. xan hus coppe egions 1 and 2 [8] e ealed he
p esence o wo o he ma ches, one ups eam o he gene o he
P1B- ype ATPase CopA and he o he ups eam o he gene
iden i ie MXAN_3427, which encodes a p o ein wi h a hea y-
me al-associa ed domain (PF00403, wi h an E- alue o 4.7e-14).
To co obo a e ha hese wo genes a e egula ed by Co E,
plasmids ha bo ing usions be ween hese wo genes and lacZ
we e in oduced in o he WT and Dco E backg ounds and b–gal
speci ic ac i i y was de e mined in hese s ains. The esul s
ob ained e ealed ha he gene o he hea y-me al-associa ed
p o ein exhibi s an exp ession p o ile in he WT s ain e y
simila o hose o cuoB and copB a e coppe supplemen a ion
(compa e Figu e 2A and 2B). Up- egula ion by coppe was
comple ely elimina ed in he Dco E mu an , demons a ing ha
his gene is also pa o he Co E egulon. In he case o copA he
esul was less clea . The exp ession p o ile o copA in he WT
s ain clea ly di e s om hose exhibi ed by he Co E- egula ed
genes (compa e panel C wi h panels A and B in Figu e 2), and
ins ead o a peak a 2 h, a pla eau is eached 24 h a e coppe
addi ion. Acco dingly, he exp ession p o ile o copA in he Dco E
mu an is qui e simila o ha o he WT s ain (Figu e 2C).
Howe e , when he exp ession le el o copA in hese wo s ains
was analyzed wi h g ea e p ecision a sho in e als (Figu e 2D),
i could be obse ed ha he apid induc ion o his gene ob ained
in he WT s ain was no longe obse ed in he mu an . This
esul sugges s ha copA is subjec o double egula ion by Co E
and ano he uniden i ied coppe -dependen egula o . Ne e he-
less, u he wo k will be equi ed o unambiguously demons a e
ha copA is egula ed by Co E. Finally, using he consensus
sequence o he p omo e s o hese ou genes, we ied o
de e mine which o he genes could also be unde con ol o
Co E. By using he app oach desc ibed in Ma e ials and
Me hods, ano he 13 simila sequences we e iden i ied in he
M. xan hus genome (Figu e S3). Howe e , he ac ha only wo o
hem con ain he se en in a iable esidues ound in he o he
p omo e s, and ha none o he p o eins encoded by he genes
loca ed downs eam o hese sequences exhibi simila i ies o
o he p o eins known o be in ol ed in coppe handling and
a icking, p e en ing us om d awing he conclusion ha hey
a e indeed egula ed by Co E.
Au ho Summa y
Coppe exe s a dual e ec on li ing o ganisms. I is
essen ial o li e, bu an excess p o okes cell damage,
o cing cells o main ain a egula ed homeos asis o his
me al. These wo an agonis ic biological e ec s o coppe
a e clea ly illus a ed by wo human gene ic diso de s,
Menkes synd ome and Wilson disease, caused by de icien-
cy o accumula ion o his me al, espec i ely. Myxococcus
xan hus, a soil-dwelling bac e ium, also has o cope wi h
changes in coppe concen a ion in i s en i onmen . The
la ge genome o his myxobac e ium encodes many genes
in ol ed in coppe homeos asis, all o which a e di e en-
ially egula ed, indica ing ha many egula o s pa icipa e
in coppe homeos asis in his p oka yo e. He e, we iden i y
one o hese egula o s (Co E), which belongs o he amily
o he ex acy oplasmic unc ion (ECF) s ac o s. We
demons a e ha Co E ep esen s a no el g oup o ECF
s ac o s and o me al egula o s, because i s ac i i y is
modula ed by he edox s a e o coppe . This abili y
esides in a Cys- ich domain, which has also been ound in
o he s ac o s o di e en bac e ial phyla. The e o e, we
p opose ha Co E is he i s membe o a mechanis ically
new g oup o ECF s ac o s.
A Coppe -Dependen Sigma Fac o
PLoS Gene ics | www.plosgene ics.o g 2 June 2011 | Volume 7 | Issue 6 | e1002106
Figu e 1. Co E is in ol ed in coppe homeos asis. (A) Coppe up- egula ion o co E.b-gal speci ic ac i i y was de e mined in cell ex ac s o he
WT (blue lines) and Dco E ( ed lines) s ains (ha bo ing he usion co E-lacZ) om CTT aga pla es con aining no coppe (open symbols) o 0.6 mM
(closed symbols) coppe sul a e. (B) E ec o coppe on M. xan hus g ow h. WT (blue line) and Dco E ( ed line) s ains we e g own in he absence o
he me al and dilu ed o an OD
600
o 0.05 in o esh CTT liquid media con aining he indica ed coppe concen a ions. The OD
600
was hen moni o ed
a e 24 h o incuba ion. E o ba s indica e s anda d de ia ions.
doi:10.1371/jou nal.pgen.1002106.g001
Figu e 2. Co E-dependen genes. (A) Regula ion o cuoB and copB by Co E. Plasmids con aining cuoB-lacZ (blue lines) and copB-lacZ ( ed lines)
usions we e in oduced in o he WT (solid symbols) o he Dco E (open symbols) backg ounds, and incuba ed on CTT aga pla es con aining 0.6 mM
CuSO
4
.b-gal speci ic ac i i y was de e mined in cell ex ac s ha es ed a he indica ed imes. The same app oach epo ed abo e was ollowed o
s udy he egula ion o MXAN_3427 (B) and copA (C and D) by Co E, al hough 0.3 mM CuSO
4
was used o ge an op imal di e ence in he copA
exp ession le els be ween he WT and he Dco E s ains a ea ly imes (panels C and D). The dashed a ow om panel C o D indica es ha in panel D
only he indica ed pa o panel C is shown. Please no e he di e ence in he scale in each panel, and he di e en ime cou se o panel D. E o ba s
indica e s anda d de ia ions.
doi:10.1371/jou nal.pgen.1002106.g002
A Coppe -Dependen Sigma Fac o
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As he ac i a ion o Co E by coppe could be caused ei he by
he gene al oxida i e s ess induced by his me al o by he di ec
binding o he p o ein o coppe in ei he o i s wo edox s a es,
cuoB exp ession in he WT s ain was es ed in he p esence o
se e al concen a ions o he oxidan s hyd ogen pe oxide and
diamide, and he Cu(II) mime ic di alen me als Cd
2+
,Ni
2+
, and
Zn
2+
. Simila ly, Ag
+
was used o mimic Cu(I). The esul s ob ained
e ealed ha only Cd
2+
and Zn
2+
could induce cuoB exp ession
(Figu e 3A and Figu e S4). The ac ha Ni
2+
does no up- egula e
cuoB is no su p ising, because he same me als canno always
mimic he coppe e ec . As an example, he M. xan hus P1B- ype
ATPase copA has been epo ed o be induced by coppe , Ni
2+
and
Co
2+
, bu no by Zn
2+
[9]. I is no able ha he exp ession le els
ob ained wi h Cd
2+
and Zn
2+
we e no only much lowe han wi h
coppe , bu also ha he exp ession p o iles we e di e en . In he
case o Cd
2+
, no peak was obse ed a 2 h; ins ead, a pla eau was
eached 24 h a e me al supplemen a ion (Figu e 3B). Al hough
Zn
2+
also yielded a apid cuoB induc ion, he peak a 2 h was no as
e iden as in he case o coppe (Figu e 3C). cuoB up- egula ion by
Cd
2+
and Zn
2+
is also dependen on Co E (Figu e 3B and 3C).
These da a indica e ha Cu(II) is he edox s a e o coppe ha
ac i a es Co E. I should be no ed ha he Cd
2+
and Zn
2+
concen a ions needed o obse e a clea cuoB induc ion a e close
o he maxima ha M. xan hus cells can ole a e [7], while 0.3 mM
coppe has almos no e ec on myxobac e ial g ow h (Figu e 1B).
I should also be no ed ha he addi ion o me als o he media no
only al e s he g ow h a es o he cul u es, bu also inhibi s cell
mo ili y, explaining why he mo phology o he cell spo s is no he
same in all o he media es ed.
Sea ching o he Co E cogna e an i-s ac o
Many ECF s ac o s unc ion wi h a cogna e an i-swhich is
gene ically linked o he ssubuni [11–16]. Analyses o he genes
loca ed in he p oximi y o co E e ealed ha hey encode ei he
p o eins loca ed in he pe iplasmic space o in he ou e
memb ane, o ha hey exhibi s iking simila i ies o well-
cha ac e ized p o eins in ol ed in speci ic unc ions, sugges ing
ha no an i-s ac o is co ansc ibed wi h co E. Howe e , he
possibili y emained ha i could be encoded in some o he egion
o he M. xan hus genome. To es o he exis ence o an an i-s
ac o , a s a egy was designed consis ing o he o e -exp ession o
co E [17]. I Co E we e p esen in highe quan i ies han an
uniden i ied an i-s ac o , i would be eleased om he
an agonis ic e ec o he an i-s, and cuoB should be exp essed
e en in he absence o any s imulus. To ollow his app oach, co E
was cloned unde con ol o he oa p omo e and in oduced in o
he Dco E mu an ha bo ing cuoB-lacZ o acili a e he analysis o
cuoB exp ession. The oa p omo e allows genes o be exp essed
cons i u i ely a high le els [18]. As a con ol, a co E’ cuoB-lacZ
s ain was also cons uc ed, in which he co E gene was unde
con ol o i s own p omo e (Figu e S2 displays he cuoB-lacZ
usions used in his s udy). Quan i a i e analyses o cuoB exp ession
in bo h s ains epo ed no exp ession o his gene in he absence
o coppe (Figu e 4A), indica ing ha an excess o Co E was no
Figu e 3.
cuoB
is only up- egula ed by coppe and o he di alen me als. (A) The WT s ain ha bo ing he cuoB-lacZ usion was spo ed on o
CTT aga pla es con aining me als o oxidan s a he concen a ions indica ed abo e each pic u e. Pla es also con ained 5-b omo-4-chlo o-3-indolyl-
b-D-galac o-py anoside o quali a i ely moni o b-gal ac i i y (blue colo de elopmen ). Pic u es we e aken a e 48 h o incuba ion. (B) Up-
egula ion o cuoB by Cd
2+
. The WT (con inuous line) and he Dco E s ains (dashed line) ha bo ing he cuoB-lacZ usion we e incuba ed on CTT aga
pla es con aining 0.1 mM Cd(NO
3
)
2
.b-gal speci ic ac i i y was de e mined in cell ex ac s ha es ed a he indica ed imes. (C) Up- egula ion o cuoB
by Zn
2+
. The app oach ollowed was he same as he one epo ed in panel B. The concen a ion o me al used was 0.4 mM Zn(NO
3
)
2
. E o ba s
indica e s anda d de ia ions.
doi:10.1371/jou nal.pgen.1002106.g003
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PLoS Gene ics | www.plosgene ics.o g 4 June 2011 | Volume 7 | Issue 6 | e1002106
su icien o ac i a e he ansc ip ion o cuoB. To co obo a e ha
Co E exp essed unde he oa p omo e was unc ional, coppe
was added o he media. In his case, up- egula ion o cuoB was
obse ed in bo h s ains and wi h simila exp ession p o iles
(Figu e 4B). Finally, o con i m ha co E was o e -exp essed when
cloned unde he oa p omo e , we cons uc ed he same wo
s ains desc ibed abo e bu in oducing a His ag a he N
e minus o Co E (hCo E’). Wes e n blo analyses using an ibodies
agains he His ag con i med ha co E was indeed exp essed a
e y high le els in he absence as well as in he p esence o coppe
(Figu e 4C and 4D). Co E mig a es as a double band, which mus
co espond o di e en o ms o he p o ein. Ac i i y o he hCo E’
p o ein was u he es ed by ollowing cuoB exp ession. The esul s
ob ained indica ed ha he p o eins holding he His ag could
p omo e cuoB ansc ip ion in he same manne as he na i e ones
(Figu e S5). Al hough i canno be comple ely uled ou ha a
cogna e an i-s ac o o Co E is encoded in he M. xan hus
genome, all o hese esul s indica e ha Co E unc ions in a
di e en manne om he one epo ed o he o he cha ac e ized
ECF s ac o s.
Co E needs coppe o bind DNA
The ac ha he o e -exp ession o co E did no lead o he
induc ion o cuoB unless coppe was added o he medium
sugges ed ha Co E migh equi e he binding o coppe o
p omo e ansc ip ion. Hence, he abili y o Co E o bind DNA in
i o was es ed by using elec opho e ic mobili y shi assays. Co E
was exp essed in E. coli wi h an N- e minal His ag and pu i ied by
a ini y ch oma og aphy. Addi ionally, a 265-bp agmen con-
aining he copB p omo e was ampli ied and labeled wi h
32
P obe
used as a p obe. As shown in Figu e 5, an elec opho e ic mobili y
shi was only obse ed in he eac ion mix u e con aining coppe
and ba hocup oine disul onic acid (BCS), a speci ic chela ing agen
o Cu(I) [19,20]. These esul s no only con i m ha Co E uses
coppe as a co ac o , bu also sugges ha Cu(I) p e en s Co E
om binding o DNA, and hence, ha Co E-Cu(II) is he ac i e
o m o his s ac o . This is also suppo ed by he ac ha only
di alen me als can mimic he e ec o coppe on cuoB up-
egula ion. No o he s ac o has so a been epo ed o equi e
any me al o bind DNA.
The edox s a e o coppe di ec s he ac i a ion/
inac i a ion o Co E
The exp ession p o iles o he Co E- egula ed genes exhibi a
peak a ound 2 h a e coppe addi ion (Figu e 2A and 2B), in spi e
o he ac ha co E exp ession is main ained o 48 h (Figu e 1A).
This obse a ion could be explained by p o eolysis o he s ac o .
Figu e 4. Sea ching o he Co E an i-s ac o . (A and B) Quan i ica ion o b-gal ac i i y (cuoB exp ession) in s ains whe e co E was cloned unde
he con ol o he oa p omo e ( ed lines) o i s own p omo e (blue lines). Ac i i ies we e de e mined in he absence o me al (A) o in he p esence
o 0.3 mM coppe (B). No e he di e ence in he scales o he wo panels. E o ba s indica e s anda d de ia ions. (C and D) Wes e n blo analyses o
con i m he o e -p oduc ion o Co E in he absence (C) o in he p esence (D) o 0.3 mM coppe in he s ains ha bo ing he gene co E cloned unde
he oa p omo e (lane 1) o i s own p omo e (lane 2). P o eins we e collec ed a 2 h a e coppe addi ion. The band o equal in ensi y in all he
lanes co esponds o an uniden i ied M. xan hus p o ein ha eac s wi h he an i-His ag an ibody used in he assay. The in ensi y o his band, which
does no change in he condi ions es ed, has been used o s anda dize he amoun o p o ein loaded in each lane.
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Figu e 5. Co E needs coppe and BCS o bind DNA. Elec opho-
e ic mobili y shi assay wi h pu i ied hCo E and a adiolabeled DNA
agmen con aining he copB p omo e was ca ied ou wi h he
addi i es indica ed in each lane. De ails a e gi en in Ma e ials and
Me hods.
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To in es iga e his op ion, Wes e n blo analyses we e ca ied ou
using he hco E’ cuoB-lacZ s ain. The da a shown in Figu e 6A
demons a e ha Co E was s able o 24 h a e coppe addi ion.
Ano he explana ion could be ha Co E unde wen a cycle o
ac i a ion/inac i a ion, whe eby he egula o would only be in
he ac i e o m o a limi ed pe iod o ime. As shown in Figu e 5,
o ob ain binding o Co E o DNA, he eac ion mix u e mus
include no only coppe , bu also a chela ing agen o Cu(I).
Mo eo e , only o he di alen me als can mimic he coppe e ec
on cuoB up- egula ion (Figu e 3 and Figu e S4). These da a sugges
ha he edox s a e o coppe could be he key in his p ocess. To
in es iga e his possibili y, he exp ession o cuoB was assayed in i o
in condi ions ha a o he o ma ion o Cu(I) and Cu(II). As
shown in Figu e 6B and 6C, cuoB up- egula ion could only be
obse ed when coppe was added o he medium. Howe e , he
maximum exp ession le els we e diminished when he educing
agen asco ba e was also included in he medium o a o he
o ma ion o Cu(I) (Figu e 6C, b own line). Simila ly, he addi ion
o Ag
+
, which mimics Cu(I), also yielded exp ession le els lowe
han hose ob ained wi h only coppe (Figu e 6C, g een line). In
con as , when coppe was added wi h he Cu(I) chela o s BCS o
bicinchoninic acid (BCA) [19,20], cuoB exp ession was a ound
h ee imes ha o he con ol (Figu e 6C, blue e sus ed and
black lines). Mo eo e , he addi ion o coppe wi h e a hiomo-
lybda e (TTM), a chela o o Cu(I) and Cu(II) [21], dec eased he
up- egula ion media ed by his me al o a e y basal le el
(Figu e 6C, o ange line). In con as , when hese h ee chela o s
we e es ed wi h Zn
2+
as he induce , he exp ession le els o cuoB
we e diminished as he concen a ions o all he chela o s
inc eased (Figu e S6), due o he ac hey can also chela e Zn
2+
,
al hough o a much lesse ex en han coppe . Acco ding o all
hese da a, Co E equi es coppe o ac i a ion, and i only
acqui es an ac i e con o ma ion in he p esence o Cu(II), while
he educed s a e o he me al leads o an inac i e con o ma ion.
This no ion ag ees well wi h he lack o a peak when up- egula ion
o cuoB is achie ed by Zn
2+
and Cd
2+
, which a e me als wi h only
one edox s a e (Figu e 3B and 3C).
To con i m he esul s ob ained in i o, he DNA-binding assay
was ca ied ou again including Ag
+
o TTM in he eac ion
mix u es. As shown in Figu e 7, hese wo addi i es o e ode he
elec opho e ic mobili y shi achie ed by he addi ion o coppe
and BCS. I should be eminded ha Ag
+
mimics Cu(I) and ha
TTM chela es Cu(II). All o he da a p esen ed in his sec ion
demons a e ha Co E ac i i y is modula ed by he edox s a e o
coppe .
This mechanism o ac ion implies ha Cu(II) mus be a ailable
in he cy osol du ing he nex 2 h a e coppe supplemen a ion.
Al hough i is assumed ha all he coppe in he educing
en i onmen o he cy oplasm is p esen as Cu(I) unde no mal
ci cums ances [2,22], i is also expec ed ha he cy oplasm will
become mo e oxidizing in he p esence o agen s such as coppe
[23], a o ing he o ma ion o Cu(II) un il he educing condi ions
a e es o ed by he pa icipa ion o he elemen s in ol ed in
coppe de oxi ica ion. Fu he mo e, as ee coppe in he cells is
es ima ed o be less han one a om pe cell [24], i is plausible o
specula e ha Co E unc ions wi h an uniden i ied Cu(II)-speci ic
me allochape one, which would e y he cup ic o m h ough he
cy oplasm o ac i a e his s ac o . Such an ac i a o wo king
ups eam o Co E would explain why he exp ession le els o cuoB
do no inc ease when co E is o e -exp essed (Figu e 4). Howe e ,
Figu e 6. Ac i a ion and inac i a ion o Co E. (A) Co E is no deg aded. M. xan hus cells ha bo ing he hCo E p o ein we e ha es ed a he
imes (h) indica ed abo e each lane a e he addi ion o 0.3 mM coppe sul a e and analyzed by Wes e n blo using an an i-His ag an ibody. (B) cuoB
is no up- egula ed by any o he addi i es indica ed in he panel. (C) Co E is ac i a ed and inac i a ed by he edox s a e o coppe . cuoB exp ession
was analyzed in he p esence o di e en addi i es ha modi y he edox s a e o coppe , ha mimic Cu(I), o ha chela e coppe in any o i s wo
edox s a es. Fo panel B and C, M. xan hus cells ha bo ing he usion cuoB-lacZ we e incuba ed on CTT aga pla es con aining he addi i es indica ed.
Samples we e ha es ed a di e en imes and b-gal speci ic ac i i y was de e mined. E o ba s indica e s anda d de ia ions.
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ano he possible explana ion o his obse a ion could be ha
Co E agg ega es when p oduced in la ge amoun s.
One pa adox is he ac ha Co E is ac i a ed by Cu(II) and
inac i a ed by Cu(I), while genes unde i s con ol encode p o eins,
such as CopA, CopB, and CuoB, ha u ilize Cu(I) as a subs a e
[7,9]. Ne e heless, his con adic ion can be explained by
conside ing wo ac s: i) Ou o he wo edox s a es o coppe ,
Cu(I) is he mos oxic o m [2]. As Co E- egula ed genes
ep esen he i s p o ec i e ba ie agains he dele e ious e ec
o coppe (please no e ha hese genes a e apidly up- egula ed
a e coppe addi ion, as shown in Figu e 2 and Figu e S1) and his
p o ein is ac i a ed by Cu(II), i is plausible o specula e ha
coppe will ini ially ge in o he cy oplasm in he o m o Cu(II),
ac i a ing Co E, and p epa ing he cells o ac on Cu(I) as soon as
i appea s. A his poin , he Co E egulon will be inac i a ed by
he p esence o Cu(I) in he cy oplasm. ii) I he p esence o coppe
pe sis s in he en i onmen , M. xan hus cells will ob ain p o ec ion
agains he me al by means o a leas wo o he mechanisms ( i s ,
by he P1B- ype ATPase CopA and he MCO CuoA, and la e , by
he Cus2 and Cus3 sys ems), which a e sequen ially induced a e
coppe addi ion [7–9] (see also Figu e 2 and Figu e S1).
Al hough many bac e ial ansc ip ional egula o s need me al
o bind DNA [25,26], none o hem ha e been epo ed o be
modula ed by he edox s a e o he me al. Mo eo e , hose ha
unc ion wi h coppe show selec i i y o Cu(I) [24,27]. Hence,
Co E ep esen s a no el ype o bac e ial coppe senso .
CRD con ols he ac i i y o Co E
Co E con ains a sho C- e minal ex ension a e he s
4
domain
consis ing o 38 esidues named CRD. Six o hese esidues a e
Cys. As di e en a angemen s o Cys ha e been p o ed o be key
elemen s in se e al me al-binding p o eins [22,27,28], we ied o
de e mine whe he CRD was in ol ed in he ac i a ion/
inac i a ion o Co E media ed by coppe . An M. xan hus in- ame
dele ion mu an was cons uc ed in which mos o he CRD egion
was dele ed. This s ain, designa ed as Dco E
CRD
, encoded a
p o ein con aining he wo domains s
2
and s
4
o Co E, bu none
o he six Cys o CRD. To analyze he ac i i y o Co E
CRD
, he
wo usions cuoB-lacZ and copB-lacZ we e in oduced in his mu an
and b-gal ac i i y was assayed in he absence and in he p esence
o coppe . The da a ob ained e ealed ha nei he cuoB o copB
we e up- egula ed by coppe (da a no shown), a esul iden ical o
ha shown in Figu e 2A, when he en i e co E gene was dele ed.
These da a demons a e ha CRD is essen ial o he coppe -
dependen ansc ip ion o he genes con olled by Co E.
To de e mine which Cys a e in ol ed in Co E ac i i y, each
esidue was indi idually mu a ed o an Ala by si e-di ec ed
mu agenesis. The six mu a ed genes we e in oduced in o he
Dco E s ain ha bo ing he usion cuoB-lacZ. The e ec o he
mu a ions was e alua ed by analyzing he exp ession o cuoB in he
absence and in he p esence o coppe . The esul s ob ained
showed di e en pa e ns. Mu a ions C181A and C206A exhibi ed
ansc ip ion p o iles e y simila o hose o he WT (Figu e 8A),
al hough some small di e ences ega ding he maximum exp es-
sion le els and iming we e obse ed, indica ing ha hese esidues
play a mino ole in Co E ac i i y.
Mo e se e e e ec s we e ob ained wi h he mu a ions C192A
and C194A. In hese mu an s, he exp ession le els o cuoB in he
absence o coppe we e highe han in he WT (Figu e 8B, dashed
lines), sugges ing ha bo h Cys play some ole in Co E
inac i a ion. Mo eo e , al hough cuoB exp ession was up- egula ed
by coppe in bo h mu an s, he apid induc ion and he peak
exhibi ed by he WT a 2 h we e no eplica ed (Figu e 8B,
con inuous lines). The e ec o he mu a ion C189A was e en
mo e d as ic, because no exp ession was obse ed in he absence
o coppe and he up- egula ion by he me al was almos
comple ely non-exis en (Figu e 8C and 8D). Acco dingly, i can
be concluded ha hese h ee esidues a e impo an in he Co E
ac i a ion p ocess.
Cys184 was clea ly equi ed o Co E inac i a ion, because
mu a ion C184A yielded a cons i u i e exp ession in he absence
o coppe (Figu e 8E) and he addi ion o coppe p o oked a apid
up- egula ion o cuoB. In e es ingly, he exp ession le el did no
peak a 2 h, bu kep inc easing un il i eached a pla eau a 24 h
(Figu e 8F).
The e ec o each poin mu a ion in cuoB exp ession was also
analyzed in cells g own on media con aining coppe plus BCS o
sil e (Figu e S7). Mu a ions C181A and C206, which in he
p esence o coppe yielded exp ession p o iles simila o ha o he
WT (Figu e 8A), also exhibi ed highe exp ession le els in he
p esence o coppe plus BCS, and lowe le els wi h coppe plus
Ag
+
(Figu e S7). In he case o subs i u ions C189A, C192A, and
Figu e 7. Sil e and TTM p e en Co E om binding o DNA. An elec opho e ic mobili y shi assay was ca ied ou as desc ibed in Figu e 5,
wi h he addi i es indica ed in each lane.
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C194A, BCS and Ag
+
ba ely a ec ed he exp ession le els
ob ained wi h only coppe (Figu e S7). Howe e , i should be
eminded ha hese h ee esidues a e impo an o ac i a ion,
and ha cuoB up- egula ion by coppe was impai ed in hese
mu an s (Figu e 8B and 8D), sugges ing ha hese h ee p o eins
migh only ha e a limi ed a ini y o he me al. Su p isingly,
howe e , he p o ein wi h he mu a ion C184A ( he mos
impo an esidue in Co E inac i a ion as shown in Figu e 8E
and 8F), can s ill be modula ed by he wo edox s a es o coppe
(Figu e S7). This esul indica es ha some o he esidues mus
also be in ol ed in he inac i a ion o Co E. As men ioned abo e,
wo o hem could be Cys192 and Cys194, because mu a ions
Figu e 8. Exp ession o
cuoB
in s ains ha bo ing poin mu a ions in he CRD egion o Co E. The mu a ed Cys a e indica ed in each
panel. Cells we e incuba ed on CTT aga pla es con aining 0.3 mM coppe (con inuous lines) o wi hou me al (dashed lines), and samples we e
ha es ed a di e en imes o de e mine b-gal speci ic ac i i y. No e ha he scale in panel F is di e en om ha used in he o he panels. E o ba s
indica e s anda d de ia ions.
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C192A and C194A yield cons i u i e exp ession o cuoB. Howe e ,
i is expec ed ha o he esidue(s) o CRD migh also be equi ed
o he p ope unc ioning o he p o ein (see below).
Taken as a whole, he esul s demons a e ha a leas ou o
he Cys o CRD o m a coo dina ion en i onmen o coppe .
This domain is able o ecognize coppe and sense i s edox s a e,
allowing he binding o Co E o DNA o ac i a e ansc ip ion in
hose condi ions ha a o he o ma ion o Cu(II), and
inac i a ing he s ac o in hose ha a o he o ma ion o
Cu(I). Howe e , how exac ly Co E dis inguishes be ween Cu(I)
and Cu(II) is no easy o p edic , because all o he esidues
iden i ied so a ha modula e he ac i i y o Co E a e Cys.
Al hough Cys a e able o coo dina e Cu(I) and Cu(II), hey equi e
he p esence o o he amino acids, such as His, Asp, Glu, o Me o
exe his unc ion [22,29]. Acco dingly, i is expec ed ha o he
esidues also pa icipa e in he coo dina ion o coppe in ei he o
he wo edox s a es. Mo eo e , hiols a e known o allow di e en
ypes o modi ica ions in an oxida i e en i onmen [30]. The exac
modi ica ion o each indi idual Cys migh also be c ucial in he
Co E ac i a ion/inac i a ion p ocess. Fu he gene ic, biochemi-
cal, and s uc u al s udies will be equi ed o elucida e his
in iguing ques ion.
The ole o CRD in Co E esembles he unc ion o he an i-s
domain p esen in many an i-s ac o s [31]. An i-sdomains
equi e Zn
2+
binding o seques e hei cogna e s ac o . Howe e ,
he an i-sdomain and CRD di e in many aspec s: i) CRD is an
ex a po ion o he s ac o ; ii) elimina ion o CRD does no
ac i a e he s ac o ; and iii) CRD senses he edox s a e o coppe
o ac i a e o inac i a e he s ac o .
ECF s ac o s wi h CRD in o he bac e ia
BLASTP analyses ha e allowed he iden i ica ion o 21 ECF s
ac o s wi h CRD, which a e dis ibu ed in only ou phyla.
Fou een belong o P o eobac e ia (9 aand 5 d), ou o Acidobac e ia,
wo o Ve ucomic obia, and one o Ni ospi a (Figu e 9). As in he case
o Co E, an i-s ac o s a e no linked o any o hese s ac o s.
The alignmen s o hese CRDs ha e e ealed ha only 4 Cys
(co esponding o esidues 181, 184, 192, and 194 in Co E) a e
absolu ely conse ed among hese s ac o s (Figu e 9A). Su p is-
ingly, Cys181, whose mu a ion causes a mino e ec on Co E
ac i i y, is conse ed in all hese egula o s. In con as , Cys189,
which is he main esidue in Co E ac i a ion by coppe , is only
p esen in 11 s ac o s. In e es ingly, howe e , se e al o he
s ains wi h s ac o s ha conse e his Cys exhibi some syn eny
in he egions whe e hey a e encoded. The su ounding genes
encode p o eins wi h high simila i ies o o he s known o be
implica ed in coppe handling and a icking (Figu e 9B).
Due o he di e si y o he ECF s ac o s, S a on
´e al. [13] ha e
p oposed a classi ica ion o his amily o egula o y p o eins in o
44 g oups based on sequence simila i ies and domain a chi ec-
u es. Howe e , Co E did no i in o any o he g oups hey
de ined and i was excluded om his classi ica ion. The da a
p esen ed in his epo suppo he no ion ha a new g oup
should be added o he lis , which will include he 21 ECF s
ac o s ha con ain CRD.
So a , se en amilies o me al de- ep esso s, me al co-
ep esso s, and me al ac i a o s a e known [25,26,32], all o
which clea ly di e mechanis ically om Co E. Hence, elucida-
ion o he exac mode o ac ion o Co E will o e new insigh s
in o ou cu en knowledge o me al senso s. Mo eo e , iden i i-
ca ion o he ac o (s) wo king ups eam o Co E will also help o
elucida e how his ype o s ac o s wo ks and how he a icking
o me als in he bac e ial cy oplasm occu s. Finally, cha ac e iza-
ion o Co E-like p o eins iden i ied in o he bac e ia will also
con ibu e o unde s anding he ole, mechanism o ac ion, and
dis ibu ion o his no el ype o egula o s.
Ma e ials and Me hods
Plasmids, bac e ial s ains, and g ow h condi ions
Geno ypes o he bac e ial s ains and plasmids used in his
s udy a e lis ed in Table S1 and Table S2, espec i ely. M. xan hus
was g own in CTT medium a 30uC, supplemen ed wi h he
addi i es indica ed in each igu e, as p e iously desc ibed [6]. E.
coli was g own on Lu ia-Be ani (LB) medium a 37uC [33].
Cons uc ion o in- ame dele ion mu an s and s ains
ha bo ing lacZ usions
The me hodologies used o ob aining he in- ame dele ion
mu an s and he ansc ip ional lacZ usion s ains used in his
s udy a e he same as p e iously epo ed [7]. To gene a e he
co esponding plasmids (lis ed in Table S2), he desi ed agmen s
we e ampli ied by polyme ase chain eac ion (PCR), using WT
ch omosomal DNA as a empla e, he oligonucleo ides lis ed in
Table S3 as p ime s, and he high- ideli y polyme ase P imeSTAR
HS (Taka a) [33]. PCR p oduc s we e liga ed o ec o s pBJ113
and pKY481 [34,35] o gene a e in- ame dele ion mu an s and
lacZ usions, espec i ely. Plasmids we e always in oduced in o M.
xan hus s ains by elec opo a ion o ob ain in eg a ion in o he
ch omosome by homologous ecombina ion. Sou he n blo
analyses we e ca ied ou o con i m he p ope ecombina ion
e en s. b-gal speci ic ac i i y in cell ex ac s ob ained by sonica ion
o he s ains ha bo ing lacZ usions was de e mined as p e iously
desc ibed [7], and i is exp essed as nmol o o-ni ophenol
p oduced pe min and mg o p o ein. Measu emen s shown a e
he a e ages o da a om iplica e expe imen s.
O e -exp ession o co E in M. xan hus using he oa
p omo e
App op ia e oligonucleo ide pai s (Table S3) we e used o
ampli y by PCR an 817-bp agmen ups eam o he oa gene
(MXAN_1450) using M. xan hus ch omosomal DNA as a empla e
[33]. Simul aneously, co E was also ampli ied by PCR. A BamHI
si e was in oduced a he s a codon o oa in ame wi h ano he
BamHI si e in oduced a he s a codon o co E. Bo h PCR
p oduc s we e cloned in a ec o de i ed om pUC19 in which
he ampicillin- esis ance gene was subs i u ed by one ha encodes
esis ance o e acycline (Te
). The esul ing plasmid, pNG06,
was in oduced by elec opo a ion in o an M. xan hus s ain wi h
he geno ype Dco E cuoB-lacZ, and se e al kanamycin- esis an
(Km
) and Te
colonies we e analyzed by Sou he n blo o
con i m he p ope ecombina ion e en . b-gal speci ic ac i i y was
de e mined o quan i y cuoB exp ession. As a con ol, he plasmid
pNG00 was cons uc ed, in which co E was cloned unde con ol
o i s own p omo e . This plasmid was also elec opo a ed in o he
Dco E cuoB-lacZ o es o e co E a i s o iginal genomic loca ion (see
Table S1 and Figu e S2). To co obo a e ha Co E was being
o e -p oduced unde he cons i u i e oa p omo e , we cons uc ed
he same s ains desc ibed abo e, bu in oducing an N-6His ag
ups eam o Co E, o ob ain he s ains hco E’ cuoB-lacZ and oa -
hco E’ cuoB-lacZ (Table S1). B ie ly, he co E gene wi h an N-6His
ag was ampli ied wi h app op ia e oligonucleo ide pai s (Table
S3) using pETTOPOCo E plasmid (see below) as a empla e. The
PCR p oduc ob ained was cloned unde con ol o he oa
p omo e and i s own p omo e as abo e, ob aining plasmids
pNG08 and pNG05, espec i ely. Plasmids we e in oduced in o
he Dco E cuoB-lacZ s ain, and Km
Te
colonies we e also
analyzed by Sou he n blo hyb idiza ion.
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