The
Myxococcus xan hus
Two-Componen Sys em Co SR
Regula es Exp ession o a Gene Clus e In ol ed in
Main aining Coppe Tole ance du ing G ow h and
De elopmen
Ma ı
´a Celes ina Sa
´nchez-Su il
1
, Juana Pe
´ ez
1
, Nu ia Go
´mez-San os
1
, Law ence J. Shimke s
2
,
Au elio Mo aleda-Mun
˜oz
1
, Jose
´Mun
˜oz-Do ado
1
*
1Depa amen o de Mic obiologı
´a, Facul ad de Ciencias, Uni e sidad de G anada, G anada, Spain, 2Depa men o Mic obiology, Uni e si y o Geo gia, A hens, Geo gia,
Uni ed S a es o Ame ica
Abs ac
Myxococcus xan hus is a soil-dwelling membe o he d–P o eobac e ia ha exhibi s a complex de elopmen al cycle upon
s a a ion. De elopmen comp ises agg ega ion and di e en ia ion in o en i onmen ally esis an myxospo es in an
en i onmen ha includes luc ua ions in me al ion concen a ions. While coppe is essen ial o M. xan hus cells because
se e al housekeeping enzymes use i as a co ac o , high coppe concen a ions a e oxic. These opposing e ec s o ce cells
o main ain a igh coppe homeos asis. A ple ho a o pa alogous genes in ol ed in coppe de oxi ica ion, all o which a e
di e en ially egula ed, ha e been epo ed in M. xan hus. The use o in- ame dele ion mu an s and usions wi h he
epo e gene lacZ has allowed he iden i ica ion o a wo-componen sys em, Co SR, ha modula es he exp ession o an
ope on e med cu A consis ing o nine genes whose exp ession slowly inc eases a e me al addi ion, eaching a pla eau.
T ansc ip ional egula ion o his ope on is complex because ansc ip ion can be ini ia ed a di e en p omo e s and by
di e en ypes o egula o s. These genes con e coppe ole ance du ing g ow h and de elopmen . Coppe induces
ca o enoid p oduc ion in a Dco SR mu an a lowe concen a ions han wi h he wild- ype s ain due o lack o exp ession
o a gene p oduc esembling subuni III o cbb3- ype cy och ome coxidase. This da a may explain why coppe induces
ca o enoid biosyn hesis a subop imal a he han op imal g ow h condi ions in wild- ype s ains.
Ci a ion: Sa
´nchez-Su il MC, Pe
´ ez J, Go
´mez-San os N, Shimke s LJ, Mo aleda-Mun
˜oz A, e al. (2013) The Myxococcus xan hus Two-Componen Sys em Co SR
Regula es Exp ession o a Gene Clus e In ol ed in Main aining Coppe Tole ance du ing G ow h and De elopmen . PLoS ONE 8(7): e68240. doi:10.1371/
jou nal.pone.0068240
Edi o : E ic Cascales, Cen e Na ional de la Reche che Scien i ique, Aix-Ma seille Uni e si e
´, F ance
Recei ed Ap il 5, 2013; Accep ed May 28, 2013; Published July 10, 2013
Copy igh : ß2013 Sa
´nchez-Su il 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 unded by he Spanish Go e nmen (g an s CSD2009-00006 and BFU2012-33248, 70% unded by FEDER). This wo k was also
suppo ed by he Na ional Ins i u e o Gene al Medical Science o he Na ional Ins i u es o Heal h unde awa d numbe R01GM095826 o LJS, and by he Na ional
Science Founda ion unde awa d numbe MCB0742976 o LJS. JMD and JP ecei ed a ellowship om Jun a de Andalucı
´a o do some wo k a Uni e si y o
Geo gia. 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 model soil bac e ium used o s udy a
unique o m o p oka yo ic de elopmen . In he p esence o
nu ien s, cells eed as coo dina ed g oups. When s a a ion
s ikes, housands o indi iduals coope a e o build a ui ing body
inside o which he elonga ed cells di e en ia e in o ound,
en i onmen ally- esis an myxospo es [1]. This complex de elop-
men al p ocess mus be accomplished in he p esence o a
luc ua ing soil en i onmen . In ou labo a o y, we dissec he
global esponse o coppe du ing he li e cycle o M. xan hus.
Coppe , a me al widely dis ibu ed in na u e, is essen ial o a
a ie y o physiological p ocesses in many o ms o li e. I is
equi ed by enzymes ha u ilize he Cu(II)/Cu(I) edox couple,
such as cy och ome c oxidases, mul icoppe oxidases (MCOs), o
Cu/Zn supe oxide dismu ases [2]. On he o he hand, excess o
coppe may lead o me al subs i u ions, oxida ion o p o eins, and
damage o memb ane lipids and DNA. This dual e ec o ces he
cells o main ain a p ecise coppe homeos asis o gua an ee p o ein
unc ion while a oiding me al oxici y. Any imbalance in hese
le els can be a al [3,4].
M. xan hus has e ol ed igh coppe homeos a ic con ol
mechanisms, mos o which a e inducible by his me al. Fo his
eason, while de eloping cells a e mo e sensi i e o coppe han
g owing cells, cells om bo h s ages each he same le el o me al
esis ance when cul u es a e p e-adap ed o coppe [5–8].
Ca o enoids ha e been epo ed o accumula e in M. xan hus in
he p esence o coppe o quench single oxygen gene a ed by his
me al. Blue ligh and coppe a e he only en i onmen al agen s
known o induce ca o enogenesis in M. xan hus. All he egula o y
elemen s known o egula e ca o enoid syn hesis by blue ligh a e
also equi ed o he esponse o coppe , wi h he excep ion o
Ca F, which uniquely esponds o ligh . Fu he mo e, induc ion o
ca o enoid biosyn hesis by coppe equi es lowe me al concen-
a ions when cells a e incuba ed a subop imal g ow h condi ions
[5].
In addi ion o genes esponsible o ca o enoid syn hesis, he M.
xan hus genome con ains many pa alogous genes ha a e in ol ed
PLOS ONE | www.plosone.o g 1 July 2013 | Volume 8 | Issue 7 | e68240
in he coppe esponse [6–8]. Th ee amilies o pa alogs ela ed o
coppe and/o o he me al homeos asis sys ems ha e been
cha ac e ized: h ee membe s o he MCO amily, h ee o he
P
1B
- ype ATPases amily, and six membe s belonging o he CBA-
e lux sys ems amily [6–8]. Exp ession p o iles o hese genes ha e
e ealed ha genes belonging o he same amily a e di e en ially
egula ed in esponse o coppe and/o o he me als. Acco dingly,
all hese sys ems unc ion sequen ially o de oxi y he cell
compa men . Al hough genes in ol ed in he coppe egulon
exhibi se e al ypes o exp ession p o iles [6–8], o he sake o
simplici y we e e he e o only wo o hem. Some genes, such as
hose encoding he MCO CuoB and he P
1B
- ype ATPase CopB,
a e pa o an immedia e esponse o coppe as hei exp ession
apidly inc eases a e coppe addi ion, eaching a peak by 2 h
[6,8]. O he genes, ep esen ed by hose o he MCO CuoA and
he P
1B
- ype ATPase CopA, a e in ol ed in he main enance o
he me al esponse as hei exp ession slowly inc eases a e coppe
addi ion, achie ing a pla eau be ween 24 and 48 h o incuba ion
wi h coppe [6,8].
Exp ession o his ne wo k o genes is o ches a ed by di e se
egula o y elemen s. We p e iously epo ed ha Co E, he i s
membe o a no el ECF (ex acy oplasmic unc ion) s ac o
g oup, egula es genes in ol ed in he immedia e esponse [9].
The Co E egulon includes, among o he s, he P
1B
- ype ATPase
CopB and he MCO CuoB.
In his epo , we iden i y an ope on e med cu A which
comp ises nine genes, including cuoA and copA. We demons a e
ha he wo-componen sys em (TCS) Co SR, also encoded in his
ope on, egula es cu A exp ession. Finally, we show ha his
clus e exhibi s complex egula ion and is in ol ed in he
main enance o coppe ole ance du ing g ow h and de elopmen .
Resul s
The Gene Clus e cu A
Genes encoding he MCO CuoA (MXAN_3420) and he P
1B
-
ype ATPase CopA (MXAN_3415) a e bo h up- egula ed by
coppe addi ion and exhibi simila exp ession p o iles [6,8]. They
a e loca ed in he coppe egion 2 o he M. xan hus ch omosome
whe e nine genes a e encoded in he same o ien a ion (Fig. 1A,
gene iden i ie s ange om MXAN_3413 o 3421). Co-exp ession
o hese genes was examined using o al RNA om ege a i e cells
g own wi h 300 mM coppe . This RNA was used as a empla e o
RT-PCR ( e e se- ansc ip ion PCR) syn hesis o cDNA as
depic ed in Fig. 1A. PCR was pe o med using his cDNA o
o al RNA as empla es and he oligonucleo ides pai s ORTR and
ORTF as p ime s (Table S1). The PCR eac ion om he cDNA
empla e ampli ied one agmen wi h he expec ed size (lane 3 in
Fig. 1B). A eac ion in which no cDNA was p e iously syn hesized
om he RNA sample yielded no PCR p oduc (lane 2 in Fig. 1B)
indica ing minimal DNA con amina ion o he RNA p ep. This
esul sugges s ha hese nine genes o m an ope on, which has
been designa ed cu A ( o Cu esis ance).
To u he e i y ha cu A exp ession is up- egula ed by coppe ,
as was p e iously demons a ed o wo genes o he ope on, cuoA
and copA [6,8], a s ain ha bo ing a usion o he s a codon o
gene MXAN_3421 wi h lacZ was gene a ed. As shown in Fig. 2,
coppe is equi ed o exp ession. Coppe up- egula es exp ession
wi h a pla eau be ween 48 and 72 h a e coppe addi ion du ing
g ow h and 24 and 48 h du ing de elopmen . This exp ession
p o iles a e simila o hose epo ed o cuoA and copA,
co obo a ing ha he nine genes o m an ope on.
The p edic ed loca ions and unc ions o he p o eins encoded
by he nine genes a e shown in Table 1. These p edic ed unc ions
we e in e ed om BLASTP simila i y sea ches and scanning o
p o ein amilies (P am) a he Sange Ins i u e [10]. Domains we e
de e mined by using ScanP osi e, Mo i Scan, and In e P oScan a
ExPASy se e (h p://expasy.o g/ ools/). Along wi h he p e i-
ously s udied genes cuoA and copA [6,8], he clus e includes wo
genes encoding a TCS, which has been e med Co S ( o coppe
esis ance senso his idine kinase) and Co R ( o coppe esis ance
esponse egula o ). Excep o he esponse egula o Co R, all
he p o eins encoded in he ope on a e p edic ed o be loca ed in
he cell en elope (Table 1).
The Coppe - esponsi e cu A Ope on is unde Con ol o
he Two-componen Sys em Co SR
Th ee genes o he cu A ope on, MXAN_3421 ( his s udy), cuoA
and copA, a e known o be up- egula ed by coppe [6,8]. They
exhibi an exp ession pla eau a e me al supplemen a ion.
A en ion was ocused on he TCS Co SR as a possible egula o .
The his idine kinase Co S was p edic ed o con ain a signal
pep ide and one ansmemb ane domain, indica ing a opology
whe e he senso domain was loca ed in he pe iplasm, while he
HAMP and he his idine kinase domains ace he cy oplasm (Fig.
S1). The esponse egula o Co R con ains a s
54
in e ac ion
domain and, he e o e, is p edic ed o unc ion as a bac e ial
enhance -binding p o ein o ac i a e exp ession o genes om
p omo e s ecognized by his s ac o (Fig. S1). A bioin o ma ics
sea ch wi h he P omScan@Queen’s p og am (h p://molbiol-
ools.ca/p omscan/) and Vi ual Foo p in so wa e (h p://
p odo ic. u-bs.de/ p/index2.php) o localize he pu a i e s
54
p omo e s in he cu A ope on e ealed he p esence o ou
sequences wi h simila i ies o hose epo ed o be ecognized by
his s ac o (Fig. S2).
An in- ame dele ion (Dco SR) was cons uc ed. This mu an and
he wild- ype (WT) s ain we e used as gene ic backg ounds o
in oduce he ansc ip ional usions MXAN_3421 ( his s udy),
cuoA-lacZ [6], copA-lacZ [8], and co S-lacZ ( his s udy). As shown in
Fig. 2, Co SR up- egula es MXAN_3421,cuoA and copA du ing
g ow h and de elopmen in he p esence o coppe . Al hough co S
exp ession ollows a p o ile simila o hose o he o he h ee genes
in he WT s ain (Fig. 2B), i should be no ed ha speci ic b-
galac osidase ac i i y le els eached in he p esence o coppe we e
signi ican ly lowe o his gene and ha maxima we e eached
la e .
While dele ion o he TCS educed mos o he coppe -
dependen exp ession o he P
1B
- ype ATPase copA, some up-
egula ion s ill emained (Fig. 2A) ha is no e iden in he plo s
shown in Fig. 2B. The up- egula ion is obse ed a a ine scale in
Fig. 3, wi h an exp ession p o ile exhibi ing a peak a e coppe
addi ion. This exp ession p o ile is ypical o hose genes egula ed
by he ECF s ac o Co E [9]. P e ious bioin o ma ics analyses
iden i ied a Co E-like p omo e ups eam o copA, and showed ha
he ea ly exp ession o copA diminished in a Dco E mu an [9]. A
Dco SR Dco E mu an was cons uc ed and he copA-lacZ usion was
in oduced in his mu an . As shown in Fig. 3, copA exp ession was
comple ely abolished in his double mu an du ing g ow h and
de elopmen . These esul s demons a e ha copA is unde con ol
o wo p omo e s. One p omo e esponds o Co E, showing a
p o ile in which ansc ip ion peaks a e coppe addi ion, while
he o he esponds o Co R-s
54
, gi ing a p o ile in which he
exp ession slowly inc eases in a ime-dependen manne , eaching
a pla eau a 24 h.
Di e en exp ession le els o genes o he cu A ope on,
iden i ica ion o ou pu a i e s
54
p omo e s and he double
egula ion o copA indica e a complex egula ion o his ope on,
whe e s ac o s o he amilies s
54
and s
70
pa icipa e.
M. xan hus Two-Componen Sys em Co SR
PLOS ONE | www.plosone.o g 2 July 2013 | Volume 8 | Issue 7 | e68240
Pheno ype o he Dco SR Mu an S ain du ing G ow h
and De elopmen
The pheno ype o he Dco SR mu an was analyzed. Fi s ,
g ow h in he p esence o di e en coppe concen a ions was
de e mined. As shown in Fig. 4A, cell yields in he p esence o
se e al coppe concen a ions we e only sligh ly lowe han hose
ob ained wi h he WT s ain. In con as , when cells we e
p e iously adap ed o li e in he p esence o 600 mM coppe , he
Dco SR mu an was mo e sensi i e (Fig. 4B). This esul co ob-
o a es a ole o his sys em in coppe de oxi ica ion.
On s a a ion media, non-p e-adap ed Dco SR cells we e
impai ed in de elopmen (Fig. 5A) e en a low coppe concen a-
ions. Howe e , he di e ence in coppe esis ance be ween he
WT s ain and he mu an was mo e d ama ic when de elopmen
was assayed wi h p e-adap ed cells. In his case, he mu an was
able o o m ui ing bodies only up o 100 mM coppe (Fig. 5B),
while he WT agg ega ed up o 2500 mM. Ne e heless, al hough
he mu an s ain was unable o o m ui ing bodies, cells
emained iable up o 2000 mM, as deduced a e s eaking cells
om hese spo s on ich media. These esul s indica e ha coppe
inhibi s he no mal de elopmen al p og am in he mu an .
In o de o elucida e why coppe p e-adap ed mu an cells we e
unable o agg ega e, we examined exp ession o h ee de elop-
men al genes ha a e sequen ially exp essed: m pC, which encodes
a ansc ip ional egula o ha egula es he exp ession o uA
[11]; uA, which encodes a esponse egula o [12,13]; and ps,
which encodes p o ein S, he majo spo e coa p o ein, whose
exp ession depends on F uA [14,15]. Plasmids ha bo ing m pC-
lacZ, uA-lacZ, and ps-lacZ usions we e in oduced in o he WT
and Dco SR s ains. Measu emen s o b-galac osidase ac i i ies
we e ca ied ou unde de elopmen al condi ions (CF media) wi h
coppe p e-adap ed cells. As shown in Fig. 6, m pC exp ession
le els we e sligh ly highe in he mu an han in he WT s ain,
e en when cells we e spo ed on o s a a ion medium con aining
200 mM coppe (Fig. 6), a coppe concen a ion ha blocks
ui ing body o ma ion in he mu an (Fig. 5B). This esul
indica es ha educed m pC exp ession is no esponsible o
inhibi ing agg ega ion in he p esence o me al. Howe e ,
exp ession o uA was clea ly educed in he Dco SR mu an
(Fig. 6). The iming o ps induc ion ei he in he absence o in he
p esence o coppe was al e ed, al hough maximal exp ession
le els a e only sligh ly lowe in he mu an han in he WT wi h 0
and 100 mM coppe . Con e sely, exp ession le els dec eased
d as ically in he mu an when CF media con ained 200 mM
coppe (Fig. 6). No ui ing bodies a e p oduced by he mu an a
his coppe concen a ion (Fig. 5B). Al oge he hese da a indica e
ha coppe a ec s de elopmen o he Dco SR mu an a a ime-
poin be ween m pC and uA induc ion.
Dele ion o MXAN_3414 Induces Ca o enoid Biosyn hesis
a Low Coppe Concen a ions du ing G ow h and
De elopmen
When he Dco SR mu an was g own in he p esence o coppe ,
cells u ned ed a lowe coppe concen a ions han WT cells
(Fig. 7A). Du ing de elopmen (Fig. 7B), ed ui ing bodies o he
mu an we e ob ained wi h jus 40 mM coppe . In con as , he
WT s ain did no u n ed e en wi h 80 mM coppe , he highes
concen a ion ha can be used wi h non-p eadap ed cells du ing
de elopmen [6]. To e i y ha he ed colo was due o
accumula ion o ca o enoids, a mu an was cons uc ed ca ying
a dele ion in bo h he TCS co SR and he ca B ope on, which
encodes all bu one o he s uc u al genes in ol ed in ca o enoid
biosyn hesis [16]. As shown in Fig. 7A and B, cells lacking ca B did
no u n ed, indica ing ha he ed colo is due o ca o enoid
accumula ion. This esul indica es ha dele ion o co SR induces
ca o enoid biosyn hesis by coppe in he p esence o low ex e nal
me al a ailabili y.
Ca o enoid-o e p oduc ion a low coppe concen a ions du -
ing g ow h and wi hin ui ing bodies was no obse ed in he WT,
o in single, double o iple dele ion mu an s o he coppe
homeos asis genes in M. xan hus [6–8]. In o de o ind he co SR-
egula ed gene in ol ed in his pheno ype, we analyzed he
sequences and mic osyn eny conse a ion o he non-p e iously
cha ac e ized genes o he cu A clus e . The in silico sea ch e ealed
ha MXAN_3414 encodes a p o ein ha belongs o P am amily
PF13442, e med ‘‘cy och ome coxidase (Cox), cbb3- ype, subuni
III’’, wi h an E- alue o 3.1e-10 (Table 1). cbb3- ype cy och ome c
oxidases (cbb3-Cox) a e mul isubuni me alloenzymes whose
assembly and ac i i y equi e he p esence o coppe in hei
ca aly ic subuni s and a e membe s o he cy och ome coxidase
Figu e 1. The
cu A
ope on includes nine genes. A. S a egy used o s udy he co-exp ession o he nine genes. cDNA is d awn as dashed line,
wi h he egion co esponding o he p ime used o syn hesize he cDNA ep esen ed by an a ow (no o scale). The name o he p ime used o
syn hesize he cDNA is indica ed on he le . PCR p oduc is d awn as a solid line abo e he cDNA used as a empla e. P ime s used o PCR a e
indica ed wi h numbe s, 1 co esponds o ORTF, and 2 co esponds o ORTR. Sequences o all p ime s a e shown in Table S1. Ve ical lines ups eam
o genes A, B, C, and G indica e he posi ion o 4 pu a i e s
54
p omo e s (see Fig. S2 o de ails). B. RT-PCR expe imen . Lane 1 con ains DNA
molecula weigh ma ke VIII (Roche). Lane 2, nega i e con ol using o al RNA as a empla e. Lane 3, PCR p oduc ob ained using cDNA as a empla e
(see panel A).
doi:10.1371/jou nal.pone.0068240.g001
M. xan hus Two-Componen Sys em Co SR
PLOS ONE | www.plosone.o g 3 July 2013 | Volume 8 | Issue 7 | e68240
supe amily [17]. cbb3-Cox a e impo an o ae obic espi a ion,
he onse o pho osyn hesis, hizobial symbiosis, and pa hogenesis
in a ious species [18]. MXAN_3414 shows homologies and
belongs o he same P am amily as he well cha ac e ized FixP
om B ady hizobium japonicum and CcoP om Rhodobac e capsula us
[19,20]. Howe e , MXAN_3414 is no gene ically linked o he es
o he Cox subuni s, sugges ing an al e na i e ole o his p o ein.
In ac , he M. xan hus genome encodes wo pa alogs o
MXAN_3414, MXAN_5539 (17% iden i y, 56% simila i y) and
MXAN_5554 (19% iden i y; 54% simila i y). Bo h o hem may
o m clus e s wi h di e en Cox subuni s, indica ing ha hey a e
he housekeeping Cox. Analysis o he mic osyn eny o he 50 bes -
Figu e 2. Genes egula ed by Co SR. A. Quali a i e analysis o he Co SR egula ed genes. Cells o he di e en s ains we e inocula ed on ich
(g ow h) o s a a ion (de elopmen ) media con aining X-gal. Pic u es we e aken a e 48 h o incuba ion. Ba s, 3 mm. B. Quan i a i e analysis o he
exp ession o MXAN_3421,cuoA,co S, and copA in he WT s ain and he Dco SR mu an . Plasmids con aining MXAN_3421-lacZ,cuoA-lacZ,co S-lacZ,
and copA-lacZ we e in oduced in o WT (blue lines) o Dco SR ( ed lines) backg ounds, and incuba ed o g ow h condi ions on CTT aga pla es
con aining 600 mM coppe . Fo de elopmen , cells we e incuba ed on CF aga pla es con aining 40 mM coppe . Speci ic b-galac osidase ac i i y in
he cell ex ac s o all he s ains was de e mined as desc ibed in Ma e ials and Me hods. No e he di e ence in he scales be ween panels. E o ba s
indica e s anda d de ia ions.
doi:10.1371/jou nal.pone.0068240.g002
M. xan hus Two-Componen Sys em Co SR
PLOS ONE | www.plosone.o g 4 July 2013 | Volume 8 | Issue 7 | e68240
i sequences showed ha 28 o hologous genes o MXAN_3414
appea ed gene ically linked o genes encoding p o eins in ol ed in
a coppe esponse (Fig. S3). The alignmen s o hese 29 p o eins
and he ep esen a i e domain a chi ec u e a e indica ed in Fig.
S4.
The esul s o hese analyses make MXAN_3414 he leading
candida e o ca o enoid induc ion obse ed in he Dco SR mu an .
As pa o a cy och ome coxidase up- egula ed by coppe ,
MXAN_3414 may gene a e ene gy in he p esence o his me al.
Acco dingly, cells whe e his gene is no up- egula ed (as in he
case o he Dco SR mu an ) would be de icien in ene gy gene a ion
when cul u ed wi h coppe . As men ioned in he In oduc ion,
ca o enoid biosyn hesis induced by coppe is mo e e icien unde
sub-op imal g ow h condi ions [5], so i is expec ed ha Dco SR
would be subjec ed o sub-op imal g ow h condi ions in he
p esence o coppe due o he lack o MXAN_3414. To examine
his hypo hesis, we cons uc ed and analyzed an in- ame dele ion
mu an o MXAN_3414. The coppe -induced ca o enogenesis
p o iles o he D3414 s ain we e simila o hose o he Dco SR
mu an du ing g ow h and de elopmen (Fig. 7B and C),
indica ing ha lack o MXAN_3414 exp ession is esponsible o
ca o enoid accumula ion in he Dco SR mu an .
Finally, we decided o es whe he ca o enoid induc ion could
be ela ed o coppe accumula ion. De e mina ion o he
in acellula coppe accumula ed by he WT s ain and he
Dco SR and D3414 mu an s e ealed ha he Dco SR mu an
accumula es mo e coppe han he WT s ain, 1.3- old du ing
g ow h and 13- old du ing de elopmen (Fig. 8). Howe e , he
D3414 mu an accumula ed he same amoun o me al as he WT
(Fig. 8) while p oducing mo e ca o enoids, so ca o enoid
biogenesis is no s ic ly ela ed o coppe accumula ion.
Discussion
The la ge numbe o genes in ol ed in coppe de oxi ica ion
mechanisms in M. xan hus and hei di e en ial egula ion sugges s
ha se e al ansc ip ional egula o s may be in ol ed. Only he
coppe -dependen ECF s ac o Co E is known o induce
exp ession o hose genes in ol ed in apid adap a ion [9]. He e
we epo he iden i ica ion o he second egula o y sys em
consis ing o he his idine kinase Co S and he esponse egula o
Co R. This TCS egula es exp ession o genes ha exhibi a slow
up- egula ion a e coppe addi ion and each a pla eau a e 24–
48 h incuba ion wi h he me al. Co SR egula es he cu A ope on,
which consis s o nine genes, including genes o he MCO CuoA,
he ATPase CopA, and he TCS Co SR.
Two lines o e idence indica e ha egula ion o he cu A ope on
is complex. Fi s , exp ession o genes loca ed a di e en posi ions
in he ope on di e s in he le els ob ained o each one as well as
he iming o when he pla eau is eached. Second, copA is no
only egula ed by Co SR, bu also by Co E. Acco dingly, ou
di e en pu a i e s
54
-dependen p omo e s ha e been iden i ied
along he cu A ope on in his s udy and one pu a i e Co E
p omo e ups eam o copA was p e iously iden i ied [9]. As h ee
pa alogous genes encoding P
1B
- ype ATPases (copA,copB, and copC)
ha e been iden i ied in he M. xan hus genome [8], copA double
egula ion sugges s ha bo h he s uc u al gene and p omo e
we e duplica ed. Acco ding o Teichmann and Babu [21],
di e gence a e duplica ion o a a ge gene can lead o he
duplica ed gene emaining unde he con ol o he same
ansc ip ion ac o o coming unde he con ol o a di e en
ansc ip ion ac o . In ou case, he da a sugges ha du ing he
duplica ion e en he p omo e o he ances al ansc ip ion
ac o has been main ained and a p omo e o a new ansc ip ion
ac o has been gained.
Pheno ypic analyses o he Dco SR mu an ha e e ealed ha he
cu A clus e is in ol ed in coppe de oxi ica ion du ing g ow h and
de elopmen . Howe e , he ac ha he mu an cells accumula ed
13- old mo e coppe han he WT s ain du ing de elopmen , bu
only 1.3- old du ing g ow h, and ha he e ec on ui ing body
o ma ion is mo e d as ic han on g ow h in he mu an in he
p esence o coppe indica e ha his TCS, and so ha he cu A
clus e , is especially impo an o coppe de oxi ica ion o
de eloping cells.
We assayed exp ession o h ee sequen ial de elopmen al genes
(mp C, uA, and ps) in he WT and mu an in he p esence and
absence o coppe . The esul s indica e ha mp C exp ession is no
educed in he Dco SR mu an . Con e sely, uA and ps exp essions
a e d as ically educed in he TCS mu an , especially in he
p esence o 200 mM o coppe . De elopmen a his coppe
concen a ion is blocked in he mu an , al hough cells a e iable.
As he ac i e o m o M pC is he p o eoly ic p oduc M pC2 [22],
a easible explana ion could be ha mp C is exp essed in he Dco SR
mu an bu no p ope ly p ocessed in he p esence o coppe ,
Table 1. In silico analysis o he nine genes o he cu A ope on.
MXAN
a
P o ein P o ein P edic ed Func ion
b
SP
c
/TM
d
3421 A P o ein wi h His- ich egion, cup edoxin1(PF13473) Yes/No
3420 B (CuoA) Mul icoppe oxidase (PF07732, PF07731, PF00394) Yes/No
3419 C (Co S) Signal ansduc ion his idine kinase (PF02518, PF00512, PF13581, PF00672, PF13589) Yes/Yes (1)
3418 D (Co R) s
54
-dependen DNA-binding esponse egula o (PF00072, PF00158, PF02954) No/No
3417 E Lipop o ein con aining wo PQQ_2 domains (PF13360) Yes/No
3416 F Lipop o ein con aining DUF305 domain (PF03713) Yes/No
3415 G (CopA) Coppe - ansloca ing P- ype ATPase (PF04945, PF00122, PF02954) No/Yes (7)
3414 H Cy och ome coxidase, cbb3- ype, subuni III (PF13442) Yes/No
3413 I Hypo he ical p o ein Yes/No
a
MXAN: gene iden i ie s in M. xan hus genome.
b
P edic ed unc ion in e ed om BLASTP, P am, ScanP osi e, Mo i Scan, and In e P oScan. The P am iden i ie s a e in pa en heses.
c
Signal pep ides (SP) we e deduced om esul s a SignalP and SOSUI se e s.
d
T ansmemb ane domains (TM) om TMHMM and SOSUI se e s. The numbe s o p edic ed ansmemb ane domains a e indica ed in pa en heses.
P og ams and da abases used a e a ailable h ough ExPASy se e (h p://www.expasy.o g/).
doi:10.1371/jou nal.pone.0068240. 001
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educing he exp ession o uA (which depends on M pC2) and ps
(which depends on F uA). The p o ease esponsible o he
p o eoly ic clea age o M pC has no been iden i ied ye , al hough
i has been p oposed ha LonD could be esponsible o his
ac i i y [22]. P ocessing o M pC in he p esence o coppe and
inhibi ion o he p o ease ac i i y by his me al emain o be
elucida ed.
Ano he in e es ing obse a ion is ha Co SR p e en s he
o ma ion o ca o enoids a low coppe concen a ions. As he
pheno ype obse ed in he Dco SR mu an is no obse ed in o he
mu an s gene a ed in genes o he cu A ope on, such as DcopA o
DcuoA [6,8], we decided o in es iga e which gene is esponsible o
his e ec . He ein, we demons a e ha he majo con ibu o o
he ca o enoid pheno ype o Dco SR a low coppe concen a ions
is he p o ein encoded by he gene MXAN_3414. The eason why
his mu an induces ca o enoid biosyn hesis is no clea . Howe e ,
i can be uled ou ha i is an e ec o in acellula coppe
accumula ion, because mu an D3414 accumula es simila
amoun s o coppe o he WT s ain, bu u ns ed a low ex e nal
coppe concen a ions du ing g ow h and o ms ed ui ing
bodies. We epo ed se e al yea s ago ha he coppe induc ion o
ca o enoid syn hesis in M. xan hus is mo e e ec i e a subop imal
g ow h condi ions [5]. One plausible explana ion o he
pheno ype o he D3414 mu an could be ha he p o ein encoded
Figu e 3. The exp ession o
copA
is egula ed by Co SR and he
ECF s ac o Co E. Plasmids con aining copA-lacZ usion we e
in oduced in o h ee di e en gene ic backg ounds: WT (blue lines),
Dco SR ( ed lines), and he double mu an Dco SR Dco E (g een lines). A.
Cells we e incuba ed on CTT aga pla es (g ow h) con aining 600 mM
coppe . B. Cells we e incuba ed on CF aga pla es (de elopmen )
con aining 40 mM coppe . Small igu es show he exp ession p o iles o
copA in he WT and Dco SR backg ounds ep esen ed in Fig. 2. No e he
di e ence in he scale be ween small and la ge g aphics. E o ba s
indica e s anda d de ia ions.
doi:10.1371/jou nal.pone.0068240.g003
Figu e 4. G ow h o WT and D
co SR
s ains in he p esence o
coppe . A. Cells g own in he absence o coppe we e dilu ed in o CTT
liquid media con aining he indica ed coppe concen a ions. B. Cells
we e adap ed o g ow in he p esence o 600 mM o coppe p io o
dilu ion in CTT liquid media con aining he indica ed coppe
concen a ions. OD
600
in bo h panels was moni o ed a e 24-h
incuba ion. E o ba s indica e s anda d de ia ions. WT, blue lines;
Dco SR, ed lines.
doi:10.1371/jou nal.pone.0068240.g004
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Figu e 5. De elopmen o he WT s ain and he D
co SR
mu an in he p esence o coppe . A. Cells g own in he absence o coppe we e
concen a ed a an OD
600
o 15 and spo ed on o CF medium wi h he coppe concen a ions indica ed. B. Cells g own in he p esence o 600 mM
coppe sul a e we e spo ed on o CF aga pla es wi h he indica ed me al concen a ions. Pho og aphs in his panel we e aken a 72 h o incuba ion.
Ba s, 1 mm.
doi:10.1371/jou nal.pone.0068240.g005
Figu e 6. Exp ession o h ee de elopmen al genes,
m pC
,
uA
and
ps
, in coppe p e-adap ed cells. WT (solid columns) o Dco SR
(dashed columns) cells ha bo ing m pC-lacZ, uA-lacZ and ps-lacZ we e g own in he p esence o 600 mM coppe , concen a ed o an OD
600
o 15,
and spo ed on o CF medium con aining he indica ed coppe concen a ions. Cells we e ha es ed a se e al ime-poin s o de e mine speci ic b-
galac osidase ac i i y.
doi:10.1371/jou nal.pone.0068240.g006
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by his gene is equi ed o ene gy p oduc ion upon coppe
addi ion. The absence o his p o ein in he D3414 mu an could
educe ene gy le els, mimicking g ow h in subop imal condi ions,
and inducing ca o enogenesis o lowe coppe concen a ions.
This explana ion is suppo ed by he homology o MXAN_3414
wi h he subuni III o cbb3-Cox. A linkage be ween coppe
esis ance, Cox biogenesis, and compe i i e i ness has p e iously
been epo ed in Pseudomonas luo escens [23] and Salmonella
yphimu ium [24]. Ou esul s ein o ce his connec ion. Suppo ing
he implica ion o MXAN_3414 and o he membe s o PF13442
in coppe ole ance is syn enic conse a ion o se e al o hologous
o his cy och ome c-like amily gene wi h genes coding o coppe -
esponse ela ed p o eins such as P
1B
- ype ATPases, MCOs, o
HRD hea y me al e lux sys ems (Fig. S3). Many bac e ial species
ha e se e al espi a o y oxidases o allow cells o adap hei
espi a o y sys ems o mee he demands o a a ie y o
en i onmen al condi ions, including di e en concen a ions o
me al ion co ac o s [23]. I is emp ing o specula e ha
MXAN_3414 could be an al e na i e Cox subuni ha con ibu es
o M. xan hus i ness in he p esence o coppe .
Ma e ials and Me hods
Bac e ial S ains, Plasmids, and G ow h Condi ions
M. xan hus and Esche ichia coli s ains, oligonucleo ides, and
plasmids used in his s udy a e lis ed in Tables S1 and S2. Lu ia-
Be ani b o h, supplemen ed wi h kanamycin (25 mg/ml) o X-gal
(5-b omo-4-chlo o-3-indolyl-b-D-galac opy anoside, 40 mg/ml)
when needed, was used o g ow E. coli s ains a 37uC.
All M. xan hus s ains we e g own a 30uC wi h igo ous shaking
(300 pm) in CTT b o h [25]. CTT aga pla es con ained 1.5%
Bac o aga (Di co). When necessa y, kanamycin (80 mg/ml), X-gal
(100 mg/ml), o galac ose (10 mg/ml) we e added. When needed,
CTT medium was supplemen ed wi h coppe a he concen a-
ions indica ed in each igu e. CTT aga pla es con aining coppe
g adien in a ange o concen a ions be ween 0 and 1000 mM
we e p epa ed acco ding o Mo aleda-Mun˜oz e al. [5].
S a a ion medium CF [25] was used o induce de elopmen ,
which was supplemen ed wi h coppe a he concen a ions
indica ed in he igu es when needed. Cells we e g own in CTT
b o h o app oxima ely 3.0610
8
cells/ml (op ical densi y a
Figu e 7. Ca o enoid accumula ion in di e en s ains du ing g ow h and de elopmen . A. WT, Dco SR, and Dco SR Dca B s ains we e
g own in he absence o coppe , concen a ed a an OD
600
o 15, and spo ed on o CTT aga pla es con aining a coppe g adien om 0 o 1000 mM.
Pic u es we e aken a e 48-h incuba ion. B. Ca o enoid accumula ion du ing de elopmen . In hese expe imen s cells g own in he absence o
coppe we e concen a ed a an OD
600
o 15 and spo ed on o CF media wi h 40 mM coppe . Pic u es we e aken a 72 h. Ba ep esen s 100 mm. C.
Ca o enoid accumula ion in he D3414 and he WT s ains du ing g ow h in he p esence o di e en coppe concen a ions. Cells we e g own in he
absence o coppe , concen a ed o an OD
600
o 15, and spo ed on o CTT aga pla es con aining he indica ed coppe concen a ions.
doi:10.1371/jou nal.pone.0068240.g007
Figu e 8. Measu emen s o in acellula coppe accumula ion
du ing g ow h and de elopmen . Cells om WT, Dco SR, and
D3414 s ains we e incuba ed on CTT (g ow h) o CF (de elopmen )
aga pla es con aining he indica ed coppe concen a ions o 72 h
p io o ha es ing o coppe de e mina ions.
doi:10.1371/jou nal.pone.0068240.g008
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600 nm [OD
600
] o 1) and esuspended o 4.5610
9
cells/ml
(OD
600
o 15) in TM bu e (10 mM T is-HCl [pH 7.6], 1 mM
MgSO
4
). Ten mic oli e s o each suspension we e spo ed on o CF
aga pla es and incuba ed a 30uC, wi h obse a ion on a Wild
Hee b ugg dissec ing mic oscope.
Nucleic Acid Manipula ions
Rou ine molecula biology echniques we e ollowed o nucleic
acid manipula ions [26]. To al RNA om cul u es con aining
300 mM coppe was p epa ed wi h he High Pu e RNA Isola ion
ki p o ided by Roche. Samples we e ea ed wi h he ki DNA-
ee (Ambion) o comple ely emo e ch omosomal DNA. The
RNA was hen subjec ed o e e se ansc ip ion using he p ime
FRTC, which anneals in gene MXAN_3413 (see Table S1). PCR
was ca ied ou wi h p ime s ORTR and ORTF, which anneal
inside gene MXAN_3421 (Fig. 1 and Table S1) using as empla es
o al RNA o cDNA syn hesized using FRTC as p ime .
Cons uc ion o In- ame Dele ion Mu an s
In- ame dele ion mu an s ha e been cons uc ed as p e iously
epo ed [6]. Concisely, in o de o gene a e he co esponding
plasmids (Table S2), he desi ed agmen s we e ampli ied by
PCR, using WT ch omosomal DNA as a empla e, he app op i-
a e oligonucleo ide pai s (Table S1), and he high- ideli y
polyme ase P imeSTARS (Taka a). PCR p oduc s we e liga ed
o ec o pBJ113 [27] o ob ain plasmids ha bo ing dele ions o
he co esponding genes (Table S2). The esul ing non- eplica ing
plasmids ca ying he dele ions we e in oduced in o M. xan hus
cells by elec opo a ion [28]. Ch omosomal in eg a ion was
selec ed by pla ing cells on o CTT pla es con aining kanamycin
(posi i e selec ion). Se e al andomly chosen kanamycin- esis an
(Km
R
) me odiploids we e analyzed by Sou he n blo hyb idiza ion
o he p ope ecombina ion e en . One posi i e s ain was hen
g own in he absence o kanamycin and pla ed on o CTT pla es
con aining galac ose o nega i e selec ion. Sou he n blo analysis
was used o sc een kanamycin-sensi i e (Km
S
) and galac ose-
esis an (Gal
R
) colonies, sea ching o he loss o he WT allele.
Cons uc ion and Assay o S ains Ha bo ing lacZ Fusions
The lacZ usion plasmids we e cons uc ed by using ec o
pKY481 [29]. F agmen s su ounding he ups eam genes egions
we e ampli ied om WT ch omosomal DNA by he use o he
oligonucleo ide pai s speci ied in Table S1. The BamHI si e in he
p ime s was in oduced a he s a codon o he co esponding
genes and in ame wi h he BamHI si e exis ing in he lacZ gene o
plasmid pKY481. PCR p oduc s we e diges ed wi h KpnI-
BamHI, XhoI-BamHI o Ps I-BamH1, and liga ed in o ec o
pKY481 diges ed wi h he same enzymes. The esul ing plasmids
we e in oduced in o M. xan hus (WT o mu an s) cells by
elec opo a ion, and he Km
R
colonies we e analyzed by Sou he n
blo ing. S ains con aining lacZ usions we e incuba ed a 30uCon
CTT and CF aga pla es con aining di e en me al concen a-
ions. Fo quali a i e analyses o b-galac osidase ac i i y, pla es
con aining X-gal we e used. Fo quan i a i e analyses, cell ex ac s
we e ob ained a di e en imes by sonica ion and assayed o
ac i i y as p e iously epo ed [5]. The amoun o p o ein in he
supe na an s was de e mined by using he Bio-Rad p o ein assay
wi h bo ine se um albumin as a s anda d. Speci ic b-galac osidase
ac i i y 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.
Coppe Accumula ion Measu emen s
Fo de e mina ions o coppe accumula ion, cul u es g own
o e nigh in CTT medium we e concen a ed o an OD
600
o 15
as desc ibed abo e. Ten d ops o 20 ml each we e spo ed on o
CTT aga pla es supplemen ed wi h 0 o 500 mM coppe (g ow h
condi ions), o on o CF aga pla es supplemen ed wi h 0 o 50 mM
coppe (de elopmen al condi ions). A e 72-h incuba ion a 30uC,
cells we e ha es ed in o a 15-ml s e ile polyp opylene cen i uge
ube and washed h ee imes wi h modi ied CTT medium (wi hou
MgSO
4
and con aining 1 mM EDTA). Pelle s we e d ied a 80uC
o e nigh and dissol ed in 5 ml o ace-me al-g ade ni ic acid by
hea ing a 80uC o 30 min acco ding o a me hod desc ibed
p e iously by Ou en e al. [30]. The me al con en o he samples
was measu ed by using The mo Ja ell Ash En i o 36 Induc i ely
Coupled Plasma-Op ical Emission Spec og aph wi h a 20
elemen sweep. The sample was nebulized and mo ed up in o a
o ch wi h bu ning a gon a o e 1370uC. The ligh emi ed om
he bu ning o he sample was e lec ed back o a g a ing ha
sp eads he ligh ou like a p ism, in o he di e en wa eleng hs,
which we e picked up by pho omul iplie ubes se a hose
wa eleng hs indica ed beside he a omic symbol on he da a shee .
The ins umen was un by he EPA me hod 6010C.
Suppo ing In o ma ion
Figu e S1 Domain a chi ec u e and loca ion o he wo-
componen sys em Co SR. A. Domain dis ibu ion o he
senso his idine kinase Co S. SP, signal pep ide ( ep esen ed as an
open box o indica e ha i will no be a pa o he ma u e
p o ein); TM, ansmemb ane domain; HAMP, linke domain
p esen in his idine kinases, adenyl cyclases, me hyl-accep ing
p o eins and phospha ases (PF00672), E- alue 1.3e-15; His Kinase
A (phosphoaccep o ) domain (PF00512), E- alue 1.1e-16, and
HATPase_c domain (PF02518), E- alue 7.5e-28, a e componen s
o his idine kinases. B. Domain dis ibu ion o he esponse
egula o Co R. Response_ eg: Response egula o ecei e
domain (PF00072), E- alue 8.5e-29; Sigma54_ac i a , sigma-54
in e ac ion domain (PF00158), E- alue 1.9e-67; HTH_8, Bac e ial
egula o y p o ein, Fis amily (PF02954), E- alue 7.4e-05. OM,
ou e memb ane; PS, pe iplasmic space; IM, inne memb ane; C,
cy oplasm.
(PDF)
Figu e S2 In silico analysis o he ups eam sequences
o he cu A genes. A. Fou di e en s
54
p omo e s we e ound
(g een ec angles). B. Sequence alignmen s o he ou pu a i e
p omo e s. Unde lined nucleo ides ma ch he E. coli s
54
consensus: TGGCACGRNNNTTGCW desc ibed by Ba ios
e al. (1999). C. LOGO ep esen a ion o he ou pu a i e
p omo e s. D. LOGO ob ained wi h he pu a i e s
54
p omo e
sequences desc ibed in o he M. xan hus genes (K oos and Inouye,
2008).
(PDF)
Figu e S3 Mic osyn eny conse a ion in se e al bac e-
ia in he genomic en i onmen o genes coding o
p o eins wi h PF13442 domains ( ed a ows). The o he
colo ed a ows ep esen genes wi h p edic ed unc ion associa ed
o coppe esponse. Da k pink, Cu
2+
-expo ing ATPase (PF00122,
PF00702); ligh g een, mul icoppe oxidase (PF07732, PF07731
and PF00394); da k g een, coppe esis ance p o ein D, CopD
(PF05425); yellow, coppe esis ance p o ein B, CopB (PF05275);
o ange, coppe esis ance p o ein C, CopC (PF04234); g ey,
coppe binding pe iplasmic p o ein CusF (PF11604); pu ple, ou e
memb ane e lux p o ein (PF02321); blue, hea y me al e lux
M. xan hus Two-Componen Sys em Co SR
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