III
Joana Ab eu Luís da Sil a San os
FE/S CLUSTER BIOGENESIS REGULATION BY THE JANUS-
FACED REGULATOR, ISCR: AN UNFORESEEN MECHANISM OF
DNA RECOGNITION AND DISCRIMINATION
Tese de Candida u a ao g au de Dou o em Ciências
Biomédicas subme ida ao Ins i u o de Ciências Biomédicas
Abel Salaza da Uni e sidade do Po o:
O ien ado – Dou o Ped o Pe ei a
Ca ego ia – In es igado P incipal
A iliação – IBMC - Ins i u o de Biologia Molecula e Celula
Co-o ien ado a – Dou o a Sand a Macedo-Ribei o
Ca ego ia – In es igado a P incipal
A iliação – IBMC - Ins i u o de Biologia Molecula e Celula
Co-O ien ado a – P o esso a Dou o a Ana Ma ga ida Damas
Ca ego ia – P o esso a Ca ed á ica
A iliação – ICBAS - Ins i u o de Ciências Biomédicas Abel
Salaza da Uni e sidade do Po o
IV
V
P ecei os Legais
De aco do com o dispos o no nº 2 do a igo 8º do Dec e o-lei nº 388/70,
nes a disse ação o am u ilizados os esul ados de abalhos publicados abaixo
indicados.
No cump imen o do dispos o e e ido Dec e o-Lei, a au o a des a
disse ação decla a que in e eio na conceção e execução do abalho
expe imen al, na in e p e ação e edação dos esul ados publicados sob o nome
San os, J. A.:
San os, J.A., Alonso-Ga cía, N., Macedo-Ribei o, S., Pe ei a, P.J.B. (2014).
"The unique egula ion o i on-sul u clus e biogenesis in a G am-posi i e
bac e ium." P oc Na l Acad Sci U S A. 111(22): E2251-E2260.
VI
O abalho ap esen ado nes a ese oi ealizado no IBMC - Ins i u o de
Biologia Molecula e Celula da Uni e sidade do Po o e oi inanciado po Fundos
FEDER a a és do P og ama Ope acional Fac o es de Compe i i idade –
COMPETE e po Fundos Nacionais a a és da FCT – Fundação pa a a Ciência e
a Tecnologia no âmbi o do p oje o PTDC/BBB-BEP/2127/2012 (FCOMP-01-0124-
FEDER-028116).
VII
“When one doo o happiness closes, ano he
opens; bu o en we look so long a he closed
doo ha we do no see he one which has
been opened o us.”
― Helen Kelle

VIII
IX
Ag adecimen os
Es as são as p imei as pala as que esc e i pa a a minha ese. As
p imei as pala as de em pe ence às pessoas que me ajuda am a chega a é
aqui, às pessoas que caminha am comigo e o na am o sucesso des a e apa,
uma ealidade.
Gos a ia de ag adece aos meus supe iso es Ped o Pe ei a e Sand a
Macedo-Ribei o po e em ac edi ado em mim e po me e em dado a
opo unidade de ealiza es e abalho. O caminho que açámos ez-me c esce
an o a ní el p o issional, como pessoal. Ob igada po e em con iado em mim e
no meu abalho. Ag adeço ambém à P o esso a Ana Ma ga ida Damas po e
acei e co-o ien a a minha ese, assim como à Annalisa Pas o e e a odos os
elemen os do seu g upo po me e em ecebido no seu labo a ó io de b aços
abe os e me e em ajudado em udo o que p ecisei. Ainda que a minha isi a
enha sido ão cu a, consegui adqui i um conhecimen o p eponde an e pa a o
desen ol imen o do abalho aqui ap esen ado.
A minha amília oi e semp e se á a minha ânco a, o meu mais ado ado
po o de ab igo. A minha mãe semp e me ez e a ealidade, semp e me ajudou
a chega mais longe e semp e econheceu o meu melho , mesmo quando as
pala as pe maneciam escondidas, e e semp e um so iso do co ação pa a me
da . O meu pai semp e me ensinou a sonha , a ac edi a no possí el do
impossí el e semp e e e eno mes demons ações de aleg ia e ca inho pa a as
minhas i ó ias, po mais pequenas que ossem. Pa a os meus pais gua do o
pedaço mais calo oso do meu co ação. A minha a ó, essa mulhe de mãos o es
e co ação aleg e, semp e me ensinou isso mesmo: a se o e e aleg e e a não
desis i . A ela de o mui o mais do que a minha educação. De o uma imensidão
de bons momen os que ize am de mim uma c iança, uma adolescen e, uma
mulhe eliz. Minha mana, minha companhei a de uma ida, u que es ás semp e
lá pa a udo, ensinas e-me que há semp e um lado bom em udo e que a é é algo
que de emos e em nós, nas pessoas que amamos e em Deus. Não há ba alhas
que não possamos ganha se ac edi a mos. Os meus que idos ios, Lina e
Fe nando, que acompanha am a minha in ância, me ajuda am a c esce e a
o na -me uma pessoa melho , ensina am-me que po mais que a ida mude,
X
podemos semp e con inua iguais a nós mesmos. Os meus sog os são um
e dadei o exemplo pa a mim, ecebe am-me de co ação abe o e em ão pouco
empo ensina am-me mais do que mui a gen e numa ida in ei a: o amo e a
amizade pode pe du a no empo e ajuda a ul apassa udo o que o des ino nos
ese e. Acima de udo, de emos alo iza o que emos e não o que não emos
ou o que não podemos e . À minha es an e amília, ag adeço odo o amo e
ca inho incondicional ao longo de odos es es anos. O meu que ido a ô não pôde
es emunha es a caminhada, mas sei que ac edi a a e o osamen e no meu
po encial e es eja onde es i e ezo pa a que es eja o gulhoso de mim.
E como pode ia eu e chegado aqui sem os meus amigos? Esses que
pa ilha am an os e bons momen os comigo? Vocês sabem quem são, mas não
posso deixa de esc e e umas pala as de ap eço a alguns que po azões
impossí eis de de ini , me ize am se maio e chega mais longe. Ri a, udo o que
passámos jun as en iqueceu-me e az-me sabe que nunca pode ei es a sozinha
con igo po pe o. So ia, ob igada po udo o que me des e ao longo des es 12
anos, con igo pa ilhei alguns dos momen os mais so iden es da minha ida.
Síl ia, minha “bina”, u és udo aquilo que semp e desejei pa a melho amiga:
so iden e, bondosa, ca inhosa e mui o mui o especial. Joaninha, sem i não me
e ia man ido minimamen e sã du an e a esc i a des a ese (assim como em
an os ou os momen os do dou o amen o!), essa é a mais pu a das e dades. Tu
ca i as e-me e con inuas a ca i a -me odos os dias e, com is o, ambas sabemos
que digo udo o que há po dize . Ana, João e Ma ga ida, a ossa amília é a
minha amília, es ou g a a po os e na minha ida, pois a nossa ligação nunca
pode á esmo ece . Gab iela, Isabel, Sa a, Iliona, Bebiana, e Rosa: ocês
ajuda am-me a c esce , ize am pa e des e caminho, de am-me a mão semp e
que p ecisei e, po isso, sei que a nossa amizade pe du a á no empo. Vilaça
Babe, ainda bem que es ás po pe o, ajudas-me mais do que imaginas. Pa a
oçês, es a ei semp e aqui. Aos meus amigos mais ecen es, mas pelos quais
nu o uma amizade incondicional: Aida, Alexand e, Ana, Tiago, Ru e, B uno e Luís
ocês são e dadei amen e os melho es companhei os pa a odos os momen os.
A minha ida é mui o mais colo ida po os e como amigos.
Nada dis o e ia sido possí el sem a ajuda, paciência e companhei ismo
dos meus colegas e amigos de labo a ó io, po quem nu o um ca inho especial.
XI
Vi emos odo o ipo de momen os, mas le o comigo os isos, as pale mices, as
demons ações de ca inho e os bons conselhos. Sem ocês não e ia chegado
onde cheguei. Desejo- os o melho des e mundo, po que pessoas especiais não
me ecem menos do que isso!
Um especial ag adecimen o à Noélia Alonso, ao F ede ico Sil a e ao Paulo
Oli ei a. À Noélia pela con ibuição p eponde an e pa a a publicação do a igo e
po me e acompanhado em odo o p ocesso, sem nunca me deixa desanima .
Ao F ede ico, pela amizade, acompanhamen o e ines imá el ajuda ao longo dos
úl imos anos. Ao Paulo, po es a semp e disponí el pa a discu i ciência e po me
e ajudado semp e que p ecisei. Ag adeço aos ês, po sen i que icam
genuinamen e elizes com as minhas i ó ias.
Rod igo, meu mais que udo, u mos as e-me a melho ace do amo :
aquela em que posso se amada, acei e e alo izada po udo aquilo que sou,
sem medos e sem es ições. Ensinas e-me que o amo é caminha de mão dada:
nunca à en e, nunca a ás, semp e lado a lado. Não pode ia e pedido melho
dádi a do que ama e se amada po alguém como u: o e, hones o e
essencialmen e único. És o meu melho amigo e odos os dias anseio pelo nosso
u u o jun os.
Um ob igado hones o e e e no a odos,
Joana
XVIII
Fig. 24 - Modula ion o apo-IscR
Tp
speci ici y by a single poin mu a ion ....... 106
Fig. 25 - Model o IscR disc imina ion be ween ype-1 and ype-2 p omo e
sequences.. ........................................................................................................ 113

XIX
Lis o Tables
Table 1 - IscR egulon in E. coli ............................................................................ 48
Table 2 - Sequence mo i s compiled om IscR and Ns R DNA-binding si es ...... 53
Table 3 - Oligonucleo ides used in binding and c ys alliza ion assays ................. 60
Table 4 - S a is ics o da a collec ion, p ocessing, and e inemen ...................... 65
Table 5 - Binding a ini ies be ween Apo-IscR
Ec
E43A and ype-1 and ype-2
p omo e sequences ............................................................................................. 81
Table 6 - S uc u al simila i y be ween T. po ens IscR and o he winged-helix
ansc ip ion egula o s ......................................................................................... 97
Table 7 - Binding a ini ies be ween IscR and ype-1 and ype-2 p omo e
sequences ......................................................................................................... 104
XX
Abb e ia ions
ATC – A- ype ca ie
CD – Ci cula dich oism
DMRB – Dissimila o y me al educing bac e ia
EMSA – Elec opho e ic mobili y shi assay
ENDOR – Elec on-nuclea double esonance
EPR – Elec on pa amagne ic essonance
DTT – Di hio h ei ol
DNA – Deoxy ibonucleic acid
DNIC – Di-ni osyl i on complex
FAD – la in adenosine nucleo ide
FADH2 – educed la in adenosine nucleo ide
Fdx – Fe edoxin
FNR – Fuma a e ni a e egula o
GPR – G am-posi i e egion
Hmp – Haemoglobin
H
2
O
2
– Hyd ogen pe oxide
IAV – In luenza A i us
IHF – In eg a ion hos ac o
IMAC – Immobilized me al a ini y ch oma og aphy
IPTG – Isop opyl β-D-1- hiogalac opy anoside
ISC – I on-sul u clus e
IscR – I on-sul u clus e egula o
MST – Mic oscale he mopho esis
MFC – Mic obial uel cell
MHC – Mul iheme c- ype cy och omes
M b – Mycobac e ium ube culosis
NDSB-201 – 3-(1-py idino)-1-p opane sul ona e
NIF – Ni ogen ixa ion
NMR – Nuclea magne ic essonance
NO – Ni ic oxide
O
2
– Molecula oxygen
XXI
O
2-
– Supe oxide
OH
-
– Hyd oxyl anion
OH – Hyd oxyl
ORE – Oxidan - esponsi e elemen
PLP – Py idoxal-5’-phospha e
RNAP – Ribonucleic acid polyme ase
RNS – Reac i e ni ogen species
RRE – Roussin’s ed es e
ROS – Reac i e oxygen species
Sº – Sul ane sul u
S
2-
– Sul ide
SOD – Supe oxide
TEV – Tobacco e ch i us
UVRR – Ul a iole esonance aman
U /Vis – Ul a iole /Visible
UP – Ups eam p omo e
XXII
23
Chap e 1
G
ene al In oduc ion

24 Chap e 1
1.1 - Fe/S clus e s as ubiqui ous and mul ipu pose co ac o s in
Na u e
I on is an essen ial biological co ac o playing a majo ole in mul iple
cellula p ocesses, such as ni ogen ixa ion and espi a ion, and can be
inco po a ed in p o eins in se e al ways: as mono o di-i on eac ion cen e s o
combined wi h elemen al sul u in he o m o Fe/S clus e s (1-3). I on-sul u (Fe/S)
clus e s a e ubiqui ous and complex p o ein co ac o s consis ing o i on and
elemen al sul u ha a e in ol ed in p ocesses as di e se as DNA eplica ion and
pho osyn hesis (4, 5). Due o hei s abili y a mul iple oxida ion s a es and hei
physiologically ele an edox po en ials ( anging om -500 o -150mV), Fe/S
clus e s con e o many enzymes he abili y o pa icipa e in elec on ans e and
edox ca alysis, as well as o unc ion as senso s ha modula e gene exp ession
acco ding o he cellula edox balance (4, 6). Thanks o hei edox p ope ies,
Fe/S clus e s a e e sa ile p os he ic g oups and he high abundance o Fe/S
clus e -con aining p o eins is p oo o he e olu iona y success o Fe/S chemis y
(7, 8). Fe/S clus e s in ub edoxins a e composed by one i on a om coo dina ed by
ou cys einyl esidues, whe eas in hombic [2Fe–2S] clus e s each i on a om is
liga ed by wo p o ein ligands (Fig. 1) (7). The combina ion o hombic cen e s
leads o he o ma ion o [4Fe–4S] and mo e complex s uc u es (5). Once
inco po a ed in o p o eins, i on in Fe/S clus e s is commonly coo dina ed by
cys eine o his idine esidues; bu aspa a e and se ine side chains o backbone
amides we e also shown o unc ion as clus e ligands (9). Se e al di e en p o ein
olds ha e been ound o coo dina e hese simple Fe/S clus e s. O he nea ly 50
olds iden i ied, o e 90% ha bo [2Fe-2S]
2+,+
o [4Fe-4S]
2+,+
clus e s, being he
la e h ee imes mo e abundan , which is consis en wi h he highe chemical
s abili y o e anuclea clus e s (10). Ne e heless, i emains di icul o in e he
p esence o Fe/S clus e s di ec ly om p o ein sequences.
Du ing he pas decades, conside able p og ess has been made owa ds
he quan i a i e and quali a i e cha ac e iza ion o i on-sul u cen e s o p o eins
(11). The p ope ies o i on-sul u clus e s a e de e mined by hei elec onic
a angemen and abili y o elec on delocaliza ion (12). I on-sul u p o eins display
a wide ange o midpoin po en ials, esul ing om se e al ac o s, such as he
25
na u e o he clus e ligands (13). Elec on pa amagne ic esonance (EPR) and
Mössbaue spec oscopies a e he echniques mos commonly used o de ec i on-
sul u clus e s in p o eins and in es iga e hei p ope ies, bu complemen a y
echniques a e also widely applied, namely Nuclea magne ic esonance (NMR)
spec oscopy, X- ay c ys allog aphy, Elec on-nuclea double esonance
(ENDOR), Magne ic ci cula dich oism and Ul a iole - isible (UV/Vis) abso p ion
(14, 15). In o ma ion ob ained om EPR analysis includes elec onic s uc u e,
me al coo dina ion-sphe e composi ion and geome y, whe eas Mössbaue
spec oscopy p o ides de ailed in o ma ion o he chemical s a e o he i on a oms,
as well as he elec on dis ibu ion in a ious edox s a es o dis inc i on-sul u
clus e ypes (16). S udies o hype ine-shi ed esonances by NMR p o ides
ele an in o ma ion abou hei s uc u es in solu ion, hei elec on dis ibu ion
om i on ions on o p o ein a oms, and he elec on- ans e eac ions in which hey
pa icipa e (17). The combina ion o hese di e en echniques p o ides ull
cha ac e iza ion o he p o ein ac i e cen e .
Fig. 1 - Common ypes o i on–sul u clus e s. S uc u al
ea angemen s o ub edoxin ype, [2Fe-2S], [3Fe-4S],
and [4Fe-4S] i on–sul u clus e s. I on is colo ed in yellow,
sul ide in o ange and coo dina ing esidues (SR) a e
indica ed.
26 Chap e 1
1.1.1. Fe/S clus e s as oxida i e s ess a ge s
Reac i e oxygen species
Al hough Fe/S inco po a ion in o p o eins p o ed o be highly bene icial
du ing e olu ion, due o hei in insic chemical eac i i y, Fe/S clus e s can be e y
agile and become haza dous elemen s o he cell (18, 19). Oxygen species
con e exposed Fe/S clus e s o uns able o ms ha quickly decompose,
ul ima ely in e e ing wi h se e al cellula p ocesses in which hey play essen ial
oles. Mo eo e , deg ada ion o Fe/S clus e s can igge he o ma ion o eac i e
oxygen species (ROS, Box 1, equa ions 1-2) ha a e de imen al o lipids, p o eins
and DNA (20). Fe/S-con aining p o eins can eac wi h supe oxide (ROS; Box 1,
equa ions 3-6) h ough hei exposed clus e s, p oducing he hyd oxyl anion and
o en leading o loss o p o ein ac i i y. The e o e, Fe/S clus e s can be bo h
a ge s and gene a o s o ROS, and bo h e en s can po en ially inhibi pi o al
me abolic pa hways (18, 19).
Ni ic oxide (NO) s ess
Ni ic oxide (NO) is a signaling and de ense molecule o majo impo ance
(21). In euka yo es and a nanomola concen a ions, NO unc ions as signal ia
e e sible coo dina ion wi h he heme g oup in soluble guanyla e cyclase o
acili a e asodila a ion, while a mic omola concen a ions wi hin mammalian
mac ophages i is used as an e ec o molecule in he de ense agains pa hogenic
in asion (22, 21). Al hough o some bac e ia (e.g. soil bac e ia) NO is a na u al
me aboli e, i s abili y o eac wi h se e al key biomolecules including DNA,
me allop o eins, hiol g oups o p o eins and low molecula weigh hiols (e.g.
glu a hione and homocys eine), means ha NO is also cy o oxic and can pose a
po en ial h ea o bac e ial su i al (23). Thus, i is c ucial o bac e ia o sense
inc eased en i onmen al NO le els and igge speci ic adap i e esponses o
neu alize i s poisonous e ec (24).
27
Box 1 – Oxida i e S ess (adap ed om (25))
Oxida i e s ess esul s om an imbalance be ween eac i e oxygen
species ( ee adicals) and an ioxidan de enses (26). This imbalance can be he
consequence o exposu e o inc eased le els o ROS: supe oxide (O
2-
), hyd ogen
pe oxide (H
2
O
2
) and he hyd oxyl adical (OH
-
), which a e p oduc s o he s epwise
educ ion o molecula oxygen (Equa ion 1)







+ 2














∙
+ 

→ 

 (1)
Du ing ae obic g ow h, bo h supe oxide and hyd ogen pe oxide a e
endogenously gene a ed upon au o-oxida ion o la in co ac o s o edox enzymes
(27). Reac ion be ween Fe
2+
and hyd ogen pe oxide yields he highly eac i e
hyd oxyl anion (Fen on eac ion, equa ion 1), linking he cellula i on le els o
oxida i e s ess.


+



+

 →  

+ 
∙
+ 

 (2)
The highly de imen al eac ion o supe oxide wi h p o ein Fe/S clus e s
p oduces hyd oxyl anion (OH) and esul s in addi ional oxida i e s ess (equa ions
3-6). The hyd oxyl anion is he s onges oxidan ha exis s in aqueous
en i onmen s and is able o cause se e e and some imes le hal DNA lesions (26)
[4 − 4]

+ 

+ 2

→ [4 − 4]

+ 



(3)
[4 − 4]

→ [3 − 4]

+ 

(4)


+ 



+ 

→ 

+ 
∙
+ 

 (5)

∙
+  → 

 +  (6)
Se e al NO- esponsi e egula o y p o eins con ain i on-sul u clus e s as
hei senso y uni and NO-media ed modi ica ion o Fe/S p o eins is well
documen ed in bo h bac e ia and mammalian cells (28-31). The eac ion o i on-
sul u clus e s in egula o y p o eins was shown o p oduce a leas wo dis inc
i on-ni osyl species: dini osyl i on complex (DNIC) and Roussin’s Res Es e
(RRE) (32). The condensa ion o wo RRE species yields a no el, e anuclea
oc oni osyl clus e . Insigh s in o he mechanisms o clus e ni osyla ion we e
p o ided by ecen s udies on he WhiD p o ein om S ep omyces coelicolo and
he WhiB-like p o eins om Mycobac e ium ube culosis (28). Since e e sible
modi ica ion o i on-sul u clus e s by ni ic oxide can se e as gene ic swi ch in a
34 Chap e 1
po en ial o SoxR upon DNA binding sugges s ha he sol en -exposed
en i onmen and elec onic s uc u es o he [2Fe–2S] clus e in SoxR may be
al e ed when he p o ein is bound o i s cogna e DNA (63). The di ec in e ac ion
be ween he senso y-domain o SoxR and he DNA-binding egion p o ides a
di ec way o communica ion o he oxida i e signal. Redox-induced changes in
he [2Fe–2S] clus e o SoxR a e ansmi ed o he DNA-binding domain and
p oduce dis o ions in a ge p omo e s, allowing ansc ip ion ac i a ion (64).
The SoxR–DNA s uc u e sugges s a easonable mechanism by which
e e sible oxida ion o he [2Fe–2S] clus e leads o an in e domain s uc u al
ea angemen equi ed o he emodeling o he -35 and -10 p omo e elemen s,
so hey a e op imally posi ioned o in e ac wi h he RNAP (Ribonucleic Acid
Polyme ase) (67). Howe e , a high- esolu ion s uc u e o he ansc ip ionally
inac i e [2Fe–2S]
+
o m o SoxR bound o DNA is essen ial o u he unde s and
how he edox signal may be p opaga ed om he [2Fe-2S] clus e o he cogna e
DNA.
Redox and ni osa i e s ess sensing by SoxR
Recen s udies in di e en bac e ia challenged he gene ally accep ed iew
ha SoxR esponded o supe oxide and sugges ed ha his egula o has a much
b oade senso ial capaci y, media ing he oxida i e s ess esponse o edox-
cycling d ugs, such as iologens, phenazines and quinones (68-70). In ac , as
men ioned p e iously, he [2Fe-2S] clus e o SoxR is sol en -exposed and poses
as a eadily a ailable pla o m o he educ ion o edox-ac i e d ugs (64).
In e es ingly, in en e ic bac e ia, SoxR senses a b oade ange o compounds han
in non-en e ic bac e ia, such as Pseudomonas ae uginosa and S ep omyces
coelicolo (69). Such di e ences in SoxR sensi i i y a e ully consis en wi h i s ole
in he gene al s ess esponse in en e obac e ia and wi h a mo e es ic ed egulon
in o he bac e ia ha lack he soxS gene. Mo e ecen ly, i was demons a ed ha
he esidues in he icini y o he [2Fe-2S] clus e a e esponsible o ine- uning
SoxR sensi i i y. In ac , a hype a iable mo i o h ee esidues wi hin he highly
conse ed Fe/S binding si e egion (69) was shown o be he p ima y modula o o
SoxR p o eins sensi i i y owa ds di e en edox compounds (69).
The SoxRS egulon can also be ac i a ed by NO, h ough di ec
ni osyla ion o he [2Fe-2S] clus e o SoxR. The eac ion be ween NO and

35
SoxR is i e e sible, wi h he concomi an o ma ion o a p o ein-bound DNIC.
Al hough ela i ely s able in i o, ni osyla ed i on-sul u cen e s in SoxR apidly
disappea in i o, p obably meaning ha such p o eins a e sca enged h ough
speci ic p o ec i e mechanisms dedica ed o coun e ac ni osa i e s ess (71).
1.3. Bac e ial Fe/S clus e biogenesis machine ies
In s iking con as o he chemical and s uc u al simplici y o Fe/S clus e s,
hei syn hesis and assembly in o apop o eins is a highly complex and
o ches a ed cellula p ocess ( e iewed in (72)). In he pas decade, di e en
machine ies in bo h bac e ia and euka yo es we e shown o be dedica ed o Fe/S-
p o eins ma u a ion, namely he NIF, ISC and SUF sys ems (73, 72, 74).
The NIF sys em, i s iden i ied in Azo obac e inelandii, is mos ly
dedica ed o he ma u a ion o he ni ogenase enzyme unde ni ogen ixa ion
condi ions (75), bu i has also been ound o ma u e o he (non-ni ogenase) Fe/S-
con aining p o eins in o ganisms ha , such as Helicobac e pylo i, do no ix
ni ogen (76). In con as , bo h ISC and SUF machine ies gua an ee he
ma u a ion o he emaining cellula Fe/S p o eins. Homologous ISC p o eins a e
ound in mi ochond ia (77) and SUF homologues a e ound in chlo oplas s (78).
Despi e he ob ious di e ences among he a ious sys ems, syn hesis o
Fe/S clus e s and hei ans e in o apo- a ge s is unde lined by he same basic
p inciples and has common molecula playe s (Fig. 4) (72). The o e all
biosyn he ic p ocess can be di ided in o wo s eps: (i) he de no o assembly o he
Fe/S clus e s on a ecipien p o ein, known as sca old, and (ii) i s subsequen
ans e in o an apo-p o ein. B ie ly, L-cys eine is con e ed in o L-alanine by a
cys eine desul u ase (named IscS, Ni S o Su S) wi h he concomi an elease o
sul u (79), which is ans e ed o he sca old p o ein ha p o ides he molecula
pla o m o Fe/S clus e assembly (80, 81). The ansien Fe/S clus e is
subsequen ly ans e ed o inal accep o s (82, 83).
36 Chap e 1
Fig. 4 - Biogenesis o Fe/S clus e s by he ISC and SUF machine ies. A) The ISC sys em is
encoded by he iscRSUA-hscBA- dx ope on. IscS con e s L-cys eine in o alanine wi h he
concomi an elease o sul u o IscU, he sca old p o ein on o which Fe and S a e ansien ly
assembled as Fe/S clus e s (depic ed as yellow and o ange sphe es, espec i ely). Deli e y o
labile [2Fe-2S] clus e s om IscU o A- ype Fe/S ca ie s (ATCs) is acili a ed h ough in e ac ion
wi h he HscBA co-chape one duo, whe eas [4Fe-4S] clus e elease om he sca old o an ATC is
no s imula ed by he p esence o hese chape ones (84) and ans e o an ATC can occu di ec ly.
F om he ATC, he Fe/S clus e is deli e ed o apo- a ge s. The iden i y o he Fe
2+
(yellow sphe e)
dono emains elusi e al hough expe imen al e idence s ongly suppo s a ole o CyaY, a a axin
homologue. Elec ons equi ed o clus e assembly a e mos p obably dona ed by e edoxin
(Fdx). B) The SUF sys em is encoded by he su ABCDSE ope on. Su S and Su E o m a
he e odime ic cys eine desul u ase complex, in which sul u is ans e ed om Su S o Su E, and
subsequen ly o he sca old Su B on o which he Fe/S clus e is assembled. Su B binds a FADH
2
co ac o and is pa o a Su BC
2
D complex, whe ein Su C is an ATPase and Su D is p esumably
in ol ed in i on acquisi ion. Rega dless o he ype o Fe/S clus e (yellow and o ange squa e)
assembled on Su B, he clus e is di ec ly deli e ed om he sca old o an ATC ha subsequen ly
ma u es inal apo- a ge s.
1.3.1. The ISC sys em
The Esche ichia coli ISC sys em, encoded by he iscRSUA-hscBA- dx (isc)
ope on, ca alyzes he ma u a ion o he majo i y o Fe/S clus e p o eins unde
non-s ess condi ions (Fig. 4). The ISC machine y exis s in bo h p oka yo es and
euka yo es and i s conside ed he housekeeping sys em o Fe/S clus e
biogenesis in p oka yo es, including E. coli and A. inelandii (85).
E. coli IscS, a PLP (py idoxal-5’-phospha e) dependen enzyme, is a
homodime o 90kDa in solu ion (86). IscS ca alyzes he con e sion o cys eine o
37
alanine wi h he concomin an p oduc ion o sul u , ia he o ma ion o a pe sul ide
on a conse ed Cys esidue (Cys328). The enzyme-bound pe sul ide can hen be
ans e ed o Cys esidues on he sca old p o ein (87). Dele ion o IscS is le hal in
A. inelandii and leads o se e e g ow h de ec s in E. coli (79, 88). Many o hese
de ec s a e a di ec consequence o he educed ac i i y o Fe/S enzymes (79).
IscU sequence is highly homologous o he N- e minal domain o Ni U, he
sca old o he NIF sys em and con ains he h ee conse ed Cys esidues known
o be in ol ed in clus e coo dina ion (89). Ex ensi e biochemical s udies led o he
con i ma ion ha IscU se es as he sca old componen o he ISC machine y: i
inco po a es bo h i on and sul u , s imula es he assembly o he Fe/S clus e , and
i s ans e o apo a ge s (89, 90, 84, 91). Analysis o he enzyma ic IscS-di ec ed
clus e assembly on IscU e ealed he sequen ial o ma ion o wo [2Fe-2S]
2+
clus e s, ollowed by he slow o ma ion o a single [4Fe-4S]
2+
clus e on each IscU
homodime (89). Impo an ly, o ma ion o he [4Fe-4S]
2+
clus e -con aining o m
was accompanied by a loss o he 2[2Fe-2S]
2+
clus e species, sugges ing ha he
[4Fe-4S]
2+
species is o med by educ i e coupling o he wo [2Fe-2S]
2+
clus e s
(89). Addi ionally, p e- o med clus e s on IscU we e shown o be e icien ly
ans e ed o apo-p o eins, such as aco inase (84), e edoxin (92), Ns R (93), and
IscR (93).
Du ing Fe/S clus e assembly, IscS in e ac s wi h and di ec ly ans e s
sul u o he IscU sca old p o ein (80). Sul u ans e was shown o occu be ween
Cys328 o IscS and Cys63 o IscU in ol ing he o ma ion o a disul ide b idge in a
co alen ly bound IscS-IscU complex (87). The c ys al s uc u e o he IscS-IscU
complex showed ha one monome o IscU in e ac s wi h one subuni o he IscS
homodime leading o a 2:2 s oichiome y (94). Following clus e assembly, E. coli
IscU was shown o in e ac wi h bo h HscA and HscB o clus e ans e o apo-
p o eins (95). A conse ed mo i in IscU, LPPVK, was iden i ied as he p ima y
HscA ecogni ion si e (95) and he co-chape one HscB was shown o s imula e
HscA-IscU in e ac ion (96), con ac ing wi h IscU h ough a conse ed pa ch o
hyd ophobic esidues (97, 98). The a e o clus e ans e om IscU o apo- a ge s
is g ea ly enhanced (>20- old) when HscA and HscB a e p esen , an e ec ha
was shown o be ATP-dependen (99).
38 Chap e 1
In summa y, Fe/S clus e assembly and subsequen deli e o apo-p o eins
is an in ica e p ocess, in ol ing a conce ed ac ion and con o ma ional changes o
he sca old, chape ones and inal a ge s.
1.3.2. The SUF sys em
In E. coli, dele ion o he en i e isc ope on p oduces g ow h de ec s and
simul aneously leads o educed ac i i y o Fe/S enzymes (100). The ac ha such
s ains emained iable aised he possibili y o unc ional compensa ion and led o
he iden i ica ion o he SUF sys em (101). The E. coli SUF sys em is composed by
six genes o ming he su ABCDSE (su ) ope on and i s unc ion in Fe/S clus e s
biogenesis was assigned a e he analysis o di e se combina ions o bo h ISC
and SUF E. coli mu an s ains (101). Supp ession o ei he he isc o su ope ons
is no le hal in E. coli ( o syn he ic le hali y bo h sys ems ha e o be inac i a ed)
and su mu a ions ha e only a mild e ec on he ac i i y o Fe/S p o eins.
Howe e , absence o he SUF sys em inc eases E. coli sensi i i y o i on
s a a ion (85), whe eas in s ains lacking he ISC pa hway and unde anae obic
condi ions, su ope on exp ession ully es o ed he ac i i y o FNR ( uma a e and
ni a e educ ion egula o y p o ein) (43)
.
Al hough he e is some edundancy
be ween he wo sys ems, he ISC sys em unc ions as he housekeeping Fe/S
clus e assembly sys em in E. coli, whe eas he su ope on is speci ically igge ed
o syn hesize Fe/S clus e s in condi ions ha lead o he dis up ion o i on o sul u
me abolism, such as i on s a a ion o oxida i e s ess (Fig. 4) (102, 85).
The SUF machine y o Fe/S clus e assembly elies on he o ma ion o wo
sepa a e complexes o su encoded p o eins: he Su BCD and he Su SE
complexes. The Su BCD complex was shown o unc ion as a sca old ha is able
o bind and ans e a [4Fe-4S] clus e o apop o eins, including Su A (83, 103, 81).
In he Su BCD complex, Su B is ega ded as he sca old con aining he Cys
esidues o coo dina e nascen Fe/S clus e s; Su C is an ATPase esembling
hose associa ed wi h ABC anspo e s (104) and Su D was sugges ed o be
in ol ed in i on en y in o he sca old complex (105). The Su SE complex poses
as a sul u dono o Fe/S clus e o ma ion. Su S is cys eine desul u ase
homologous o IscS, whose ac i i y is g ea ly enhanced h ough in e ac ion wi h
Su E (106, 107). In i o expe imen s showed ha he cys eine desul u ase ac i i y
39
o he Su S-Su E complex is subs an ially s imula ed in p esence o he Su BCD
complex and Su E in u n in e ac s wi h Su B, leading o he p oposal ha sul u
ans e om Su S o Su B is media ed by Su E (108).
The Su BCD complex exis s p edominan ly as a Su BC
2
D s able o m,
which is he mos e icien complex in Fdx ma u a ion and he e o e is p oposed o
se e as he e minal sca old (109). A wo h no icing ea u e o he Su BC
2
D
complex is i s abili y o bind one equi alen o la in adenosine nucleo ide (FAD)
only in i s educed s a e (FADH
2
) (81). This co ac o can e en ually p o ide he
elec ons o mobilize he e ic i on equi ed o Fe/S clus e assembly om e ic
ci a e, e i ins o CyaY.
1.3.3. The deli e y s ep – A- ype p o eins
Once a clus e is assembled on a sca old, i mus be deli e ed o an apo-
a ge . In E. coli, he o al numbe o Fe/S-con aining p o eins is p edic ed o be
close o 150 (8) and expe imen al da a indica es ha A- ype p o eins media e he
Fe/S clus e s deli e y p ocess (Fig. 4) (110, 111). E. coli possesses h ee A- ype
p o eins, namely IscA, Su A and E pA ha sha e 30% sequence iden i y. IscA and
Su A belong o he isc and su ope on, espec i ely, whe eas E pA is loca ed
elsewhe e in he ch omosome (111). A- ype p o eins we e ini ially p oposed o ac
as complemen a y sca olds gi en he p esence o h ee highly conse ed cys eine
esidues on hei C- e minal egion, as i was obse ed o he IscU sca old (112,
113). This p oposal was unde sco ed by he obse a ion ha he p e- o med
clus e could be ans e ed o apo- a ge s (113, 114). Howe e , his iew was
p o oundly challenged by se e al obse a ions. Fi s , A- ype p o eins a e no able
o in e ac wi h cys eine desul u ase enzymes, an essen ial s ep o sul u ans e
and subsequen clus e assembly on a sca old (94). Second, while mu a ions on
IscA o Su A we e ound o be almos neu al, an e pA mu a ion was ound o be
le hal unde espi a o y g ow h condi ions and nei he o hese p o eins was able o
compensa e o a mu a ion on IscU (115, 100). Finally, bo h IscU and Su BCD
we e able o ma u e IscA and Su A, espec i ely, bu he e e se eac ion was no
possible (83, 114). Taken oge he , hese esul s es ablished A- ype p o eins as
“Fe/S clus e s ca ie s” (ATCs, A-Type Ca ie s) a he han sca olds (109, 116).
Mo eo e , unc ion o A- ype p o eins was shown o equi e unc ional Su B o

40 Chap e 1
IscU, u he p o ing ha ATCs a e unlikely o ha e a sca old unc ion (111).
Unde his pe spec i e, nascen clus e s would be ans e ed om hei sca olds
o ATCs ha would deli e hem o apo-p o eins. Howe e , phylogenomic and
gene ic analyses sugges ha ATCs unc ion enables mul iple ou es o Fe/S
clus e s a icking and ha he choice o ma u a ion ou e is modula ed by g ow h
condi ions, such as oxygen le els (111).
1.3.4. CyaY as an i on dono o Fe/S clus e biogenesis
CyaY, he bac e ial homolog o euka yo ic a axin, is he p ima y candida e
as i on dono o Fe/S clus e biosyn hesis (117). In humans, educed le els o
a axin we e linked o he neu odegene a i e disease F ied eich’s a axia (118).
This pa hology is a consequence o Fe/S clus e biogenesis dis up ion,
mi ochond ial i on accumula ion and oxida i e s ess, which a e known
con ibu o s o educed ac i i y o Fe/S p o eins (119). In con as o a axin
dele ion in euka yo es o ganisms, supp ession o CyaY, he bac e ial homolog o
a axin, does no al e i on con en o sensi i i y o oxida i e s ess (120).
None heless, he CyaY E. coli mu an had educed amoun s o Fe/S clus e -
con aining espi a o y Complex I and Complex II, which is p obably a
consequence o dec eased ma u a ion o Fe/S p o eins (121, 122). In i o s udies
p o ided u he insigh s in o CyaY unc ion: CyaY can bind i on speci ically
o ming a s able complex, albei wi h some dis inc p ope ies om o he i on-
binding p o eins (123).
CyaY in e ac s s ongly wi h he IscU-IscS complex, o ming a
he e o ime ic assembly (124) and was able o dona e i on o Fe/S clus e
assembly on IscU in he p esence o bo h IscS and cys eine (125). Fu he
biochemical s udies, sugges ed ha CyaY can ac as an i on-dependen inhibi o
o clus e o ma ion, whose unc ion is exe ed h ough in e ac ion wi h IscS and
modula ed by i on bioa ailabili y o adap Fe/S clus e biogenesis o he pool o
Fe/S accep o p o eins (126). Ve y ecen ly, e edoxin (Fdx) was shown o bind o
IscS (127, 128), while supplying elec ons o educe sul ane sul u (S
0
) o sul ide
(S
2-
) ollowing he enzyma ic con e sion o cys eine (127). Mo eo e , Fdx and
CyaY compe e o o e lapping binding si es on IscS and holo-Fdx binding o IscS
does no pe u b he a ini y o IscS o IscU (127, 128). These esul s ein o ce he
41
p oposal o Fdx and CyaY ac ing, in a s epwise ashion, as elec ons and i on
dono s o Fe/S clus e biosyn hesis, espec i ely,.
1.3.5. ISC and SUF sys ems unde s ess condi ions
Fe/S p o eins a e easily damaged by oxida i e s ess due o he inhe en
eac i i y o hei Fe/S clus e s (18, 129). Hence, such en i onmen al s ess
condi ions a e expec ed o in luence he unc ion o p o ein machine ies
esponsible o he de no o syn hesis and deli e y o Fe/S clus e s o apo-
p o eins. Al hough he ISC and he SUF sys ems sha e he same basic p inciples,
combined gene ic and biochemical s udies pinpoin ed se e al di e ences in he
abili y o unc ion unde s ess condi ions (102, 85). The ISC sys em is sensi i e o
oxida i e s ess, being ully inac i a ed by ROS (102). Indeed, he Fe/S clus e
assembled on IscU is exposed and hus in insically p one o oxida ion (89, 130).
Such clus e accessibili y p omo es coppe o cobal a ack ha , due o hei
hiophilici y (i.e abili y o bind sul u ), can eplace i on and o m mixed clus e s.
Al hough his was ue o coope (131), cobal eac s p e e en ially wi h deg aded
o ansien ly syn he ized clus e s, a he han a ack hem di ec ly in Fe/S
p o eins. Indeed, a ansien i on-cobal -sul u complex buil on IscU could be
ans e ed o an apo-p o ein (129). Thus cobal oxici y is in ima ely linked o he
poisoning o he en i e Fe/S clus e biogenesis pa hway.
In E. coli, submic omola concen a ions o H
2
O
2
we e de imen al o he
ISC machine y and, unde such condi ions, he su ope on was ac i a ed o
compensa e he lack o Fe/S clus e biosyn hesis (102). Likewise, su ope on
exp ession was igge ed unde i on s a a ion condi ions (85). These esul s
made e iden ha o ganisms exposed o oxidan s ely on he SUF sys em o
media e Fe/S clus e biogenesis a he han he ISC pa hway. Addi ionally, he ac
ha Su B has FADH
2
as a co ac o may be indica i e ha he SUF sys em is be e
equipped o i on mobiliza ion when i on is sca ce, gi en ha his co ac o can
p obably p o ide he necessa y educing powe o mobilize i on om di e se
sou ces (105, 81). Mo eo e , he E. coli Su S-Su E complex is mo e esis an o
oxida i e s ess han he IscS-IscU complex (132) and he SUF sys em e icien ly
epai ed damaged clus e s (102). Finally, ansc ip omic analysis showed ha
42 Chap e 1
cobal induced su ope on exp ession, linking cobal oxici y o he impai men o
Fe/S clus e biosyn hesis, i on bioa ailabili y and oxida i e s ess (133).
1.3.6. Di e si y o ISC and SUF sys ems
Homologs o E. coli ISC and SUF p o eins a e ound in a wide ange o
o ganisms, bu some species con ain dis inc pa e ns o Fe/S clus e biosyn hesis
sys ems. Fo ins ance, in con as o E. coli and mos G am-nega i e bac e ia,
cyanobac e ia (e.g Synechocys is spp.) possess only an incomple e SUF sys em
and no ISC sys em (134) and Mycobac e ium ube culosis (M b), he causa i e
agen o ube culosis, con ains a simpli ied Su BCDS ope on (135). A simila
scena io is encoun e ed in mos G am-posi i e bac e ia ha ca y only a su
ope on (136), a pa icula case ha will be desc ibed in mo e de ail in he ollowing
sec ion.
Fe/S clus e biogenesis in G am-posi i e bac e ia
Fe/S clus e biogenesis sys ems ha e been unde in ensi e sc u iny in
G am-nega i e bac e ia, such as E. coli ( e iewed in (137)), bu a e poo ly
unde s ood in G am-posi i e bac e ia. In silico analyses iden i ied a highly
conse ed SUF machine y in G am-posi i e bac e ia ha is ela i ely di e en om
he E. coli SUF sys em (138). Bioin o ma ic cha ac e iza ion iden i ied cis-ac ing
elemen s on he su p omo e egion simila o hose loca ed ups eam o he su
ope on in E. coli. Simila o wha was obse ed in E. coli, he Fu and OxyR
egula o s, as well as he DNA-bending p o ein IHF (in eg a ion hos ac o ),
p obably ecognize hese egions (136). Howe e , in con as o E. coli, no
o hologs o IscR, Su E o Su A we e ound in he G am-posi i e bac e ium
En e ococcus aecalis (138).
Fu he mo e, G am-posi i e bac e ia code o an IscU-like p o ein, named
Su U, ha lacks he conse ed HscA ecogni ion mo i LPPVK (138). Such ea u e
is ully consis en wi h he absence o a HscA in G am-posi i e o ganisms. Su U is
essen ial in Bacillus sub ilis and was shown o in e ac wi h Su S, om which i
ecei es sul u , o subsequen ly ma u a e apo- a ge s (139). Mo eo e , Su U is
highly homologous o he N- e minal domain o A. inelandii Ni U, including he
esidues known o coo dina e he clus e . Besides lacking he speci ic LPPKV
43
mo i , Su U also con ains a cha ac e is ic 19 amino acid inse ion ha is appa en ly
es ic ed o Su U homologues om G am-posi i e bac e ia. This inse ion was
named G am-posi i e egion (GPR) and may be in ol ed in he in e ac ion o Su U
wi h o he mac omolecules (136). Howe e , conside ing ha Su B lacks some o
he cys eine esidues ha we e ound o coo dina e he Fe/S clus e in i s E. coli
homolog (81), addi ional expe imen s a e equi ed o es ablish i Su BCD is indeed
a sca old in G am-posi i e bac e ia and, i so, unde s and he p esence o wo
sca old p o eins. In ac , i is possible ha he e is no sca old edundancy, gi en
ha Su U was shown o ac as desul u ase ac i a o in B. sub ilis (140). Su C and
Su B a e highly simila o hei E. coli o hologues, bu unc ional and biochemical
da a is equi ed o ully unde s and he Su BCD complex ole in Fe/S clus e
biogenesis in G am-posi i e bac e ia.
1.4. IscR, he Fe/S clus e biogenesis egula o
1.4.1. Conce ed egula ion o ISC and SUF sys ems by IscR
Fe/S p o eins a e widely dis ibu ed in Na u e and essen ial o many
cellula p ocesses (8). The syn hesis o many o hese p o eins is egula ed o
accommoda e changes in en i onmen al condi ions ha can be de imen al o
Fe/S clus e s (141). Consis en wi h his no ion, bo h Fe/S clus e biogenesis
machine ies, ISC and SUF, a e igh ly egula ed by he [2Fe-2S] clus e -con aining
ansc ip ion ac o IscR (142). IscR, encoded by he i s gene o he iscRSUA-
hscBA- dx (isc) ope on, ac s as a ep esso o he Isc pa hway and was shown o
coo dina e a [2Fe-2S]
+1
clus e upon anae obic isola ion (142). Mu a ions on ei he
iscA o hscA genes signi ican ly educed he ac i i y o IscR, showing ha
ep ession by IscR is dependen o unc ional ISC machine y and is closely linked
o i s Fe/S clus e (142). Biochemical cha ac e iza ion o pu i ied holo-IscR ([2Fe-
2S]-IscR) p o ided insigh s in o he mechanism by which IscR is able o sense he
cellula Fe/S clus e s a us. Mössbaue expe imen s using whole cell ex ac s
e ealed ha [2Fe-2S]
+1
-IscR is he p edominan o m in i o, which becomes
u he oxidized e en upon anae obic isola ion (143). Howe e , clus e oxida ion
did no a ec he binding a ini y o IscR owa ds he isc p omo e (143). Si e-
speci ic mu agenesis e ealed ha he h ee highly conse ed cys eine esidues
50 Chap e 1
The abili y o IscR o ecognize di e en binding mo i s was unexpec ed,
gi en ha he IscR monome con ains a single DNA binding domain (142).
Regula ion o ype-2 p omo e s by IscR, such as he hyaA p omo e , was
subsequen ly cha ac e ized and p o ided insigh s in o he di e en ial DNA
ecogni ion by IscR (159). Su p isingly, binding o IscR o ype-2 p omo e s does
no equi e he [2Fe-2S] clus e , a ea u e ha is no linked o whe he IscR ac s as
an ac i a o o as a ep esso (144). Fu he cha ac e iza ion o he in e ac ion
be ween IscR and i s a ge s, demons a ed ha p esence o he Fe/S clus e
g ea ly enhances he a ini y o IscR owa ds ype-1 p omo e s and is essen ial o
hei egula ion in i o (147, 144, 142), whe eas bo h apo-IscR and holo-IscR bind
wi h simila a ini ies o ype-2 p omo e s bo h in i o and in i o (144). Ex ensi e
analysis o he ype-2 sequences allowed he iden i ica ion o an impe ec
palind ome mo i , con aining se e al bases a conse ed posi ions, including a CC
dinucleo ide (Fig. 7). Al hough he posi ion o he binding si es wi hin he hyaA,
su A and ydiU p omo e s is nea ly he same ela i e o hei p edic ed -35
elemen s, IscR ep essed he hya ope on exp ession, bu induced he exp ession
o bo h su and ydiU ope ons. This obse a ion may esul om mino di e ences
in he space egions o he -35 hexame and/o he s ong in e ac ion o IscR wi h
he hyaA p omo e in compa ison o o he IscR- a ge p omo e s (144).
Fig. 7 - Compa ison be ween IscR-binding si es and he IscR binding mo i s p oposed om
phylogene ic sequence conse a ion. Si es ecognized exclusi ely by [2Fe-2S]-IscR (iscb, yadR
and yhgI) we e compiled as ype-1 p omo e s, whe eas ecogni ion o ype-2 p omo e sequences
was shown o be independen o IscR clus e occupancy. S ic ly conse ed bases a e highligh ed
in ed and esidues conse ed be ween a leas wo sequences a e colo ed in yellow. In he binding
mo i s: R and Y a e puRine and pY imidine, espec i ely; S is G o C; W is A o T; K is T o G; M is
A o C and n is any nucleo ide (147). Alignmen p epa ed wi h Clus alW (161) and colo ed wi h
Aline (162).

51
Gi en ha bo h holo- and apo- o ms we e shown o in e ac simila ly wi h
ype-2 p omo e s, holo-IscR binding o hese p omo e s unde anae obiosis is
p obably p e en ed h ough he p esence o o he ansc ip ion ac o s ha
compe e wi h IscR o he same binding si e. This was ound o be he case o
bo h su A and hyaA p omo e s, whe e he IscR binding si e o e laps wi h he
binding sequence o Fu and A cA, espec i ely (154, 159). This in e play be ween
IscR and o he ansc ip ion ac o s ep esen s a mechanism by which hese
p omo e s a e igh ly egula ed in esponse o en i onmen al condi ions.
Thus, in con as o o he well cha ac e ized Fe/S-con aining egula o s ha
ha e only one ansc ip ionally ac i e p o ein o m, bo h apo- and holo-IscR en oll
in speci ic egula o y oles, being able o di e en ially ecognize wo ypes o DNA
sequences acco ding o i s clus e occupancy (146, 144).
1.4.4. The R 2 amily o egula o s
IscR belongs o he widesp ead R 2 amily o egula o s (PF02082, P am
da abase). R 2 membe s a e ela i ely small p o eins (12-18 kDa) wi h a
cha ac e is ic helix- u n-helix (HTH) domain nex o hei N- e minus. Besides IscR,
his amily con ains he global cys eine egula o , CymR (163); he NO-sensing
ep esso , Ns R (164); and he hizobial i on egula o A, Ri A (165). Wi h he
excep ion o CymR, all R 2 membe s wi h an assigned unc ion we e shown o
coo dina e a Fe/S clus e (142, 165, 166).
In B. sub ilis, CymR is he egula o o he cys eine biosyn hesis pa hway
(167) and i s ac i i y is posi i ely egula ed by he in e ac ion wi h he o-
ace ylse ine (OAS)- hiol-lyase, CysK (168). Complex o ma ion (CymR-CysK)
s abilizes binding o CymR o DNA. Th ough binding o i s OAS subs a e, a di ec
pe cu so o cys eine, CysK is able o ac as an indi ec senso o he cellula
cys eine concen a ion and ansmi his in o ma ion o CymR. When cys eine is
p esen , OAS concen a ions a e low and o ma ion o he CymR-CysK complex is
a o ed, which in u n leads o ep ession o cys eine biosyn hesis (168). In
S aphylococcus au eus, CymR was shown o indi ec ly egula e bio ilm o ma ion
and s ess esponse, playing a key ole in i ulence (169-171). The c ys al
s uc u e o bo h CymR om B. sub ilis and S. au eus e eals a biologically ac i e
dime , whe e each monome olds in o wo igh ly packed domains: a DNA-binding
52 Chap e 1
domain, ha bo ing he cha ac e is ic winged helix– u n–helix (wHTH) mo i ; and a
long dime iza ion domain, which places he wHTH mo i s a he ex emes,
opposing each o he (Fig. 8A). This a chi ec u e explains how hese small
egula o s can ecognize DNA a ge s encompassing 23 o 27 bp (169, 163).
No ably, Ri A, IscR and Ns R con ain h ee conse ed C- e minal cys eine
esidues (Fig. 8B), bu hese conse ed esidues a e no ound in all R 2 amily
p o eins. Some R 2 amily membe s comp ise wo, one o no cys eines and, wi h
he excep ion o IscR, Ns R and CymR (146, 169, 172), he ligands and sensing
mechanisms ha ac i a e o inac i a e hese p o eins a e unknown.
Ns R is a ela i ely well-cha ac e ized R 2 egula o , pa icula ly in B.
sub ilis, whe e i was shown o con ol he ansc ip ion o genes in ol ed in NO
de oxi ica ion (172). Fo NO de oxi ica ion, B. sub ilis makes use o bo h
la ohemoglobin (hmp) and ni i e educ ase (encoded by he nasDEF ope on)
p o eins (173). Ns R is a mas e egula o o NO me abolism in bo h G am-
nega i e and G am-posi i e bac e ia, ep essing he ansc ip ion o he nasDEF
ope on and hmp gene unde anae obic e men a i e g ow h condi ions (174).
T ansc ip ion o hese genes is con olled by he memb ane-bound his idine kinase
ResE and he cy oplasmic ResD egula o (172). Ns R ecognizes he -35 elemen
o he nasD p omo e , leading o he dis up ion o he RNA polyme ase-ResD-DNA
complex (172). Unde NO-exposu e, bo h nasD and hmp a e de ep essed due o
he elease o Ns R om he nasD p omo e h ough di ec ni osyla ion o i s [4Fe-
4S] clus e (172, 175). A se o Ns R-con olled genes iden i ied by ansc ip mic
analysis belongs o he Fu egulon and is in ol ed in i on homeos asis (176).
The e is some con o e sy ega ding he ype o clus e bound by Ns R. Bo h S.
coelicolo (166) and Neisse ia gono hoeae Ns R (177) coo dina e [2Fe-2S]
clus e s, whe eas isola ion o B. sub ilis Ns R yielded a p o ein con aining a [4Fe-
4S] clus e . (175). Rega dless o he ype o clus e , Ns R o hologues bea a Fe/S
clus e ha eac s di ec ly wi h NO, leading o he o ma ion o DNIC and
de ep ession o he nasD p omo e (166, 175). Ns R binding si es compiled om
he nasD and hmp p omo e s display an impe ec dyad symme y ha was no
ound in o he p omo e egions known o be con olled by Ns R and whose
ecogni ion by Ns R is NO insensi i e (Table 2) (174, 172). Mo eo e , in B. sub ilis,
binding o Ns R was modula ed by ResD and Fu in co- egula ed p omo e s (174)
and i s Fe/S clus e was eac i e owa ds o he molecules such as cyanide,
53
di hio h ei ol, and O
2
(175). Thus, Ns R se es as a senso o en i onmen al NO
concen a ions and is in ol ed in he global s ess esponse o ni osa i e s ess
(173).
IscR and Ns R sha e low sequence simila i y wi hin he helix- u n-helix
domain, ha ing only h ee iden ical amino acids (unde lined, 28-LADISER-QGIS-
LSYLEQLFSRLRK-51), and he e o e ecognize e y dis inc DNA binding mo i s
(Table 2) (147, 178). Al hough i is p edic ed ha R 2 egula o s con ain a
common s u u al signa u e cha ac e ized by a N- e minal helix– u n–helix DNA-
binding domain, membe s o his amily display di e en DNA-binding p ope ies
ha a e ansla ed in dis inc cellula unc ions.
Fig. 8 - S uc u e o he R 2 amily egula o CymR and he Fe/S clus e binding egion in
o he R 2 egula o s. A) S uc u e o he B. sub ilis CymR monome , showing he
dime iza ion helix and he ypical HTH DNA-binding domain [PDB en y 2Y75 (163)].
The p edic ed loca ion o he clus e -coo dina ing cys eine esidues ound in
homologous Fe/S clus e -con aining R 2 p o eins (e.g., IscR, Ns R, and Ri A) is
delimi ed by lines. B) Amino acid sequence alignmen o B. sub ilis CymR wi h Fe/S-
con aining R 2- ype egula o s. The sequences o IscR and Ns R o E. coli, Ri A o
Rhizobium leguminosa um (R. leguminosa um) and CymR o B. sub ilis, a e aligned.
S ic ly conse ed amino acids a e highligh ed in ed, and inc easing esidue
conse a ion is ep esen ed by a colo g adien om g een o ed. Alignmen
p epa ed wi h Clus alW (162). The h ee cys eines assigned as clus e ligands in
IscR, Ns R, Ri A a e deno ed wi h open iangles.
Table 2 – Sequence mo i s compiled om IscR and Ns R DNA-binding si es
P o ein
DNA-binding mo i * Re .
IscR
ATASYYGACTRwwwYAGTCRRSTAT o
AWARCCCYTSnGTTTGMnGKKKTKWA (147)
Ns R
gATGyATTTxAAAT CA c (178)
*R and Y a e puRine and pY imidine, espec i ely; S is G o C; W is A o T; K is T o G; M is
A o C and n is any nucleo ide
54 Chap e 1
1.4.5. Implica ions o IscR in hos -pa hogen in e ac ions
The in e ac ion be ween pa hogenic mic obes and hei hos s is de e mined
by su i al s a egies on bo h sides (179). Due o i s edox p ope ies, i on holds a
cen al posi ion a he hos -pa hogen in e ace, being i al o he g ow h o i ually
all o ganisms, including pa hogenic bac e ia. I on u iliza ion by pa hogens equi es
speci ic mechanisms ha allow he in e ac ion wi h and he acquisi ion o i on om
i on-binding p o eins (180). Howe e , high concen a ions o i on lead o oxida i e
s ess, while low concen a ions subs an ially a ec bac e ial g ow h. Hence, o
gua an ee hei su i al, pa hogens ha e o igh ly egula e bo h i on acquisi ion
and anspo (180). Addi ionally, o coun e ac bac e ial in asion, he hos inna e
immune sys em gene a es ROS and eac i e ni ogen species (RNS) esponsible
o oxida i e s ess (179). Bo h i on s a a ion and oxida i e s ess ha e a
de imen al e ec on he unc ion and biogenesis o Fe/S enzymes (18). Thus, he
abili y o adap o changes in O
2
and i on bioa ailabili y is i al o many bac e ial
pa hogens, as many niches wi hin a hos a e hypoxic (181) and i on deple ed
(180). Pa hogenic bac e ia ha e e ol ed ansc ip ional egula o y sys ems ha
pe cei e such hos ile condi ions and espond by ep og amming gene exp ession
(182, 183). Fo his eason, de ensi e mechanisms ha de oxi y ROS and RNS
and epai damaged cell componen s essen ial o he in acellula su i al o he
pa hogen du ing in ec ion a e s ic ly egula ed in pa hogens (179, 182).
Gi en he ole o IscR is as a senso o he cellula Fe/S clus e s pool ha
di ec ly modula es he exp ession o a di e se se o genes, i is no su p ising ha
IscR unc ion di ec ly a ec s he abili y o some mic obial pa hogens o main ain
i on homeos asis and esis oxida i e s ess du ing hos in ec ion (184-187). The
plan pa hogen E winia ch ysan hemi aces bo h i on s a a ion and oxida i e
s ess a he onse o in ec ion and consequen ly bo h oxida i e-s ess esis an
and Fe/S biogenesis sys ems a e essen ial o i ulence (186). A abidopsis
haliana p oduces ROS o coun e ac in ec ion, which a e de imen al o Fe/S
clus e s. The deg ada ion o Fe/S clus e s is sensed by E. ch ysan hemi IscR,
whose ac i i y is essen ial o main ain he app op ia e le els o Fe/S clus e
biogenesis in such ad e se en i onmen s (186). Mo eo e , IscR was equi ed o
pe oxide esis ance in Pseudomonas ae uginosa PA14, an oppo unis ic human
pa hogen esponsible o le hal in ec ions in immunocomp omised indi iduals
55
(188). P. ae uginosa iscR mu an was shown o be hype sensi i e o H
2
O
2
and
pa aqua , due o dec eased ca alase A (Ka A) enzyme ac i i y (184). Recen ly, i
was shown ha IscR egula ed he P. ae uginosa isc ope on unde bo h
physiological and s ess condi ions, con e ing esis ance o oxida i e s ess and
con ibu ing o i on homeos asis (184). In Shigella lexne i, a acul a i e
in acellula pa hogen and he causa i e agen o bac e ial dysen e y, he
exp ession o bo h su and isc p omo e s inc eased when he pa hogen was li ing
in acellula lly due o i on sca ci y. IscR was ound o be a posi i e egula o o su
exp ession unde oxidan condi ions and a nega i e egula o o isc exp ession in
he absence o hyd ogen pe oxide, p o ec ing S. lexne i agains oxida i e s ess
(187). IscR was also ound o modula e sensi i i y o oxida i e s ess h ough
ep ession o Fe/S clus e biogenesis du ing hos coin ec ion wi h he
bac e ium Haemophilus in luenza and in luenza A i us (IAV) (189). Fu he mo e,
IscR unc ion on Vib io ulni icus highly impac ed he mo ili y and adhesion o hos
cells, i s hemoly ic ac i i y, and he su i al o his pa hogen unde oxida i e s ess
du ing in ec ion (185). Thus, V. ulni icus IscR, whose exp ession is up egula ed
by he ansc ip ional egula o Aph, unc ions as a global egula o con ibu ing o
he o e all e iciency o pa hogenesis by egula ing no only he Fe/S clus e
biogenesis genes bu also he exp ession o a wide ange o i ulence and su i al
ela ed genes (185, 190).
Ve y ecen ly, IscR was also linked o he i ulence o ood-bo ne
pa hogen Ye sinia pseudo ube culosis possibly h ough he con ol o he Type III
sec e ion sys ems (T3SS), an injec isome ha deli e s bac e ial e ec o p o eins
di ec ly in o he hos cell cy oplasm (191).
In conclusion, apa om i s e iden biological impo ance in he egula ion
o bo h ISC and SUF machine ies, IscR may be o pha macological in e es . In
plan s, egula ion o Fe/S clus e biogenesis is p oposed o be achie ed by
glu a edoxins ha could ac as Fe/S clus e ‐con aining senso o he SUF
machine y (192), whe eas in mammals, IRP1 and IRP2 p o eins ac pos -
ansc ip ionally o adjus he cellula equi emen s o i on (193). Hence, since
IscR s uc u al homologs can be ound in he pa hogens men ioned abo e, bu a e
absen in hei hos s o choice, IscR is a po en ial a ge o no el an imic obial
agen s.

56 Chap e 1
57
Chap e 2
M
e hods
58 Chap e 2
P o ein Exp ession and Pu i ica ion
A syn he ic isc gene, encoding he same amino acid sequence as
The JR_1914 om he T. po ens genome, excep o a Gly-Leu inse ion
immedia ely downs eam om he N- e minal me hionine and con aining he NcoI
and Acc65I es ic ion si es, was o de ed om Eu o ins MWG Ope on. The E. coli
isc gene (b2531) agmen spanning nucleo ides +4 o +489 o he IscR ORF was
ampli ied om an E. coli K12 colony using speci ic p ime s (see Table A.1 in
Appendix). IscR
Tp
-w ORF was cloned in o he NdeI and XhoI si es o he
exp ession ec o pET30a o in o he Acc65I and NcoI si es o he exp ession
ec o pETZ2_1a, whe eas IscR
Ec
-w ORF was cloned in o HindIII and NcoI si es
o he exp ession ec o pETZ2_1a (194). The la e cons uc s we e used o
ob ain he iple mu an s (C92/101/107S o T. po ens o C92/98/104S o E. coli)
co esponding o he clus e less o ms o he p o eins (apo-IscR
Tp
and apo-IscR
Ec
)
by si e-di ec ed mu agenesis (see Table A.2 in Appendix). All cons uc s we e
e i ied by DNA sequencing. The N- e minal His
6
- agged apo-IscR
Tp
was
o e exp essed in E. coli BL21 (DE3) cells and he E. coli p o ein in E. coli BL21
S a (DE3) (Li e Technologies). B ie ly, cells we e g own in LB medium a 37 °C
un il OD
600
= 0.7. A his poin , he empe a u e was dec eased o ei he 25 °C
(apo-IscR
Ec
) o 30 °C (apo-IscR
Tp
), and he exp ession was induced wi h he
addi ion o 0.5 mM isop opyl β-D-1- hiogalac opy anoside (IPTG). Cells we e
ha es ed by cen i uga ion a e 4 h and lysed by incuba ion (60 min on ice wi h
shaking) wi h 25 µg/mL chicken egg whi e lysozyme (Sigma). Cla i ied p o ein
ex ac s in 20 mM sodium phospha e (pH 7.5), 0.5 M NaCl, 10 mM imidazole, 5%
( ol/ ol) glyce ol, 150 mM a ginine, and 2.5 mM β-me cap oe hanol (bu e A)
we e loaded on o a HisT ap HP column (GE Heal hca e) p eequilib a ed in he
same bu e , and bound p o eins we e elu ed wi h bu e A con aining 125 mM
imidazole. The IscR-con aining ac ions we e pooled, and he His6 and he
solubili y ags we e emo ed by incuba ion wi h obacco e ch i us (TEV) p o ease
a 4 °C concomi an ly o an o e nigh dialysis agains 20 mM sodium phospha e
(pH 7.5), 0.2 M NaCl, 10 mM imidazole, 5% ( ol/ ol) glyce ol, 150 mM a ginine,
and 2.5 mM β-me cap oe hanol. A e o e nigh dialysis, he NaCl was es i u ed o
app oxima ely 0,5M by adding he necessa y olume o a 5M NaCl solu ion and
pu e ecombinan IscR was sepa a ed om he exp ession ag and nonclea ed
59
ma e ial by a second immobilized-me al a ini y ch oma og aphy (IMAC) s ep, in
he same condi ions as desc ibed abo e. The bu e was u he exchanged o 10
mM Hepes (pH 7.5), 800 mM KCl, and 5% ( ol/ ol) glyce ol using a HiP ep 26/10
(GE Heal hca e) desal ing column. Following concen a ion in a cen i ugal de ice,
he p o ein was ei he used immedia ely o lash- ozen in liquid ni ogen and
s o ed a −80 °C un il needed. Final p o ein concen a ions we e es ima ed by
measu ing he abso bance o he samples a 280 nm. Poin mu an s apo-IscR
Tp
-
E43A, apo-IscR
Ec
E43A, and apo-IscR
Tp
-P40S we e gene a ed by si e-di ec ed
mu agenesis o he pETZ2_1a cons uc s (see Table A.2 in Appendix). All IscR
p o ein a ian s used o biochemical and c ys alliza ion expe imen s we e
exp essed and pu i ied as desc ibed o apo-IscR
Tp
, excep o selenome hionyl
apo-IscR
Tp
ha was p oduced in a me hionine auxo oph s ain (E. coli B834,
S a agene), using SelenoMe medium (Molecula Dimensions) ollowing
manu ac u e ’s ins uc ions and o apo-IscR
Tp
-w ha was pu i ied by a single
IMAC s ep ollowed by desal ing on a HiP ep 26/10 column (GE Heal hca e). The
E. coli cys eine desul u ase IscS used in econs i u ion assays was exp essed and
pu i ied as desc ibed p e iously (195).
Elec opho e ic Mobili y-Shi Assay
Complemen a y oligonucleo ides (Sigma) con aining he sequence o he E.
coli hya o o he T. po ens isc p omo e (Table 3) we e annealed in o double-
s anded DNA by hea ing a 50 µM solu ion o 95 °C o 5 min in a wa e ba h,
ollowed by slowly (o e nigh ) cooling o oom empe a u e. Fo elec opho e ic
mobili y-shi assay (EMSA) analysis using he comple e sequence o he T.
po ens su p omo e egion (Table 3), he sequence ups eam o he su C gene
(The JR_0923) was ampli ied by PCR using a syn he ic empla e (Eu o ins). DNA
solu ions (1 µM) we e incuba ed wi h 7.5–10 µM pu i ied p o ein a oom
empe a u e o 20 min in binding bu e [40 mM T is·HCl (pH 8.0), 100–150 mM
KCl, 5% ( ol/ ol) glyce ol, and 1 mM DTT (Di hio h ei ol)], and he esul ing
complexes we e esol ed on 8% (w / ol) nondena u ing polyac ylamide gels using
1× TAE (40 mM T is·HCl, 20 mM ace ic acid, and 1 mM EDTA) as unning bu e .
DNA was de ec ed by ei he e hidium b omide s aining o chemiluminescen
de ec ion. Fo chemiluminescen de ec ion, annealed DNA p obes we e end-
66 Chap e 2

67
Chap e 3
The E. coli Fe/S clus e biogenesis
egula o , IscR – s uc u al ine- uning o
DNA disc imina ion
68 Chap e 3
3.1. Summa y
The ubiqui ous i on-sul u (Fe/S) clus e -con aining p o eins a e in ol ed in
coun less biological ou es and play c ucial oles o he unc ioning o bo h
p oka yo ic and euka yo ic cells. The ansc ip ion ac o IscR was i s implica ed
in he ep ession o he ISC (I on Sul u Clus e ) biogenesis pa hway and was
shown o ha bo a [2Fe–2S] clus e . P omo e s con olled by IscR belong o wo
dis inc sequence g oups and he [2Fe-2S] clus e o IscR was shown o be
essen ial o egula ion o ype-1 p omo e s (isc, yadR, yhgI), while apo-IscR is
esponsible o he egula ion o ype-2 p omo e s (su A, hyaA, hybO, napF, ydiU).
IscR egula ion o he ISC machine y elies on a eedback mechanism ine- uned
by he cellula Fe/S clus e s a us, while i ac i a es he SUF pa hway o
compensa e o oxida i e damage o Fe/S-con aining p o eins. Despi e ecen
ad ances in unde s anding he egula ion o isc and su ope ons by en i onmen al
signals, he ea u es o IscR modula ing sequence disc imina ion and he s uc u al
changes associa ed wi h DNA binding speci ici y upon liga ion o he [2Fe-2S]
clus e emain poo ly unde s ood. He e we epo a de ailed s uc u al and
biochemical cha ac e iza ion o IscR sugges ing ha un a o able in e ac ions wi h
Glu43 impai ecogni ion o ype-1 p omo e s by apo-IscR. Ou indings sugges
ha binding o a Fe/S co ac o leads o he eshaping o he DNA-IscR in e ace o
speci ic ecogni ion o ype-1 si es.
69
3.2. In oduc ion
I on-sul u (Fe/S) p o eins a e widely dis ibu ed in Na u e and a e key
componen s in se e al physiological p ocesses including espi a ion,
pho osyn hesis, DNA epai , me abolism and egula ion o gene exp ession (6,
208). Relying on hei chemical e sa ili y, Fe/S p o eins can se e as egula o y
senso s o di e se small molecules (36). FNR is one o he bes cha ac e ized
Fe/S-con aining egula o s, wi h an ac i a ion mechanism ans e sal o he
majo i y o me allo egula o s: he swi ch o a ansc ip ionally p oduc i e mode
equi es binding o a speci ic me al (209, 40). In con as , he ansc ip ional
egula o IscR has wo ac i e ansc ip ional o ms: he clus e less (apo-IscR) and
he [2Fe-2S]-bound o ms ha exhibi al e ed DNA ecogni ion speci ici y and
he eby con ol di e en se s o genes acco ding o en i onmen al condi ions (146,
147, 144).
IscR belongs o he R 2 amily o egula o s, displaying he ypical winged
helix– u n–helix mo i close o he N- e minus and a C- e minal Fe/S clus e
coo dina ed by h ee cys eine and one his idine ligands (143). I is now known ha
he exp ession o ~40 genes is unde he di ec o indi ec con ol o IscR, which is
he key egula o o Fe/S biogenesis: he [2Fe-2S]- o m o IscR ep esses he
ansc ip ion o he isc ope on encoding he ISC Fe/S biogenesis pa hway and,
unde s ess condi ions, he apo- o m ac i a es he exp ession o he su ope on
(147, 142, 145). In E.coli, Fe/S homeos asis is main ained h ough a nega i e
eedback loop based on he abili y o [2Fe-2S]-IscR o sense he cellula demand
o Fe/S clus e s and ac i ely egula e he ansc ip ion o he iscRSUA–hscBA– dx
ope on (146). Thus, due o i s in insic senso p ope ies, IscR adjus s he
syn hesis o p o eins in ol ed in he Fe/S clus e biogenesis pa hway and allows
E. coli o cope wi h a ying Fe/S clus e equi emen s.
Two dis inc p omo e sequences we e compiled om IscR- egula ed
genes (147), e med ype-1 and ype-2 DNA-binding mo i s, and whose ecogni ion
by IscR is dependen o i s clus e occupancy (144). Type-1 p omo e s a e
ecognized exclusi ely by [2Fe-2S]-IscR, whe eas bo h [2Fe-2S]-IscR and apo-
IscR in e ac simila ly wi h ype-2 sequences, sugges ing ha he Fe/S clus e is
dispensable o ecogni ion o he la e mo i s (144). Gi en ha he s uc u e o
70 Chap e 3
IscR is p edic ed o ha bo a single wHTH DNA-binding domain, i is unexpec ed
ha i can in e ac and dis inguish wo DNA binding si es. To un eil he ea u es
unde lying p omo e disc imina ion by IscR, he h ee-dimensional s uc u e o
apo-IscR
Ec
in complex wi h a ype-2 a ge sequence he hya p omo e om he
hyd ogenase-1 ope on, was sol ed. This expe imen al model combined wi h
biochemical s udies e ealed some o he molecula de ails in ol ed in he unusual
en i onmen ally modula ed ecogni ion o wo dis inc p omo e consensus
sequences by IscR, using a single p edic ed helix– u n–helix DNA binding mo i .
Ou s udies highligh he ole o he Glu43 esidue on DNA sequence
disc imina ion by IscR and how s uc u al changes aking place upon liga ion o a
[2Fe-2S] clus e con ibu e o b oaden DNA speci ici y o IscR o include ype-1
p omo e s.
71
3.3. Resul s
Apo-IscR
Ec
induces s uc u al changes on ype-2 binding mo i s
I emains unclea how clus e liga ion enables IscR o ecognize wo e y
dis inc DNA mo i s (Fig. 7) using a single wHTH domain (146, 144). To add ess
his ques ion, DNA-binding assays using an IscR a ian (apo-IscR
Ec
) wi h he
pu a i e clus e -coo dina ing cys eine esidues mu a ed o se ines (C92/98/104S)
we e pe o med. Conse a i e eplacemen o he cys eine ligands ensu es a
minimal s uc u al and unc ional impac , as well as a homogeneous apo-p o ein
p epa a ion lacking he Fe/S clus e . The CD spec a ob ained o he hya binding
sequence (hyaEc, Table 3) display he cha ac e is ic ea u es o B- o m DNA wi h
a nega i e peak a ≈240nm and a posi i e peak cen e ed a ≈277nm (blue line,
Fig. 9A) (210). Con o ma ional changes in he DNA can be moni o ed wi hin his
egion because he e is no ellip ici y om he in insic Co on e ec o he p o ein
(211). A la ge inc ease in he magni ude o he CD signal (≈85% inc ease in he
∆ε alue a sa u a ion) wi h a sligh wa eleng h shi o 272 nm ollowing sequen ial
addi ions o apo-IscR
Ec
indica i e o p oduc i e binding o apo-IscR
Ec
o he hya
binding si e. Al hough he shi blue shi o he signal maximum is no eadily
in e p e ed, i can be a consequence o , o example, DNA unwinding induced by
p o ein binding (212). Bo h DNA and p o ein concen a ions we e kep well abo e
he p e iously de e mined appa en K
d
o apo-IscR
Ec
binding o he hya sequence
(≈44±3 nM) o ensu e ha binding would occu in a 1:1 a io and allow
s oichiome ic de e mina ion (144). I is clea ha he ellip ici y changes (∆∆ε)
inc ease o a pla eau a app oxima ely 2:1 mola a io o apo-IscR
Ec
:hya (Fig. 9B).
Since E. coli IscR was shown o be a dime in solu ion, his means ha each hya
duplex is ecognized by one dime o apo-IscR
Ec
. This obse a ion is in con as
wi h p e ious esul s indica ing ha wo dime s o E. coli IscR (bo h wild- ype IscR
and IscR-C92/98/104A)
bind o one hya si e (144).
DNA bending is an impo an s uc u al ea u e o base ead-ou in p o ein-
DNA in e ac ions and e en small dis o ions can ine- une sequence ecogni ion by
a o ing he o ma ion o speci ic con ac s (213). To u he in es iga e he ea u es
modula ing DNA ecogni ion by IscR, we pe o med a DNA bending assay using
he pBend5 ec o ha con ains duplica e se s o es ic ion si es (214). This assay

72 Chap e 3
allows an empi ical es ima ion o he magni ude o bending induced by a speci ic
DNA-binding p o ein, such as IscR. Following he hya p omo e (hya_bend, Table
3) cloning in o he pBend5 ec o , a se o DNA agmen s wi h iden ical sizes bu
displaying dis inc ela i e posi ions o he p o ein-binding si e we e gene a ed by
diges ion (Fig. 9C). The elec opho e ic mobili y o ben DNA is dependen upon
he loca ion o he bend; mig a ion delay is g ea es when he DNA is ben a i s
cen e and smalles when nea i s end (215). Gel e a da ion analysis clea ly
shows ha he agmen wi h he mos cen ally posi ioned hya binding sequence
(EcoRV, Fig. 9C, 9D) has minimum mobili y and ha mobili y inc eases as he
binding si e is placed nea e he ends o he agmen (BamHI and MluI, Fig. 9C,
9D). F agmen s ob ained a e diges ion wi h BamHI and MluI ha e i ually
iden ical mobili y in he absence o apo-IscR
Ec
, sugges ing ha he hya si e did no
con ain signi ican in insic DNA bending p io o complex o ma ion. Taking in o
conside a ion he mobili y o each hya agmen in complex wi h apo-IscR
Ec
, i was
es ima ed ha he hya sequence is dis o ed app oxima ely 24º±2º om linea i y
(196). Howe e , his analysis is in luenced no only by p o ein-induced bends, bu
also by o he dis o ions in DNA s uc u e as a consequence o p o ein shape o
DNA lexibili y (212). Thus, he alue ob ained can e lec mo e han one ype o
DNA s uc u al change and is be e desc ibed as DNA lexu e angle.
Ne e heless, hese esul s sugges ha binding o one apo-IscR
Ec
dime o he
hya p omo e sequence is accompanied by s uc u al de o ma ions, hough hei
na u e could no be dis inguished by he echniques used.
73
Fig. 9 - Apo-IscR
Ec
binding o ype-2 p omo e sequences. (A, B) Apo-IscR
Ec
binds as a dime
and induces s uc u al de o ma ions in he hya p omo e sequence. A - CD spec a ollowing apo-
IscR
Ec
binding o he hyaEc sequence (hyaEc, Table 3). Scans we e aken om 230 o 360 nm
ollowing sequen ial addi ions o apo-IscR
Ec
(235 µM). Rep esen a i e spec a co esponding o
p o ein:DNA a ios o 0, 0.34, 0.68, 1.02, 1.37, 1.71, 2.05, 2.39, 2.73, 3.07 3.42, 3.76, 4.10 a e
shown. Expe imen s we e ca ied ou in 40mM T is-HCl pH 7.5, 150mM KCl, 1mM DTT, 5%
glyce ol ( ol/ ol). Inse shows an elec opho e ic mobili y shi assay assessing complex o ma ion
immedia ely a e he CD i a ion. The band-shi co esponding o he apo-IscR
Ec
:hya complex is
deno ed by an a ow. (B) The change in he CD spec um o he hyaEc sequence ollowing
sequen ial addi ions o apo-IscR
Ec
is indica i e o a 2:1 apo-IscR
Ec
:hya complex. Values o ∆ε we e
aken a he peak maxima wa eleng h o he CD spec a (272 nm) shown in A. (C-E) The hya
binding si e is ben by apo-IscR
Ec
. C - Schema ic ep esen a ion o he hya si e (hya_bend, Table 3)
inse ed be ween he MluI–BamHI es ic ion si es o he pBend5 ec o . The egion delimi ed by
he MluI–BamHI ecogni ion sequences con ains es ic ion si es in duplica e o gene a e DNA
agmen s ha a e iden ical in leng h bu con ain he p o ein-binding sequence (black ec angle,
hyaEc) a di e en posi ions. D - Gel elec opho esis o pe mu ed agmen s con aining he hyaEc
ecogni ion si e. Apo-IscR
Ec
p o ein was mixed sepa a ely wi h 6 di e en agmen s. The DNA
agmen s used we e gene a ed by diges ion es ic ion enzymes, which, om le o igh , a e MluI,
BglII, XhoI, EcoRV, S uI, and BamHI. 100-bp DNA ladde s we e used o compa ison and he
74 Chap e 3
250bp ladde band is indica ed. E - Calcula ion o he lexu e angle o he hyaEc sequence induced
by apo-IscR
Ec
. The mobili y o he apo-IscR
Ec
:hya complexes (R
bound
) was no malized o he
mobili y o he co esponding ee p obe (R
ee
). Flexu e displacemen co esponds o he a io
be ween he dis ance om he 5’ end o each agmen o he cen e o he hyaEc binding si e and
he ull size o he p obe. The plo ed poin s we e i ed wi h a quad a ic unc ion: y = 0.1445x
2
-
0.1450x + 0.6941 (
2
= 0.983). The i s and second o de pa ame e s o he equa ion a e in close
ag eemen and es ima e a hyaEc lexu e angle o 24°±2.
S uc u e o apo-IscR
Ec
bound o a ype-2 p omo e
In o de o be e g asp he ine molecula de ails o speci ic p omo e
sequences ecogni ion by IscR, he s uc u e o apo-IscR
Ec
in complex wi h one o
i s ype-2 a ge sequences, he hya p omo e , was de e mined using
o ho o hombic (P
444
) c ys als di ac ing o 2,5 Å (Table 4). The asymme ic uni
con ained he apo-IscR
Ec
biological dime bound o a 26-base double s anded
oligonucleo ide wi h a single nucleo ide o e hang a he 5’-end o each s and
(hya_26_OH, Table 3, Fig. 10A). This s uc u e is highly simila o he ecen ly
epo ed model o apo-IscR
Ec
(C92/98/104A mu an ) in complex wi h DNA (PDB
en y 4HF1; (216)), supe posing wi h a .m.s.d. o 0.5 Å o 124 aligned Cα a oms.
The apo-IscR
Ec
monome is mos ly α-helical and encompasses wo cen al
s uc u al ea u es: a DNA-binding domain and a dime iza ion helix (Fig. 10B,
10C). Residues 88 o 103, encompassing pa o he pu a i e i on-sul u clus e -
binding egion (Fig. 10B), a e diso de ed in bo h monome s and could no be
modeled (Fig. 10A-C). The s uc u es o he monome s a e nea ly iden ical,
supe posing wi h an .m.s.d. o 1,2 Å o 115 aligned Cα a oms. The cha ac e is ic
wHTH domain o R 2 egula o s in ol ed in DNA- ecogni ion is o med by helices
α2, α3, β-s ands β1 and β2 and he wing (Fig. 10B, 10C). The leng h o he
dime iza ion helix allows o he placemen o each DNA-binding domain a he
ex emes o he dime , as well as o he DNA- ecogni ion helix (α3) o each
monome o speci ically ecognize he majo g oo e and he wing o in e ac wi h
adjacen mino g oo es (Fig. 10A). Such s uc u al a chi ec u e accoun s o he
somehow unexpec ed abili y o IscR o ecognize DNA sequences ha a e
ela i ely long impe ec palind omes (Fig. 7) (147).
75
Fig. 10 – The 3D s uc u e o apo-IscR
Ec
bound o he hya p omo e . (A) In e ac ions a he
DNA- ecogni ion in e ace. One o he monome s is colo ed om N- (blue) o C- e minal ( ed).
Residues making con ac s wi h he DNA a e highligh ed as s icks and basic esidues as sphe es.
Hyd ogen bonds be ween apo-IscR
Ec
and DNA a e ep esen ed as do ed lines. (B) Ribbon
ep esen a ion o he apo-IscR
Ec
monome . (C) Topology diag am o he apo-IscR
Ec
monome .
Seconda y s uc u e elemen colo s ma ch hose on B.
Apo-IscR
Ec
dime o ma ion in ol es mos ly in e ac ions be ween esidues
om helix α5 o one monome and helix α6 o he neighbo ing subuni , bu
esidues om helices α1 and α2 also con ibu e o s abilize he homodime (Fig.
11). O e all, 42 amino acids om monome A and 40 amino acids om monome
B a e ound a he in e -subuni in e ace. Each monome bu ies ~17% (1692 Å
2
)
o i s o al sol en accessible su ace. The main chain o A g116 c osslinks he
dime iza ion helix α5 o one monome wi h he C- e minal helix α6 o he opposing
subuni (Fig. 11). A g116 om one monome o ms a sal b idge wi h Glu128 OE1
and OE2, whe eas A g116 om he adjacen monome hyd ogen bonds o Asn132
OD1 and he main chain oxygens o Gln133 and Glu134. The in e -monome
associa ion o helices α5 and α6 is u he achie ed by an in e ac ion be ween he
82 Chap e 3
Fig. 15 – In e ac ion o apo-IscR
Ec
E43A wi h ype-1 and ype-2 binding si es. (A) Glu43 is
ully conse ed among IscR p o eins om G am-nega i e bac e ia. Amino acid sequence alignmen
o ep esen a i e IscR p o eins om G am-nega i e bac e ia: Esche ichia coli (P08AGK), E winia
ch ysan hemi (E0SAX8), Ye sinia pes is (Q0WD07), Shigella lexne i (P0AGL1), Azo obac e
inelandii (O69219). The Glu43 esidue is highligh ed wi h a ed iangle, o he s ic ly conse ed
amino acids a e colo ed in ed and ecogni ion helix α‐‐is unde lined. Inc easing esidue
conse a ion is ep esen ed by a colo g adien om g een o ed. Alignmen p epa ed wi h
Clus alW (161) and colo ed wi h Aline (162). (B) Apo-IscR
Ec
E43A in e ac s simila ly wi h ype-2
hyaEc and ype-1 iscbEc sequences. Cu es co espond o i a ion o he hyaEc and iscbEc
sequences (Table 3) wi h ei he apo-IscR
Ec
( iangles) o apo-IscR
Ec
E43A (squa es). Values o ∆ε
we e aken a 272 nm o sequen ial addi ions o p o ein.
Gi en he speci ic biden a e in e ac ion o he Glu43 esidue wi h ype-2
sequences, he ac ha subs i u ion o his amino acid by an alanine had li le
e ec on he DNA-binding ac i i y o IscR was unexpec ed. One easonable
explana ion is ha elimina ion o he Glu43 side chain in luences speci ici y, a he
han binding a ini y. Indeed, he apo-IscR
Ec
E43A bound o he hya si e wi h
simila a ini y as he wild- ype p o ein (216). Howe e , i did no disc imina e
be ween nucleo ide bases a posi ions 6 o 7, since cold sequences mu a ed a he
C6-C7 posi ions (Table 3) we e able o compe e wi h he hya sequence o binding

83
o his a ian bu no o he apo-IscR
Ec
p o ein (Fig. 16B). The e o e, subs i u ion
o Glu43 o an alanine does no pe u b subs an ially he ecogni ion o he hya
p omo e no he s oichiome y o he complex. Du ing he cou se o his wo k, he
s uc u e o he IscR
Ec
E43A a ian bound o he hya sequence
(C92/C98/C104/E43A, PDB en y 4HF2, (216)) was sol ed and, despi e losing
speci ici y o he symme ical CC dinucleo ide, his a ian was o he wise simila ly
bound o he hya si e and e ained all o he speci ic in e ac ions (216).
Fig. 16 - Glu43 ole in he DNA sequence disc imina ion by E. coli IscR. (A) Recogni ion o he
hya p omo e by IscR is una ec ed by elimina ion o he Glu43 side chain. Compe i ion assay using
a 100x old mola excess o cold compe i o s, ei he speci ic (cold hyaEc) o non-speci ic (cold
andom sequence, RS). (B) Apo-IscR
Ec
E43A looses disc imina ion agains he well-conse ed CC
dinucleo ide in ype-2 binding si es. Cold hyaEc sequences mu a ed a ei he he C6 (C6G, C6A o
C6T, Table 3) o he C7 (C7G, C7A o C7T, Table 3) bases compe e signi ican ly wi h he wild ype
hyaEc sequence o binding o apo-IscR
Ec
E43A (le panel), whe eas binding o apo-IscR
Ec
is
mos ly una ec ed by he p esence o cold compe i o s ( igh panel). (C) Type-2 CC dinucleo ide
(g een) is eplaced by a TT dinucleo ide ( ed) in ype-1 binding si es. Conse ed nucleo ides a e
colo ed in black. DNA sequence alignmen was pe o med wi h Clus alW (161) and colo ed wi h
Aline (162). (D) The single E43A mu a ion allows apo-IscR
Ec
o speci ically ecognize ype-1 mo i s.
84 Chap e 3
Compe i ion assay using a 100x old mola excess o cold compe i o s, ei he speci ic (cold iscbEc)
o unspeci ic (cold RS, andom sequence). DNA sequences used in his g oup o expe imen s a e
lis ed in Table 3.
Glu43 disc imina es agains Type-1 p omo e s
Unlike ype-2 sequences, in ype-1 p omo e s he C6-C7 bases a e
eplaced by a TT dinucleo ide (Fig. 16C). Gi en ha apo-IscR ecognizes ype-2
bu no ype-1 p omo e sequences, a possible inhibi o y in e ac ion o Glu43 wi h
he T6-T7 bases ha could somehow p e en ecogni ion o ype-1 p omo e s by
apo-IscR was p oposed (147). Indeed, Apo-IscR
Ec
E43A speci ically ecognized
he ype-1 iscbEc sequence (Table 3, Fig. 16D). These esul s we e u he
subs an ia ed by mic oscale he mopho esis measu emen s ha yielded a K
d
o
154 nM o Apo-IscR
Ec
E43A binding o he iscbEc sequence, a alue ha is in
close ag eemen wi h he K
d
ob ained o he hyaEc sequence (Table 3, Table 5)
and is indica i e ha he Glu43 esidue does no con ibu e subs an ially o he
binding ene gy o ei he ype o si e. In line wi h p e ious esul s linking he Fe/S
clus e o IscR o he ecogni ion o ype-1 p omo e s (147, 159, 142), apo-IscR
Ec
was unable o ecognize he iscb sequence (Fig. 16D).
In summa y, hese indings sugges ha elimina ion o he Glu43 side chain
p obably li s he un a o able in e ac ions wi h sequences con aining hymine a
posi ions 6 and 7, due o lack o sui able hyd ogen-bond dono s. Hence, Glu43
esidue is a c ucial selec i i y il e ha nega i ely a ec s binding o E. coli apo-
IscR o ype-1 binding mo i s, he eby linking speci ic ecogni ion o hese
sequences by E. coli IscR o con o ma ional ea angemen s ollowing binding o
he [2Fe-2S] clus e . These esul s a e subs an ia ed by he indings o o he
esea ch g oup (216) and demons a ed he key ole o Glu43 in sequence
disc imina ion.
85
3.4. Discussion
In E. coli, IscR is a mas e ansc ip ional egula o con olling he
exp ession o sys ems dedica ed o he biogenesis o Fe/S clus e s, as well as he
O
2
-dependen exp ession o se e al Fe/S-con aining p o eins (147). Acco ding o
i s clus e occupancy, which is modula ed by O
2
a ailabili y in i o, IscR con ols
he exp ession o wo se s o a ge p omo e s con aining dis inc DNA mo i s (146,
144). The biochemical and s uc u al analysis o IscR a ian s epo ed he e
un eils de e minan ea u es unde lying DNA ecogni ion ha explain how clus e
liga ion enables IscR o speci ically disc imina e be ween wo ypes o binding si es
h ough a single DNA-binding domain, an abili y undesc ibed o any o he
ansc ip ion ac o .
The s uc u e o he apo-IscR
Ec
:hya complex esembles hose desc ibed o
o he egula o s o he R 2 amily, con aining a wHTH DNA-binding domain ha
g ea ly con ibu es o speci ic ecogni ion o he ype-2 binding si e (169, 163).
Binding o he hya sequence is accompanied by mild s uc u al changes and
induces a ela i ely small DNA bend, sugges ing ha apo-IscR is na u ally shaped
o ecognize ype-2 DNA mo i s. The wing esidue A g59 in e ac s ex ensi ely wi h
he AT- ich mino g oo e egion and p o ides an ancho ing poin o IscR’s
accu a e placemen and concomi an DNA sequence ead-ou . Speci ic binding is
s eng hened by in e ac ions be ween majo g oo e cy osines (C6C7 and C7’),
pu ines (A19 and G20’) and hymines (T18 and T19’) and he side chains o
Glu43, Se 40 and Gln44, espec i ely. The con ac be ween Glu43 and wo
consecu i e bases is somewha s iking because biden a e in e ac ions a e
no mally es ic ed o single base posi ions (223). Despi e he biden a e con ac
be ween he highly conse ed Glu43 esidue and he C6C7 and C7´A8´
dinucleo ides in each hya hal -si e, his in e ac ion con ibu es less han expec ed
o he o e all complex a ini y. Thus, he in e ac ion be ween Glu43 and he CC
dinucleo ide poses as a cen al ea u e in he DNA-p o ein in e ace esponsible o
si e-speci ic in e ac ion o apo-IscR
Ec
wi h ype-2 sequences.
While in ol ed in speci ic ecogni ion o ype-2 mo i s, he Glu43 esidue is
used o disc imina e agains ype-1 sequences. A physiological pH, his esidue is
expec ed o ha e hyd ogen accep o g oups bu no dono g oups, and he e o e
86 Chap e 3
can only o m hyd ogen bonds wi h cy osine and adenine bases (224). Lack o
ene ge ically a ou able in e ac ions be ween Glu43 and he conse ed T6T7
dinucleo ide in one hal -si e o ype-1 si es may nega i ely a ec he placemen o
he ecogni ion helix wi hin he DNA majo g oo e, he eby limi ing p oduc i e
binding o ype-1 mo i s. Consis en wi h his hypo hesis, speci ic in e ac ion o
apo-IscR wi h he ype-1 isc p omo e sequence was possible h ough he
eplacemen o he Glu43 esidue by an apola and smalle esidue, such as
alanine. Consequen ly, one can specula e ha a leas pa o he ole o Fe/S
clus e binding is he emo al o he Glu43 nega i e e ec on high-a ini y binding
o ype-1 p omo e s.
By using a s uc u e-guided mu agenesis app oach, a key sequence
disc imina o y ole o Glu43 could be un eiled. Reloca ion o his esidue upon
clus e binding is c ucial o ype-1 p omo e s speci ic ecogni ion, sugges ing ha
e e sible Fe/S clus e liga ion enables IscR o ac as a senso o Fe/S clus e
homeos asis h ough a swi ch in i s a ge -si e speci ici y.
87
.
Chap e 4
The unique egula ion o Fe/S clus e
biogenesis in a
G am-posi i e bac e ium

88 Chap e 4
4.1. Summa y
I on-sul u clus e s unc ion as co- ac o s o a wide ange o p o eins, wi h
di e se molecula oles in bo h p oka yo ic and euka yo ic cells. Dedica ed
machine ies assemble he clus e s and deli e hem o he inal accep o
molecules in a igh ly egula ed p ocess. In he p o o ypical G am-nega i e
bac e ium E. coli, he wo exis ing i on-sul u clus e assembly sys ems, ISC and
SUF, a e closely in e connec ed. The ISC pa hway egula o , IscR, is a
ansc ip ion ac o o he helix- u n-helix ype ha can coo dina e a [2Fe-2S]
clus e . Redox condi ions and i on o sul u a ailabili y modula e he liga ion s a us
o he labile IscR clus e , which in u n de e mines a swi ch in DNA sequence
speci ici y o he egula o : clus e -con aining IscR can bind o a amily o gene
p omo e s ( ype-1), while he clus e -less o m only ecognizes a second g oup o
sequences ( ype-2).
Howe e , i on-sul u clus e biogenesis in G am-posi i e bac e ia is no so
well cha ac e ized, and mos o ganisms o his g oup display only one o he i on-
sul u clus e assembly sys ems. A no able excep ion is he unique G am-posi i e
dissimila o y me al educing bac e ium The mincola po ens, whe e genes om
bo h sys ems could be iden i ied, albei wi h a di e ging o ganiza ion om ha o
G am-nega i e bac e ia. We demons a ed ha one o hese genes encodes a
unc ional IscR homologue, and is likely in ol ed in he egula ion o i on-sul u
clus e biogenesis in T. po ens. S uc u al and biochemical cha ac e iza ion o T.
po ens and E. coli IscR e ealed a s ikingly simila a chi ec u e and un eiled an
un o eseen conse a ion o he unique mechanism o sequence disc imina ion
cha ac e is ic o his dis inc i e g oup o ansc ip ion egula o s.
89
4.2. In oduc ion
I on-sul u (Fe/S) p o eins play c ucial oles o he unc ioning o bo h
p oka yo ic and euka yo ic cells, being equi ed o biological unc ions anging
om elec on anspo o edox and non- edox ca alysis, and om DNA syn hesis
and epai o sensing in egula o y p ocesses (7). The main ole o he Fe/S clus e
assembly machine ies is o mobilize i on and sul u a oms om hei s o age
sou ces, assemble he wo componen s in o a Fe/S clus e , and hen ans e he
newly o med clus e o he inal p o ein accep o s (73). In Esche ichia coli, he e
a e wo o hese Fe/S clus e ‘‘ ac o ies’’, he ISC (I on Sul u Clus e ) and SUF
(SUlFu assimila ion) sys ems whose co esponding genes a e o ganized in wo
ope ons, iscSUA-hscBA- dx and su ABCDSE, espec i ely (73, 225). Dele ion
mu an s o he ISC sys em display a a ie y o g ow h de ec s due o loss o Fe/S
clus e -con aining enzyme ac i i y and dis up ion o sul u me abolism, whe eas
ailu e o bo h he ISC and SUF sys ems leads o syn he ic le hali y (226, 101).
In E. coli, he ISC machine y is conside ed he housekeeping sys em
esponsible o he ma u a ion o a la ge a ie y o Fe/S p o eins, whe eas he SUF
sys em is igge ed unde s ess condi ions, such as oxida i e s ess o i on
s a a ion (85). IscR is a [2Fe-2S] clus e -con aining ansc ip ion ac o wi h a
single p edic ed helix– u n–helix mo i , i s iden i ied o i s ole in egula ing
exp ession o he ISC biogenesis pa hway (142) and subsequen ly ound o con ol
he exp ession o mo e han 40 genes in E. coli (147, 142). Acco ding o he
cu en ly accep ed model o Fe/S clus e biogenesis, unde condi ions
un a o able o Fe/S clus e o ma ion he labile IscR clus e is los and IscR-
media ed ep ession o he isc ope on is alle ia ed. A he same ime, apo-IscR
ac i a es he SUF ope on o u he compensa e o damage o loss o Fe/S
clus e s (154, 145). Once he demand o Fe/S biogenesis is me , highe le els o
clus e -con aining holo-IscR exis , causing an inc eased ep ession o he ISC
pa hway. Mo eo e , unde i on limi a ion he ISC and SUF machine ies a e unable
o main ain he le els o holo-IscR and he e o e his eedback mechanism allows
IscR o sense Fe/S demand and enables E. coli o espond app op ia ely o s ess
condi ions (146).
The e a e wo classes o IscR binding si es in he E. coli genome: a ype-1
90 Chap e 4
si e deduced om iscR, yadR, and yhgI p omo e egions, and a ype-2 si e
compiled om he IscR si es ups eam o he hyaA, ydiU, and su A p omo e s
(147). In e es ingly, IscR binds ype-1 p omo e s solely in i s holo- o m, while
binding o ype-2 p omo e s was shown o be independen o he p esence o he
Fe/S clus e (144). In E. coli, IscR mu a ion E43A enabled speci ic ecogni ion o
ype-1 p omo e s by apo-IscR, likely mimicking he in e ac ion mode o he clus e -
bound o m o he p o ein (see Chap e 3, (216)).
Al hough a molecula le el unde s anding o he complex p ocesses o Fe/S
clus e biosyn hesis in se e al o ganisms is now eme ging om he combina ion o
in i o and in i o app oaches, hese machine ies a e s ill poo ly unde s ood in
G am-posi i e bac e ia. While homologs o he E. coli ISC o SUF sys ems a e
p esen in se e al o ganisms, some species exhibi unusual Fe/S clus e
biosyn he ic machine ies. Mos G am-posi i e bac e ia ca y only a su ope on,
con aining genes coding o Su U and he Su BCD complex (227, 138), bu no
su E o su A- ela ed genes, e en i in some cases su A can be ound elsewhe e in
he genome (139, 138).
The mincola po ens (s ain JR) is an anae obic, he mophilic, G am-posi i e
dissimila o y me al educing bac e ium (DMRB), isola ed om a he mophilic
mic obial uel cell (MFC) (228). I is o he i s G am-posi i e DMRB o which
he e is a comple e genome sequence, which e ealed an unusual abundance o
mul iheme c- ype cy och omes (228, 229). Using homology sea ches, we iden i ied
a se ies o genes wi h sequence simila i y o bo h E. coli SUF and ISC
machine ies in he T. po ens genome, including a gene locus coding o a pu a i e
IscR p o ein. Taken oge he , ou esul s bo h iden i y and cha ac e ize a unique
Fe/S biogenesis egula o in G am-posi i e bac e ia. Th ough s uc u al and
biochemical analysis o bo h T. po ens and E. coli apo-IscR p o eins and hei
E43A mu an s, we we e able o un eil sub le s uc u al ea u es impo an o DNA
ecogni ion and binding speci ici y.
91
4.3. Resul s
Unique Fe/S clus e biogenesis in The mincola po ens
In G am-posi i e bac e ia he e is conse a ion o he su ope on, o en
p esen as su CDSUB, which is he only machine y o Fe/S clus e biosyn hesis in
he majo i y o hese o ganisms (136, 138). Su p isingly, homology sea ches on
he G am-posi i e DMRB T. po ens JR genome (228) allowed iden i ying wo gene
loci wi h sequence simila i y o E. coli SUF and ISC machine ies (Fig. 17A). In T.
po ens, he e a e ORFs coding o homologues o he ansc ip ion ac o IscR
(The JR_1914, 37% iden ical o he E. coli p o ein (142)), he cys eine desul u ase
IscS (The JR_1913 (226, 88)) and he sca old IscU (The JR_1912 (230)) om he
ISC pa hway. An addi ional su -like ope on in T. po ens comp ises homologues o
su C (The JR_0923), su B and su D (The JR_0924) om he E. coli SUF pa hway,
and hcsA (The JR_0925) and hcsB (The JR_0926) om he E. coli isc ope on
(104, 105, 96).
When compa ed o o he G am-posi i e bac e ia, namely om he
Fi micu es phylum, some unique ea u es o he T. po ens su ope on become
e iden . In T. po ens, he su ope on does no code o cys eine desul u ase (Su S)
homologues, al hough he e a e elsewhe e in he T. po ens genome wo addi ional
genes coding o pu a i e cys eine desul u ases (The JR_0460 and The JR_3003)
homologous o CsdA/Su S, which can unc ion as complemen a y sul u sou ces
o Fe/S clus e biogenesis, possibly h ough he ec ui men o he SUF machine y
(231). The T. po ens su ope on is also de oid o homologues o Su U, ecen ly
epo ed o be a zinc-dependen sul u ans e ase in B. sub ilis (140), bu encodes
a su BD p o ein, which oge he wi h su C was shown o ac as sca old in G am-
nega i e bac e ia (83, 103). Fu he mo e, he su ope on in T. po ens includes he
hscA and hscB genes coding o he chape ones esponsible o ans e ing p e-
o med clus e s om he sca old IscU o inal accep o s and ha , in E. coli, a e co-
ansc ibed wi h he isc and no wi h he su ope on (226). Addi ionally, genes
coding o A- ype ca ie s a e absen om he T. po ens genome.
IscU is a highly conse ed p o ein ha unc ions as sca old o clus e
assembly and subsequen ans e . P ese ed ea u es include he clus e ligands
( h ee cys eines and one his idine), an aspa a e esidue ha plays a c i ical ole in
98 Chap e 4
DNA-binding su ace (Glu33, Asp30 and Glu43; Fig. 13) a e s uc u ally equi alen
o he acidic esidues iden i ied on he equi alen side o apo-IscR
Tp
. Howe e ,
only Glu43 con ac s di ec ly he bound oligonucleo ide. Toge he wi h Gln44
(conse ed) and Se 40 ( a iable), Glu43 is in ol ed in base-speci ic ecogni ion
wi hin he majo g oo e (Fig. 20A) and was shown o speci ically disc imina e
agains ype-1 p omo e sequences in E. coli (see Chap e 3, (216)). O e all, he e
is a s icking conse a ion o he DNA-binding in e ace (Fig. 17D; Fig. 20A). Apo-
IscR
Tp
di e s om he E. coli homologue only a ou posi ions wi hin he
in e ac ion su ace, which could esul in al e ed DNA binding a ini y and
speci ici y: Se 27 (P o27 in apo-IscR
Ec
), P o40 (Se 40 in apo-IscR
Ec
) and Ala61-
Gln62 (P o61-Gly62 in apo-IscR
Ec
). The eplacemen o P o27 by a se ine is likely
o inc ease he lexibili y o he linke be ween he i s wo α-helices, al hough a
la ge change in DNA a ini y is no p edic able. In con as , he subs i u ion o
P o61-Gly62 by an Ala-Gln dipep ide can impac he con o ma ion o he wing β-
hai pin and in e e e wi h he igh packing o his s uc u al elemen wi hin he
mino g oo e. In pa icula , he esidue a posi ion 40 is likely o play a key ole in
sequence-speci ic ecogni ion o DNA (Fig. 20B).
In he IscR
Ec
-DNA complex he p o ein packs e y igh ly wi hin he majo
g oo e, lea ing limi ed space o bulkie esidues (Fig. 20A, 20B). Al hough a
p oline could be accommoda ed a he N- e minus o helix α3 wi hou helical
dis up ion (Fig. 20B), he esul ing s e ic hind ance migh p e en he placemen
and base eadou o he conse ed Glu43-Gln44 and/o con ac s o he esidues
in e ac ing wi h he phospha e backbone (Ty 9, Se 38, Ty 41). Subs i u ion o he
pu ines in e ac ing wi h Se 40 (G20’ and A19) p e en s binding o E. coli apo-IscR
o he hya p omo e sequence, highligh ing he impo ance o his esidue o base-
speci ic ecogni ion (144). Fu he , mu a ion o Se 40 o alanine in E. coli IscR
dec eases binding o he hya p omo e by 90% when compa ed o he wild ype
p o ein, a dec ease ha is sequence-dependen and mo e p onounced o ype-2
si es (216).

99
Fig. 20 – Sequence conse a ion a he IscR-DNA in e ace (A) Biden a e binding o apo-IscR
Ec
(C92/98/104S) o he hya p omo e DNA sequence. In one o he monome s, esidues a e colo ed
acco ding o conse a ion, whe e ed co esponds o posi ions s ic ly conse ed be ween E. coli
and T. po ens IscR. Residues a he DNA-in e ac ing in e ace a e ep esen ed as balls and s icks.
Hyd ogen bonds be ween apo-IscR
Ec
and DNA a e ep esen ed as do ed lines. (B) Apo-IscR
Ec
S40 in e ac s speci ically wi h he hya p omo e and is subs i u ed by a p oline in apo-IscR
Tp
.
S e eoscopic iew o key esidues in ol ed in DNA ecogni ion by apo-IscR
Ec
(magen a) and
co esponding esidues in apo-IscR
Tp
(g een) a e ep esen ed as s icks and colo -coded (ni ogen
blue, oxygen ed).
The in luence o P o40 in apo-IscR
Tp
in e ac ion wi h DNA is e idenced by
i s inabili y o bind he E. coli hya p omo e sequence (Fig. 21A). Replacemen o
P o40 in IscR
Tp
by he s uc u ally equi alen amino acid in E. coli IscR (apo-
IscR
Tp
-P40S) is su icien o allow binding o he he e ologous p omo e (Fig. 21A).
The p esence o a se ine esidue a posi ion 40 is likely o alle ia e he igh
packing o IscR wi hin he majo g oo e o DNA, educing s e ic hind ance and
allowing binding. Acco dingly, he IscR
Tp
-E43A mu an , whe e he sho e alanine
side chain can p o ide oom o posi ional adjus men s o his egion, also
ecognized he hya sequence (Table 3, Fig. 21A) wi h an a ini y compa able o
100 Chap e 4
ha o he E. coli p o ein, as assessed by mic oscale he mopho esis (Table 7).
Taken oge he , hese esul s sugges ha subs i u ion o Se 40 by a p oline in
apo-IscR
Tp
p e en s base ecogni ion h ough s e ic hind ance, an impai men
li ed by in oducing less bulky esidues a ei he posi ion 40 o 43.
Fig. 21 - Binding o IscR o ype-2 p omo e sequences. (A) Elec opho e ic mobili y-shi assay
analysis o apo-IscR binding o he E. coli hya p omo e . A ows deno e obse ed band-shi s. (B)
S e eoscopic iew o he in ica e ne wo k o hyd ogen bonds in apo-IscR
Tp
(one monome o he
unc ional dime is colo ed g een and he o he one blue) cen e ed on he clus e -binding esidue
107 (ligh g ay). The 2Fo − Fc elec on densi y map a ound esidue 107 is ep esen ed as an
o ange mesh. Wa e molecules and s ic ly conse ed esidues in closely ela ed IscR molecules
a e colo ed ed. (C) In apo-IscR
Ec
(C92/98/104S), se ine 104 (ball and s ick) pa icipa es in a
ne wo k o pola in e ac ions wi h neighbo ing esidues (s icks), c oss-linking helices α1, α2, and
α5. The co esponding cys eine esidue in he wild- ype p o ein could be pa o a sensing
mechanism o he p esence o he Fe/S clus e .
Posi ion o he clus e -binding esidues
In con as o p e ious s udies wi h E. coli IscR, whe e all pu a i e clus e -
binding cys eine esidues we e mu a ed o alanine in o de o ob ain homogeneous
clus e -less p o ein (144, 216, 145), in T. po ens IscR he co esponding esidues
101
we e mu a ed o se ine, which is a close s uc u al ma ch. In bo h E. coli IscR and
apo-IscR
Tp
s uc u es, he egion in ol ed in i on-sul u clus e associa ion is
pa ially diso de ed, bu he se ine esidues eplacing Cys107 in apo-IscR
Tp
and
Cys104 in apo-IscR
Ec
a e clea ly isible in he elec on densi y maps (Fig. 21B,
21C). In con as o wha is obse ed o he Cys- o-Ala mu an s uc u e o E. coli
ee apo-IscR whe e he wo isible clus e ligands (Ala104 and His107) a e on he
ou e ace o he longe dime iza ion helix α5 (216), in apo-IscR
Tp
he equi alen
Se 107 is pa o he coil egion p eceding helix α5 and he c ys al s uc u e shows
i pa icipa es in a wa e -media ed ne wo k o hyd ogen bonds connec ing his
s uc u al segmen o helices α1 and α2 (Fig. 21B). In pa icula , he Se 107 side-
chain is hyd ogen-bonded o Th 109 OG1 wi hin each monome . Bo h esidues
also es ablish pola in e ac ions wi h o de ed sol en molecules ha pa icipa e in
a hyd ogen bond ne wo k in e acing he wo monome s o he unc ional dime
and in ol ing he side chains o Gln19, Asp16, and Gln35 om he adjacen
monome ( he las wo esidues s ic ly conse ed ac oss IscR molecules, Fig.
17D). O pa icula ele ance is he in ol emen o Asp16 side chain in a sal
b idge wi h A g34 wi hin he DNA-binding helix- u n-helix mo i . The clus e -binding
segmen is u he s abilized by a pola con ac wi h Gln140 o he adjacen
monome , which also connec s he co esponding helices α1 and α6. Al oge he ,
his pola in e ac ion ne wo k igh ly connec s he clus e -binding segmen a he N-
e minal po ion o helix α5 om one monome wi h he N- e minal helix α1, helix
α6 and helix α2 om he adjacen monome . Pa icula ly, his ne wo k sugges s an
in e connec ion be ween s uc u al changes in he clus e -binding segmen and
unc ional e ec s a he DNA-binding in e ace.
The geome y o he hyd ogen bonds es ablished by he mu a ed Se 107 in
apo-IscR
Tp
, and he o a ional eedom o Th 109, e idenced by he wo disc e e
con o ma ions o i s side-chain in he cu en c ys al s uc u e, a e compa ible wi h
he exis ence o simila hyd ogen bonds in ol ing Cys107 in he clus e - ee wild-
ype IscR. Indeed, he cys eine side chain hiol g oup is a mode a ely good
hyd ogen bond dono , some imes c ucial o p o ein ac i i y and unc ion (240-
243).
In he c ys al s uc u e o he apo-IscR
Ec
-DNA complex, Se 104 (s uc u ally
equi alen o Se 107 in T. po ens IscR) is also well de ined in he elec on densi y
102 Chap e 4
maps. In one o he monome s, Se 104 is pa o helix α5, as p e iously obse ed
(216). Howe e , in he o he monome o he apo-IscR
Ec
dime his esidue
hyd ogen bonds o he conse ed Th 106 (Th 109 in T. po ens IscR), which in u n
engages in a ne wo k o di ec pola con ac s c osslinking he dime iza ion helix α5
o he C- e minus o he adjacen helix α2 (A g34, Gln35) and o helix α1 (Asp16)
(Fig. 21C). This hyd ogen bond ne wo k in ol es di ec in e ac ions be ween he
amino acid side chains, in con as o wha is obse ed in apo-IscR
Tp
, whe e
sol en molecules media e some o he con ac s. In he Cys- o-Ala iple mu an o
E. coli IscR his a angemen o pola con ac s is p ese ed, wi h he expec ed
excep ion o esidue 104, which is he e an alanine (216).
The p edic ed unc ion o esidue 107/104 (in T. po ens and E. coli,
espec i ely) in i on-sul u clus e binding, as well as i s loca ion be ween he
dime iza ion helix o one monome and he i s helix o he helix- u n-helix DNA-
binding mo i o he neighbo ing subuni , sugges a possible ole as a cen al
nanoswi ch, whe eby clus e binding-induced mo emen could igge a global
mo ion in ol ing bo h he dime in e ace and he DNA-binding egion om he
opposi e monome . The esul ings uc u al changes could explain he obse ed
al e a ion in DNA binding speci ici y upon clus e associa ion (216).
A single mu a ion allows apo-IscR
Tp
o ecognize ype-1 p omo e
sequences om T. po ens and E. coli
In E. coli, holo-IscR was shown o in e ac wi h bo h ype-1 and ype-2 DNA
mo i s in a simila manne , while apo-IscR bound solely o ype-2 p omo e
sequences (144). Recen ly, i was also demons a ed ha eplacemen o Glu43
by alanine in E. coli IscR C92/98/104A emo ed un a o able in e ac ions wi h
ype-1 mo i s, allowing ecogni ion o hese p omo e s (216). The T. po ens isc
p omo e egion displays cis- egula o y elemen s simila o hose iden i ied in he
ype-1 E. coli iscRSUA-hscBA- dx, including he -35 hexame and he -10 elemen
sequences (142). In ac , i is possible o delimi a segmen (iscTp_1; Fig. 22A,
Table 3) displaying 48% iden i y o he E. coli isc p omo e sequence and
con aining a -10 elemen and a consensus -35 hexame o he Eσ
70
-binding si e
wi h a 20-bp space egion (216).
103
Fig. 22 - The T. po ens isc p omo e egion con ains wo binding si es o IscR
Tp
. (A)
IscR
binding
si es in ype-1 [T. po ens isc (iscTp) and E. coli isca (iscaEc) and iscb (iscbEc)]
and ype-2 [E. coli
hya
(hyaEc)] p omo e s. Numbe s e e o he mos ups eam base o each
IscR si e ela i e o
he
co esponding s a codon. Conse ed bases be ween he isc
p omo e s a e highligh ed in ed
whe eas
bases conse ed be ween he i e T. po ens isc
p omo e sequences a e shaded black. The
highly
conse ed CC mo i in ype-2 p omo e s is
colo ed g een (12). (B) DNA ecogni ion was assessed
by
elec opho e ic mobili y shi assay
o he complexes o med be ween apo-IscR
Tp
o apo-IscR
Tp
E43A
and
ei he he ull (iscTp_1)
o immed (iscTp_2) isc p omo e sequence. DNA band-shi s a e deno ed
by
a ows.
Simila o wha is obse ed o E. coli apo-IscR and isc, apo-IscR
Tp
does no
bind iscTp_1 (Fig. 22B). Using an enzyma ic sys em unde oxygen-deple ed
a mosphe e (195), a Fe/S clus e could be e e sibly econs i u ed in wild- ype
apo-IscR
Tp
yielding he holo o m o he p o ein, as judged by he appea ance o
an abso p ion maximum a 420 nm (Fig. 23A). A dose-dependen s uc u al
change o iscTp_1 DNA could be iden i ied by ci cula dich oism spec oscopy,
upon holo-IscR
Tp
-w binding (Fig. 23B). These esul s demons a e ha , as
expec ed o a bona ide IscR, he enzyma ically econs i u ed Fe/S clus e -bound
o m o IscR
Tp
-w binds o he T. po ens isc p omo e egion (Fig. 23B). As seen o
E. coli IscR (216), he single poin mu an apo-IscR
Tp
E43A binds speci ically o he
iscTp_1 sequence, seemingly o ming wo dis inc complexes – wi h ei he one o
wo IscR dime s binding o he a ge sequence – as sugges ed by he wo
obse ed DNA band shi s (Fig. 22B). This is u he suppo ed by he obse a ion

104 Chap e 4
o a single complex wi h he 3’- immed iscTp_1 sequence, e med iscTp_2 (Fig.
22A, 22B; Table 3). In E. coli, DNase oo p in ing led o he iden i ica ion o wo
IscR binding si es wi hin he isc p omo e egion, isc a and isc b (147). Two highly
homologous egions could be iden i ied in he T. po ens isc p omo e , iscTp_3 and
iscTp_4 (Fig. 22A, Table 7), o which apo-IscR
Tp
E43A displays speci ic binding
(Fig. 24A, Table 7). Fu he , emo al o he wo 3’-end nucleo ides o iscTp_3,
yielding he sho e iscTp_5 (Fig. 22A, Table 7), e ec i ely p e en s binding o
apo-IscR
Tp
E43A (Fig. 24A, Table 7), in good ag eemen wi h he obse ed
biden a e binding o IscR o he mino g oo e o AT- ich segmen s a he e mini o
i s ecogni ion sequence (Fig. 20A).
Table 7 - Binding a ini ies be ween IscR and ype-1 and ype-2
p omo e sequences de e mined by mic oscale he mopho esis
DNA sequence
IscR a ian
Dissocia ion cons an
K
d
(nM)
hyaEc Apo-IscR
Tp
E43A 340 ± 52
Apo-IscR
Tp
n. d.
Apo-IscR
Tp
P40S 11900 ± 3340
iscbEc Apo-IscR
Tp
E43A 97 ± 6
Apo-IscR
Tp
n. d.
iscTp_3 Apo-IscR
Tp
E43A 320 ± 19
Apo-IscR
Tp
n. d.
iscTp_4 Apo-IscR
Tp
E43A 905 ± 87
(a)
Apo-IscR
Tp
n. d.
iscTp_5 Apo-IscR
Tp
E43A n. d.
n. d. – binding no de ec ed
(a)
The K
d
alue migh be o e es ima ed, since sa u a ion was no eached.
105
Fig. 23 -
T. po ens IscR is a Fe/S-con aining p o ein ha binds he isc p omo e sequence
(A)
Uppe
panel: Time cou se o Fe/S clus e assembly on apo-IscR
Tp
-w . The e is a ime-
dependen inc ease o
he
cha ac e is ic Fe/S clus e abso p ion peak a 420nm o econs i u ed
apo-IscR
Tp
-w (R-IscR
Tp
-w ;
blue
ci cles), while no no iceable a ia ion could be obse ed o he
assay pe o med in he absence
o
cys eine (NR-IscR
Tp
-w , black ci cles). A he end o he assay,
he eac ion con aining
R-IscR
Tp
-w
displayed a cha ac e is ic b own colou ( op cu e e; R), which
was essen ially absen in he
con ol
eac ion (bo om cu e e; NR). Lowe panel: UV/Visible
abso p ion spec a o R-IscR
Tp
-w . The
spec um o
pu i ied R-IscR
Tp
-w (blue cu e) displays local
maxima a 420 nm and 320 nm ha a e
cha ac e is ic o
Fe/S clus e s, which disappea ed upon
educ ion wi h 2 mM di hioni e ( ed cu e). (B) Fe/S
clus e
binding modula es ecogni ion o
ype-1 p omo e DNA sequences by IscR. Ci cula dich oism spec a
o
iscTp_1 DNA sequence
(Table 3) wi h inc easing concen a ions o R-IscR
Tp
-w ( op panel) o
NR-IscR
Tp
-
w
(bo om
panel). The spec a we e eco ded upon successi e addi ions o each pu i ied p o ein o
an
iscTp_1 solu ion (2 µM). A signi ican change in ellip ici y a he cha ac e is ic B-DNA peak a
285
nm
(4) can only be obse ed upon R-IscR
Tp
-w addi ion, indica ing ha only he Fe/S clus e -
con aining
o m o
IscR
Tp
-w is able o ecognize he The mincola po ens isc p omo e sequence
and induce local
DNA
s uc u al changes. Expe imen s we e ca ied ou a 20°C in 40 mM T is
pH 8, 150 mM KCl, 5%
( / )
glyce ol, 1mM
DTT.
In line wi h he conside able conse a ion be ween E. coli and T. po ens
IscR p o eins and isc p omo e sequences, he e is c oss- ecogni ion be ween he
ansc ip ional egula o o T. po ens and he E. coli p omo e . While apo-IscR
Tp
does no bind E. coli isc b (iscbEc, Table 7), his sequence is speci ically
ecognized by he E43A mu an (Fig. 24B; Table 7), as obse ed o E. coli apo-
IscR (216). The e o e, he unique mechanism o p omo e sequence disc imina ion
by IscR seems o be conse ed be ween hese o ganisms.
106 Chap e 4
Fig. 24 - Modula ion o apo-IscR
Tp
speci ici y by a single poin mu a ion. (A) The e a e
wo
independen binding si es o apo-IscR
Tp
E43A in he T. po ens isc p omo e . Pu i ied
apo-IscR
Tp
E43A
(7.5 µM) was incuba ed wi h iscTp_3, iscTp_4 and iscTp_5 sequences,
analyzed by nondena u ing
PAGE,
and isualized by e hidium b omide s aining. An a ow
deno es he DNA band-shi upon
complex
o ma ion. (B) C oss- ecogni ion o he E. coli isc
p omo e by T. po ens IscR. Pu i ied p o eins
(apo-
IscR
Tp
, apo-IscR
Tp
E43A, o apo-IscR
Ec
E43A) we e incuba ed wi h DIG-labeled isc b p omo e
sequence
(iscbEc) (Table 3) and
analyzed by nondena u ing PAGE. An a ow deno es bands indica i e o
DNA–
IscR complex
o ma ion. Whe e indica ed, cold isc b o a simila ly sized andom sequence ( andom)
was
added in 100- old mola excess as
compe i o
.
107
4.4. Discussion
We pe o med a de ailed analysis o he p oduc o gene The JR_1914 om
T. po ens, undoub edly es ablishing i s unc ional ela ionship wi h he Fe/S
clus e -binding ansc ip ion egula o , IscR, known o con ol Fe/S clus e
biogenesis in se e al G am-nega i e bac e ia. The iden i ica ion o an IscR
homologue in T. po ens was unp eceden ed: mos o he G am-posi i e bac e ia
s udied so a do no code o any IscR-like p o eins no ha e an isc ope on, and
he a e cases whe e an isc ope on is p esen (e.g. he DMRB Desul obac e ium
ha niense o he bac e ium Clos idium pe ingens (233)) lack he SUF machine y.
The combina ion o biochemical and s uc u al s udies, on T. po ens IscR
and i s homologue om E. coli, e ealed also an un o eseen conse a ion o he
unique mode o IscR p omo e sequence ecogni ion and disc imina ion. Despi e
ex ensi e conse a ion o he DNA-binding su ace, apo-IscR
Tp
was unable o
ecognize he he e ologous hya p omo e om E. coli. Residue a posi ion 40
played a pi o al ole in his p ocess, since elie o s e ic hind ance (P40S mu an )
was su icien o p omo e binding. These sub le di e ences in speci ici y highligh
he p ecise ailo ing o each IscR molecule o i s cogna e pa ne s, despi e o e all
conse a ion o he ecogni ion mechanism.
Simila o he E. coli molecule (see Chap e 3, (144)), he clus e -less o m
o he p o ein binds o he he e-iden i ied T. po ens su ( ype-2) p omo e , while he
no el ype-1 p omo e (isc) is ecognized by holo-IscR. In he absence o he Fe/S
clus e , he s ic ly conse ed Glu43 esidue is pi o al o disc imina ing be ween
ype-1 and ype-2 p omo e s by es ablishing speci ic in e ac ions wi h an in a ian
CC dinucleo ide in ype-2 sequences. In ac , mu a ion o his esidue o an
uncha ged alanine seems o mimic he clus e -induced speci ici y swi ch o IscR,
allowing he clus e -less egula o o ecognize and o bind o ype-1 p omo e
sequences (216). In apo-IscR
Tp
, he E43A
mu a ion p omo es binding o wo
sequences ups eam o he T. po ens iscRSU ope on. These egions a e highly
homologous o he E. coli isc sequences ecognized by bo h holo- and mu an
apo-IscR E43A. Gi en ha one o hese sequences (iscTp_4) con ains a -35-
elemen sequence, we p opose ha T. po ens holo-IscR may ac as a ep esso o
Fe/S biogenesis by hinde ing RNA polyme ase binding. As a whole, ou esul s
114 Chap e 5
un a o able in e ac ion wi h he TT dinucleo ide has an inhibi o y e ec on ype-1 si es ecogni ion
(b,e). Al hough binding o [2Fe-2S]-IscR o ype-2 sequences is possible in i o, i is p e en ed in
i o by addi ional ansc ip ion ac o s bound a a ge p omo e s (c). Upon clus e binding, a
con o ma ional change li s he un a o able in e ac ions wi h he ype-1 TT dinucleo ide h ough
displacemen o he Glu43 side chain, which is deno ed by a cu ed a ow (d).
In summa y, ou indings un eil an un o eseen conse a ion o co ac o -
based ansc ip ion egula ion mechanism. Unde anae obiosis, s uc u al changes
induced by clus e liga ion allow he speci ic ecogni ion o ype-1 p omo e s by
IscR. Al hough he e is a wide ange o ansc ip ion ac o s whose unc ion
equi es a co ac o , o ou knowledge, IscR is he only ac i e egula o in bo h apo
and holo- o ms whose DNA-binding speci ici y was ound o be modula ed by
co ac o binding.

115
Chap e 6
F
u u e Pe spec i es
116 Chap e 6
Fu u e pe spec i es
The eme gence and sp ead o an ibio ic esis ance in pa hogenic bac e ia is
now a se ious h ea o global public heal h (250). The disco e y o d ugs wi h
no el modes o ac ion will be i al o he eplacemen o cu en d ugs o which
esis ance is widesp ead. Iden i ying a sui able a ge is he i s s ep in a s uc u e-
based d ug design app oach. When choosing a po en ial an imic obial d ug a ge ,
h ee equi emen s mus be con ened: he a ge is i) essen ial o pa hogen
su i al; ii) i s unc ion is es ic ed o he pa hogen; and iii) small molecules can be
used o modi y i s ac i i y (251). Du ing he pas decade, IscR was iden i ied as a
po en ial a ge o no el an imic obial agen s due o i s ole as a global egula o
con ibu ing o he o e all i ulence o se e al human and plan pa hogens (185,
191, 186). Howe e , addi ional expe imen s a e equi ed o assess i non- oxic
an imic obial agen s can modula e IscR ansc ip ional ac i i y. Ne e heless,
s uc u e-based d ug design p o ides an excellen pla o m o he iden i ica ion o
no el an ibac e ial agen s and he s uc u al and biochemical cha ac e iza ion o
IscR desc ibed he ein is o u mos impo ance o pu sue such line o esea ch.
Ou s udies un eil a no el pa adigm on Fe/S clus e biogenesis wi h he
iden i ica ion o he isc and su ope ons in a G am-posi i e bac e ium, T. po ens,
as well as he disco e y o i s unc ional IscR egula o . To gain u he insigh s
in o T. po ens Fe/S clus e biogenesis, mul iple ques ions need o be add essed.
Fi s , i is necessa y o cha ac e ize T. po ens IscR in i o and i s unc ion unde
di e en en i onmen al condi ions, namely oxida i e s ess. The ou come o his
cha ac e iza ion could p o ide expe imen al e idence o he link be ween igh
egula ion o Fe/S clus e biogenesis and he physiology o his bac e ium.
Second, gi en he con o e sy on Su U unc ion in G am-posi i e bac e ia, whe e i
was shown o ac as an enhance o he cys eine desul u ase ac i i y a he han
as a sca old, i would be o in e es o assign he unc ional ole o he
The JR_1912 gene p oduc , whose amino acid sequence is closely ela ed o i s
G am-nega i e bac e ia sca old coun e pa s (139, 227, 140). Thi d, gi en he lack
o a Su S encoding gene, one would expec his unc ion o be compensa ed by
addi ional T. po ens cys eine desul u ases homologous o CsdA/Su S p o eins, bu
117
he in e ac ion be ween hese p o eins and he p edic ed T. po ens Su BCD
sca old p o ein needs o be es ed in i o.
Ul ima ely, he wo k desc ibed in his hesis con ibu es o a deepe
unde s anding o Fe/S clus e biogenesis egula ion by IscR and demons a es he
conse a ion o i s unique mechanism o sequence disc imina ion. Desc ip ion o
ea u es go e ning DNA ecogni ion links he s uc u e o IscR wi h in i o
sequence disc imina ion, which is essen ial o accu a e con ol o a ge
p omo e s acco ding o en i onmen al condi ions. Gi en he ele ance o IscR o
pa hogen i ulence and he p ospec i e use o T. po ens in bio emedia ion and
enewable ene gy p oduc ion, ou indings c ea e exci ing no el a enues o
esea ch wi h po en ial he apeu ic and indus ial applica ions.
118 Chap e 6
119
A
ppendix

120 Appendix
Table A. 1 – Fo wa d and e e se p ime s used o cons uc gene a ion
P o ein Vec o
Gene
Acession
Numbe
Res ic ion
si es
Fo wa d
p ime
(5′→3′)
Re e se
p ime
(5′→3′)
IscR
Ec
-w pETZ2_1a P0AGK8 NcoI and
HindIII
cca gggaaga
c gaca c aaa
ggg
a caagc a
agcgcg aac
IscR
Tp
-w pET30a D5X843 NdeI and
XhoI
cagggcca a g
ggcc aaag c
ag acgaaagg
c cgaa cgg
a ccgg c cg
agga g ag a
ca g accc
Table A. 2 Fo wa d and e e se p ime s used o si e-di ec ed mu agenesis. The
mu a ed nucleo ides a e unde lined
P o ein Subs i u ion
Fo wa d p ime
(5′→3′)
Re e se p ime
(5′→3′)
Apo-IscR
Tp
(C92/101/107S)
C92S ca gc cc g cgag
a
g
g g c cagga ga ca cc gagacacac c
cgacaggagcaa g
C101S c cagga ga ccgga
aca agcc gaaa cg cgaa caggc a g cc
gga ca cc gaga
C107S ggaaca gcc gaaa
cgac agcg gacga
aa c
ga cg cacgc aaag
cgaa caggcaa g c
c
Apo-IscR
Ec
(C92/98/104S)
C92S g aga gccacccg
a
g
caggg aaaggcg cgcc accc gac acg
gg ggca c ac
C98S
caggg aaaggcggc
a
g
ccagggcggcg
cgccgccc ggc gccgcc
accc g
C104S gccagggcggcga aa
aagcc gaccc ggg caggc a cgcc
gccc ggc
Apo-IscR
Tp
E43A
(C92/101/107S/ E43A)
E43A g cagaaccg a gg
c
acagc ga gccg ac g acggcaa cagc g g
ccaaa acgg c gac
Apo-IscR
Ec
E43A
(C92/98/104S/E43A) E43A a ccc c a c ggc
acaac g cccg c g
cagacgggaaaacag
g gccaga aagaaagg
gaaa
Apo-IscR
Tp
P40S
(C92/101/107S/P42S) P40S cggcaaggc g caga
a cg a c ggaacag c g ccaga acgc c g
acaagcc gccg
121
B
ibliog aphy
122 Bibliog aphy
1. Bagg A & Neilands JB (1987) Fe ic up ake egula ion p o ein ac s as a ep esso ,
employing i on (II) as a co ac o o bind he ope a o o an i on anspo ope on in
Esche ichia coli. Biochemis y 26(17):5471-5477.
2. Huynh BH, Pa il DS, Mou a I, Teixei a M, Mou a JJ, De Va anian DV,
Czechowski MH, P ick il BC, Peck HD, J ., & LeGall J (1987) On he ac i e si es
o he [NiFe] hyd ogenase om Desul o ib io gigas. Mossbaue and edox- i a ion
s udies. J Biol Chem 262(2):795-800.
3. No dlund P & Eklund H (1995) Di-i on-ca boxyla e p o eins. Cu Opin S uc
Biol 5(6):758-766.
4. Beine H & Kiley PJ (1999) Fe-S p o eins in sensing and egula o y unc ions.
Cu Opin Chem Biol 3(2):152-157.
5. Johnson DC, Dean DR, Smi h AD, & Johnson MK (2005) S uc u e, unc ion, and
o ma ion o biological i on-sul u clus e s. Annu Re Biochem 74:247-281.
6. Kiley PJ & Beine H (2003) The ole o Fe-S p o eins in sensing and egula ion in
bac e ia. Cu Opin Mic obiol 6(2):181-185.
7. Beine H, Holm RH, & Munck E (1997) I on-sul u clus e s: na u e's modula ,
mul ipu pose s uc u es. Science 277(5326):653-659.
8. Py B & Ba as F (2010) Building Fe-S p o eins: bac e ial s a egies. Na Re
Mic obiol 8(6):436-446.
9. Moulis JM, Da asse V, Golinelli M-P, Meye J, & Quinkal I (1996) The
coo dina ion sphe e o i on-sul u clus e s: lessons om si e-di ec ed mu agenesis
expe imen s. J Biol Ino g Chem 1(1):2-14.
10. Meye J (2008) I on-sul u p o ein olds, i on-sul u chemis y, and e olu ion. J
Biol Ino g Chem 13(2):157-170.
11. Cammack R (2013) I on–Sul u P o eins. Encyclopedia o Biological Chemis y,
eds Lenna z WJ & Lane MD (Academic P ess, Wal ham), pp 657-664.
12. Noodleman L, Lo ell T, Liu T, Himo F, & To es RA (2002) Insigh s in o
p ope ies and ene ge ics o i on-sul u p o eins om simple clus e s o
ni ogenase. Cu Opin Chem Biol 6(2):259-273.
13. Moo e GR, Pe ig ew GW, & Roge s NK (1986) Fac o s in luencing edox
po en ials o elec on ans e p o eins. P oc Na l Acad Sci U S A 83(14):4998-
4999.
14. Banci L, Be ini I, Fe e i S, Luchina C, & Piccioli M (1993) The s uc u e o
i on-sul u clus e s in p o eins as moni o ed by NMR, mössbaue , EPR and
molecula dynamics. J Mol S uc 292(0):207-219.
15. Solomon EI, Pa el EG, Loeb KE, & Campochia o C (1995) Magne ic ci cula
dich oism spec oscopy as a p obe o he geome ic and elec onic s uc u e o non-
heme e ous enzymes. Coo d Chem Re 144(0):369-460.
16. Johnson CE, Cammack R, Rao KK, & Hall DO (1971) The in e p e a ion o he
EPR and Mossbaue spec a o wo-i on, one-elec on, i on-sulphu p o eins.
Biochem Biophys Res Commun 43(3):564-571.
17. Cheng H & Ma kley JL (1995) NMR spec oscopic s udies o pa amagne ic
p o eins: i on-sul u p o eins. Annu Re Biophys Biomol S uc 24:209-237.
18. Imlay JA (2006) I on-sulphu clus e s and he p oblem wi h oxygen. Mol Mic obiol
59(4):1073-1082.
19. Imlay JA (2008) Cellula de enses agains supe oxide and hyd ogen pe oxide. Annu
Re Biochem 77:755-776.
20. Schiebe M & Chandel NS (2014) ROS Func ion in Redox Signaling and Oxida i e
S ess. Cu Biol 24(10):R453-R462.
123
21. T ipa hi P (2007) Ni ic oxide and immune esponse. Indian J Biochem Biophys
44(5):310-319.
22. De byshi e ER & Ma le a MA (2012) S uc u e and egula ion o soluble
guanyla e cyclase. Annu Re Biochem 81:533-559.
23. Poole RK & Hughes MN (2000) New unc ions o he ancien globin amily:
bac e ial esponses o ni ic oxide and ni osa i e s ess. Mol Mic obiol 36(4):775-
783.
24. Spi o S (2006) Ni ic oxide-sensing mechanisms in Esche ichia coli. Biochem Soc
T ans 34(P 1):200-202.
25. G een J & Page MS (2004) Bac e ial edox senso s. Na Re Mic obiol 2(12):954-
966.
26. C oss CE, Halliwell B, Bo ish ET, P yo WA, Ames BN, Saul RL, McCo d JM, &
Ha man D (1987) Oxygen adicals and human disease. Ann In e n Med 107(4):526-
545.
27. Messne KR & Imlay JA (1999) The iden i ica ion o p ima y si es o supe oxide
and hyd ogen pe oxide o ma ion in he ae obic espi a o y chain and sul i e
educ ase complex o Esche ichia coli. J Biol Chem 274(15):10119-10128.
28. C ack JC, Smi h LJ, S aple on MR, Peck J, Wa mough NJ, Bu ne MJ, Bux on RS,
G een J, Oganesyan VS, Thomson AJ, & Le B un NE (2011) Mechanis ic insigh
in o he ni osyla ion o he [4Fe-4S] clus e o WhiB-like p o eins. J Am Chem Soc
133(4):1112-1121.
29. C ack JC, S aple on MR, G een J, Thomson AJ, & Le B un NE (2013) Mechanism
o [4Fe-4S](Cys)4 clus e ni osyla ion is conse ed among NO- esponsi e
egula o s. J Biol Chem 288(16):11492-11502.
30. C uz-Ramos H, C ack J, Wu G, Hughes MN, Sco C, Thomson AJ, G een J, &
Poole RK (2002) NO sensing by FNR: egula ion o he Esche ichia coli NO-
de oxi ying la ohaemoglobin, Hmp. EMBO J 21(13):3235-3244.
31. Ding H & Demple B (2000) Di ec ni ic oxide signal ansduc ion ia ni osyla ion
o i on-sul u cen e s in he SoxR ansc ip ion ac i a o . P oc Na l Acad Sci U S A
97(10):5146-5150.
32. Lewandowska H, Kalinowska M, B zoska K, Wojciuk K, Wojciuk G, &
K uszewski M (2011) Ni osyl i on complexes--syn hesis, s uc u e and biology.
Dal on T ans 40(33):8273-8289.
33. Yang W, Roge s PA, & Ding H (2002) Repai o ni ic oxide-modi ied e edoxin
[2Fe-2S] clus e by cys eine desul u ase (IscS). J Biol Chem 277(15):12868-12873.
34. C ack JC, G een J, Hu chings MI, Thomson AJ, & Le B un NE (2012) Bac e ial
i on-sul u egula o y p o eins as biological senso -swi ches. An ioxid Redox Signal
17(9):1215-1231.
35. G een J, C ack JC, Thomson AJ, & LeB un NE (2009) Bac e ial senso s o oxygen.
Cu Opin Mic obiol 12(2):145-151.
36. C ack JC, G een J, Thomson AJ, & Le B un NE (2012) I on-sul u clus e senso -
egula o s. Cu Opin Chem Biol 16(1-2):35-44.
37. Unden G & Bongae s J (1997) Al e na i e espi a o y pa hways o Esche ichia
coli: ene ge ics and ansc ip ional egula ion in esponse o elec on accep o s.
Biochim Biophys Ac a 1320(3):217-234.
38. Gonzalez R, Tao H, Pu is JE, Yo k SW, Shanmugam KT, & Ing am LO (2003)
Gene a ay-based iden i ica ion o changes ha con ibu e o e hanol ole ance in
e hanologenic Esche ichia coli: compa ison o KO11 (pa en ) o LY01 ( esis an
mu an ). Bio echnol P og 19(2):612-623.
130 Bibliog aphy
clus e assembly: e idence o i s implica ion in he pa hogen's su i al. J Bac e iol
187(17):6137-6146.
136. Riboldi GP, de Ma os EP, & F azzon J (2013) Biogenesis o [Fe-S] clus e in
Fi micu es: an unexploi ed ield o in es iga ion. An onie Van Leeuwenhoek
104(3):283-300.
137. Roche B, Aussel L, Ez a y B, Mandin P, Py B, & Ba as F (2013) Rep in o :
I on/sul u p o eins biogenesis in p oka yo es: o ma ion, egula ion and di e si y.
Biochim Biophys Ac a 1827(8-9):923-937.
138. Riboldi GP, Ve li H, & F azzon J (2009) S uc u al s udies o he En e ococcus
aecalis Su U [Fe-S] clus e p o ein. BMC Biochem 10:3.
139. Alb ech AG, Ne z DJ, Mie hke M, Pie ik AJ, Bu ghaus O, Peucke F, Lill R, &
Ma ahiel MA (2010) Su U is an essen ial i on-sul u clus e sca old p o ein in
Bacillus sub ilis. J Bac e iol 192(6):1643-1651.
140. Selbach BP, Chung AH, Sco AD, Geo ge SJ, C ame SP, & Dos San os PC
(2014) Fe-S clus e biogenesis in G am-posi i e bac e ia: Su U is a zinc-dependen
sul u ans e p o ein. Biochemis y 53(1):152-160.
141. Pa schowski T, Ba es DM, & Kiley PJ (2000) Mechanism o sensing and
esponding o oxygen dep i a ion in p oka yo ic o ganisms. Bac e ial S ess
Responses, eds S o z G & Hengge-A onis R (ASM P ess).
142. Schwa z CJ, Giel JL, Pa schkowski T, Lu he C, Ruzicka FJ, Beine H, & Kiley
PJ (2001) IscR, an Fe-S clus e -con aining ansc ip ion ac o , ep esses exp ession
o Esche ichia coli genes encoding Fe-S clus e assembly p o eins. P oc Na l Acad
Sci U S A 98(26):14895-14900.
143. Fleischhacke AS, S ubna A, Hsueh KL, Guo Y, Te e SJ, Rose JC, B unold TC,
Ma kley JL, Munck E, & Kiley PJ (2012) Cha ac e iza ion o he [2Fe-2S] clus e
o Esche ichia coli ansc ip ion ac o IscR. Biochemis y 51(22):4453-4462.
144. Nesbi AD, Giel JL, Rose JC, & Kiley PJ (2009) Sequence-speci ic binding o a
subse o IscR- egula ed p omo e s does no equi e IscR Fe-S clus e liga ion. J
Mol Biol 387(1):28-41.
145. Yeo WS, Lee JH, Lee KC, & Roe JH (2006) IscR ac s as an ac i a o in esponse o
oxida i e s ess o he su ope on encoding Fe-S assembly p o eins. Mol Mic obiol
61(1):206-218.
146. Giel JL, Nesbi AD, Me e EL, Fleischhacke AS, Wan a BT, & Kiley PJ (2013)
Regula ion o i on-sulphu clus e homeos asis h ough ansc ip ional con ol o
he Isc pa hway by [2Fe-2S]-IscR in Esche ichia coli. Mol Mic obiol 87(3):478-
492.
147. Giel JL, Rodiono D, Liu M, Bla ne FR, & Kiley PJ (2006) IscR-dependen gene
exp ession links i on-sulphu clus e assembly o he con ol o O2- egula ed genes
in Esche ichia coli. Mol Mic obiol 60(4):1058-1075.
148. Bo ukho S & Se e ino K (2002) Role o he RNA polyme ase sigma subuni in
ansc ip ion ini ia ion. Res Mic obiol 153(9):557-562.
149. Gou se RL, Ross W, & Gaal T (2000) UPs and downs in bac e ial ansc ip ion
ini ia ion: he ole o he alpha subuni o RNA polyme ase in p omo e
ecogni ion. Molecula Mic obiology 37(4):687-695.
150. F azzon J & Dean DR (2001) Feedback egula ion o i on-sul u clus e
biosyn hesis. P oc Na l Acad Sci U S A 98(26):14751-14753.
151. Lee JH, Yeo WS, & Roe JH (2004) Induc ion o he su A ope on encoding Fe-S
assembly p o eins by supe oxide gene a o s and hyd ogen pe oxide: in ol emen o
OxyR, IHF and an uniden i ied oxidan - esponsi e ac o . Mol Mic obiol
51(6):1745-1755.

131
152. Zheng M, Åslund F, & S o z G (1998) Ac i a ion o he OxyR T ansc ip ion Fac o
by Re e sible Disul ide Bond Fo ma ion. Science 279(5357):1718-1722.
153. Lee JH, Yeo WS, & Roe JH (2003) Regula ion o he su ABCDSE ope on by u . J
Mic obiol 41(2):109-114.
154. Lee KC, Yeo WS, & Roe JH (2008) Oxidan - esponsi e induc ion o he su
ope on, encoding a Fe-S assembly sys em, h ough Fu and IscR in Esche ichia
coli. J Bac e iol 190(24):8244-8247.
155. Va ghese S, Wu A, Pa k S, Imlay KR, & Imlay JA (2007) Submic omola
hyd ogen pe oxide dis up s he abili y o Fu p o ein o con ol ee-i on le els in
Esche ichia coli. Mol Mic obiol 64(3):822-830.
156. Masse E, Vande pool CK, & Go esman S (2005) E ec o RyhB small RNA on
global i on use in Esche ichia coli. J Bac e iol 187(20):6962-6971.
157. Desnoye s G, Mo isse e A, P e os K, & Masse E (2009) Small RNA-induced
di e en ial deg ada ion o he polycis onic mRNA iscRSUA. EMBO J
28(11):1551-1561.
158. Richa d DJ, Sawe s G, Sa gen F, McWal e L, & Boxe DH (1999)
T ansc ip ional egula ion in esponse o oxygen and ni a e o he ope ons
encoding he [NiFe] hyd ogenases 1 and 2 o Esche ichia coli. Mic obiology 145 (
P 10):2903-2912.
159. Nesbi AD, Fleischhacke AS, Te e SJ, & Kiley PJ (2012) A cA and AppY
an agonize IscR ep ession o hyd ogenase-1 exp ession unde anae obic
condi ions, e ealing a no el mode o O2 egula ion o gene exp ession in
Esche ichia coli. J Bac e iol 194(24):6892-6899.
160. S ewa V, Bledsoe PJ, & Williams SB (2003) Dual o e lapping p omo e s con ol
napF (pe iplasmic ni a e educ ase) ope on exp ession in Esche ichia coli K-12. J
Bac e iol 185(19):5862-5870.
161. Thompson JD, Gibson TJ, & Higgins DG (2002) Mul iple sequence alignmen
using Clus alW and Clus alX. Cu P o oc Bioin o ma ics Chap e 2:Uni 2 3.
162. Bond CS & Schu elkop AW (2009) ALINE: a WYSIWYG p o ein-sequence
alignmen edi o o publica ion-quali y alignmen s. Ac a C ys allog D Biol
C ys allog 65(P 5):510-512.
163. Shepa d W, Sou ou ina O, Cou ois E, England P, Haouz A, & Ma in-Ve s ae e I
(2011) Insigh s in o he R 2 ep esso amily-- he s uc u e o CymR, he global
cys eine egula o o Bacillus sub ilis. FEBS J 278(15):2689-2701.
164. Tucke NP, Le B un NE, Dixon R, & Hu chings MI (2010) The e's NO s opping
Ns R, a global egula o o he bac e ial NO s ess esponse. T ends Mic obiol
18(4):149-156.
165. Todd JD, Wexle M, Sawe s G, Yeoman KH, Poole PS, & Johns on AW (2002)
Ri A, an i on- esponsi e egula o in he symbio ic bac e ium Rhizobium
leguminosa um. Mic obiology 148(P 12):4059-4071.
166. Tucke NP, Hicks MG, Cla ke TA, C ack JC, Chand a G, Le B un NE, Dixon R, &
Hu chings MI (2008) The ansc ip ional ep esso p o ein Ns R senses ni ic oxide
di ec ly ia a [2Fe-2S] clus e . PLoS One 3(11):e3623.
167. E en S, Bu guie e P, Auge S, Sou ou ina O, Danchin A, & Ma in-Ve s ae e I
(2006) Global con ol o cys eine me abolism by CymR in Bacillus sub ilis. J
Bac e iol 188(6):2184-2197.
168. Tanous C, Sou ou ina O, Raynal B, Hullo MF, Me ele P, Gilles AM, Noi o P,
Danchin A, England P, & Ma in-Ve s ae e I (2008) The CymR egula o in
complex wi h he enzyme CysK con ols cys eine me abolism in Bacillus sub ilis. J
Biol Chem 283(51):35551-35560.
132 Bibliog aphy
169. Ji Q, Zhang L, Sun F, Deng X, Liang H, Bae T, & He C (2012) S aphylococcus
au eus CymR is a new hiol-based oxida ion-sensing egula o o s ess esis ance
and oxida i e esponse. J Biol Chem 287(25):21102-21109.
170. Sou ou ina O, Dub ac S, Poupel O, Msadek T, & Ma in-Ve s ae e I (2010) The
pleio opic CymR egula o o S aphylococcus au eus plays an impo an ole in
i ulence and s ess esponse. PLoS Pa hog 6(5):e1000894.
171. Sou ou ina O, Poupel O, Coppee JY, Danchin A, Msadek T, & Ma in-Ve s ae e I
(2009) CymR, he mas e egula o o cys eine me abolism in S aphylococcus
au eus, con ols hos sulphu sou ce u iliza ion and plays a ole in bio ilm
o ma ion. Mol Mic obiol 73(2):194-211.
172. Kommineni S, Yukl E, Hayashi T, Delepine J, Geng H, Moenne-Loccoz P, &
Nakano MM (2010) Ni ic oxide-sensi i e and -insensi i e in e ac ion o Bacillus
sub ilis Ns R wi h a ResDE-con olled p omo e . Mol Mic obiol 78(5):1280-1293.
173. Kommineni S, Lama A, Popescu B, & Nakano MM (2012) Global ansc ip ional
con ol by Ns R in Bacillus sub ilis. J Bac e iol 194(7):1679-1688.
174. Hena es B, Kommineni S, Chumsakul O, Ogasawa a N, Ishikawa S, & Nakano
MM (2014) The ResD Response Regula o , h ough Func ional In e ac ion wi h
Ns R and Fu , Plays Th ee Dis inc Roles in Bacillus sub ilis T ansc ip ional
Con ol. J Bac e iol 196(2):493-503.
175. Yukl ET, Elbaz MA, Nakano MM, & Moenne-Loccoz P (2008) T ansc ip ion
Fac o Ns R om Bacillus sub ilis Senses Ni ic Oxide wi h a 4Fe-4S Clus e
(dagge ). Biochemis y 47(49):13084-13092.
176. Bsa N, He big A, Casillas-Ma inez L, Se low P, & Helmann JD (1998) Bacillus
sub ilis con ains mul iple Fu homologues: iden i ica ion o he i on up ake (Fu )
and pe oxide egulon (Pe R) ep esso s. Mol Mic obiol 29(1):189-198.
177. Isabella VM, Lapek JD, J ., Kennedy EM, & Cla k VL (2009) Func ional analysis
o Ns R, a ni ic oxide-sensing R 2 ep esso in Neisse ia gono hoeae. Mol
Mic obiol 71(1):227-239.
178. Rodiono DA, Dubchak IL, A kin AP, Alm EJ, & Gel and MS (2005)
Dissimila o y me abolism o ni ogen oxides in bac e ia: compa a i e
econs uc ion o ansc ip ional ne wo ks. PLoS Compu Biol 1(5):e55.
179. Eisen eich W, Heesemann J, Rudel T, & Goebel W (2013) Me abolic hos
esponses o in ec ion by in acellula bac e ial pa hogens. F on Cell In ec
Mic obiol 3:24.
180. Nai z M, Sch oll A, Sonnwebe T, & Weiss G (2010) The s uggle o i on - a
me al a he hos -pa hogen in e ace. Cell Mic obiol 12(12):1691-1702.
181. Die z I, Je chel S, Szaszak M, Shima K, & Rupp J (2012) When oxygen uns sho :
he mic oen i onmen d i es hos -pa hogen in e ac ions. Mic obes In ec
14(4):311-316.
182. G een J, Rol e MD, & Smi h LJ (2014) T ansc ip ional egula ion o bac e ial
i ulence gene exp ession by molecula oxygen and ni ic oxide. Vi ulence 5(4).
183. T oxell B & Hassan HM (2013) T ansc ip ional egula ion by Fe ic Up ake
Regula o (Fu ) in pa hogenic bac e ia. F on Cell In ec Mic obiol 3:59.
184. Kim SH, Lee BY, Lau GW, & Cho YH (2009) IscR modula es ca alase A (Ka A)
ac i i y, pe oxide esis ance and ull i ulence o Pseudomonas ae uginosa PA14. J
Mic obiol Bio echnol 19(12):1520-1526.
185. Lim JG & Choi SH (2014) IscR is a global egula o essen ial o pa hogenesis o
Vib io ulni icus and induced by hos cells. In ec Immun 82(2):569-578.
133
186. Rincon-En iquez G, C e e P, Ba as F, & Py B (2008) Biogenesis o Fe/S p o eins
and pa hogenici y: IscR plays a key ole in allowing E winia ch ysan hemi o adap
o hos ile condi ions. Mol Mic obiol 67(6):1257-1273.
187. Runyen-Janecky L, Daughe y A, Lloyd B, Welling on C, Eskanda ian H, &
Sag ansky M (2008) Role and egula ion o i on-sul u clus e biosyn hesis genes
in Shigella lexne i i ulence. In ec Immun 76(3):1083-1092.
188. Young LS (1984) The clinical challenge o in ec ions due o Pseudomonas
ae uginosa. Re In ec Dis 6 Suppl 3:S603-607.
189. Wong SM, Be nui M, Shen H, & Ake ley BJ (2013) Genome-wide i ness p o iling
e eals adap a ions equi ed by Haemophilus in coin ec ion wi h in luenza A i us
in he mu ine lung. P oc Na l Acad Sci U S A 110(38):15413-15418.
190. Lim JG, Pa k JH, & Choi SH (2014) Low cell densi y egula o AphA up egula es
he exp ession o Vib io ulni icus iscR gene encoding he Fe-S clus e egula o
IscR. J Mic obiol.
191. Mille HK, Kwuan L, Schwiesow L, Be nick DL, Me e E, Rami ez HA, Ragle
JM, Chan PP, Kiley PJ, Lowe TM, & Aue buch V (2014) IscR Is Essen ial o
Ye sinia pseudo ube culosis Type III Sec e ion and Vi ulence. PLoS Pa hog
10(6):e1004194.
192. Bandyopadhyay S, Gama F, Molina-Na a o MM, Gualbe o JM, Clax on R, Naik
SG, Huynh BH, He e o E, Jacquo JP, Johnson MK, & Rouhie N (2008)
Chlo oplas mono hiol glu a edoxins as sca old p o eins o he assembly and
deli e y o [2Fe-2S] clus e s. EMBO J 27(7):1122-1133.
193. Lill R, Ho mann B, Molik S, Pie ik AJ, Rie zschel N, S ehling O, Uza ska MA,
Webe H, Wilb ech C, & Mühlenho U (2012) The ole o mi ochond ia in
cellula i on–sul u p o ein biogenesis and i on me abolism. Biochimica e
Biophysica Ac a (BBA) - Molecula Cell Resea ch 1823(9):1491-1508.
194. Olichon A & Su ey T (2007) Selec ion o gene ically encoded luo escen single
domain an ibodies enginee ed o e icien exp ession in Esche ichia coli. J Biol
Chem 282(50):36314-36320.
195. P ischi F, Pas o e C, Ca oni M, Iannuzzi C, Adinol i S, Temussi P, & Pas o e A
(2010) O he ulne abili y o o phan complex p o eins: he case s udy o he E.
coli IscU and IscS p o eins. P o ein Exp Pu i 73(2):161-166.
196. Fe a i S, Ha ley VR, Pon iggia A, Good ellow PN, Lo ell-Badge R, & Bianchi
ME (1992) SRY, like HMG1, ecognizes sha p angles in DNA. EMBO J
11(12):4497-4506.
197. Ca pen e ML & Kneale GG (1994) Ci cula dich oism o he analysis o p o ein-
DNA in e ac ions. Me hods Mol Biol 30:339-345.
198. Je abek-Willemsen M, Wienken CJ, B aun D, Baaske P, & Duh S (2011)
Molecula in e ac ion s udies using mic oscale he mopho esis. Assay D ug De
Technol 9(4):342-353.
199. Flo D, Mai s T, Gi aud T, Guija o M, Lesou d M, Rey V, an B ussel D, Mo awe
C, Bo el C, Higne e O, Cha anne J, Nu izzo D, McSweeney S, & Mi chell E
(2010) The ID23-2 s uc u al biology mic o ocus beamline a he ESRF. J
Synch o on Radia 17(1):107-118.
200. Kabsch W (2010) XDS. Ac a C ys allog D Biol C ys allog 66(2):125-132.
201. Kabsch W (2010) In eg a ion, scaling, space-g oup assignmen and pos -
e inemen . Ac a C ys allog D Biol C ys allog 66(2):133-144.
202. Collabo a i e Compu a ional P ojec N (1994) The CCP4 sui e: p og ams o
p o ein c ys allog aphy. Ac a C ys allog D Biol C ys allog 50(P 5):760-763.
134 Bibliog aphy
203. Sheld ick GM (2010) Expe imen al phasing wi h SHELXC/D/E: combining chain
acing wi h densi y modi ica ion. Ac a C ys allog D Biol C ys allog 66(P
4):479-485.
204. Pape T & Schneide TR (2004) HKL2MAP: a g aphical use in e ace o
mac omolecula phasing wi h SHELX p og ams. J Appl C ys allog
37(5):843-844.
205. McCoy AJ, G osse-Kuns le e RW, Adams PD, Winn MD, S o oni LC, & Read RJ
(2007) Phase c ys allog aphic so wa e. J Appl C ys allog 40(P 4):658-674.
206. Emsley P & Cow an K (2004) Coo : model-building ools o molecula g aphics.
Ac a C ys allog D Biol C ys allog 60(P 12 P 1):2126-2132.
207. Adams PD, A onine PV, Bunkoczi G, Chen VB, Da is IW, Echols N, Headd JJ,
Hung LW, Kap al GJ, G osse-Kuns le e RW, McCoy AJ, Mo ia y NW, Oe ne
R, Read RJ, Richa dson DC, Richa dson JS, Te willige TC, & Zwa PH (2010)
PHENIX: a comp ehensi e Py hon-based sys em o mac omolecula s uc u e
solu ion. Ac a C ys allog D Biol C ys allog 66(P 2):213-221.
208. Lill R (2009) Func ion and biogenesis o i on-sulphu p o eins. Na u e
460(7257):831-838.
209. Fleischhacke AS & Kiley PJ (2011) I on-con aining ansc ip ion ac o s and hei
oles as senso s. Cu Opin Chem Biol 15(2):335-341.
210. Ca y PD & Kneale GG (2009) Ci cula dich oism o he analysis o p o ein-DNA
in e ac ions. Me hods Mol Biol 543:613-624.
211. Blou ER, Schmie I, & Simmons NS (1962) New Co on E ec s in Polypep ides
and P o eins. J Am Chem Soc 84(16):3193-3194.
212. Ke ppola TK & Cu an T (1991) DNA bending by Fos and Jun: he lexible hinge
model. Science 254(5035):1210-1214.
213. Dicke son RE (1998) DNA bending: he p e alence o kinkiness and he i ues o
no mali y. Nucleic Acids Res 26(8):1906-1926.
214. Kim J, Zwieb C, Wu C, & Adhya S (1989) Bending o DNA by gene- egula o y
p o eins: cons uc ion and use o a DNA bending ec o . Gene 85(1):15-23.
215. Wu HM & C o he s DM (1984) The locus o sequence-di ec ed and p o ein-
induced DNA bending. Na u e 308(5959):509-513.
216. Rajagopalan S, Te e SJ, Zwa PH, B ennan RG, Phillips KJ, & Kiley PJ (2013)
S udies o IscR e eal a unique mechanism o me al-dependen egula ion o DNA
binding speci ici y. Na S uc Mol Biol 20(6):740-747.
217. Zheng G, Lu XJ, & Olson WK (2009) Web 3DNA--a web se e o he analysis,
econs uc ion, and isualiza ion o h ee-dimensional nucleic-acid s uc u es.
Nucleic Acids Res 37(Web Se e issue):W240-246.
218. an Dijk M & Bon in AM (2009) 3D-DART: a DNA s uc u e modelling se e .
Nucleic Acids Res 37(Web Se e issue):W235-239.
219. Rohs R, Wes SM, Sosinsky A, Liu P, Mann RS, & Honig B (2009) The ole o
DNA shape in p o ein-DNA ecogni ion. Na u e 461(7268):1248-1253.
220. Wes SM, Rohs R, Mann RS, & Honig B (2010) Elec os a ic in e ac ions be ween
a ginines and he mino g oo e in he nucleosome. J Biomol S uc Dyn 27(6):861-
866.
221. Ha an TE & Mohan y U (2009) The unique s uc u e o A- ac s and in insic DNA
bending. Q Re Biophys 42(1):41-81.
222. Zeng J, Zhang X, Wang Y, Ai C, Liu Q, & Qiu G (2008) Glu43 is an essen ial
esidue o coo dina ing he [Fe2S2] clus e o IscR om Acidi hiobacillus
e ooxidans. FEBS Le 582(28):3889-3892.
135
223. Luscombe NM, Laskowski RA, & Tho n on JM (2001) Amino acid-base
in e ac ions: a h ee-dimensional analysis o p o ein-DNA in e ac ions a an a omic
le el. Nucleic Acids Res 29(13):2860-2874.
224. Kondo J & Wes ho E (2011) Classi ica ion o pseudo pai s be ween nucleo ide
bases and amino acids by analysis o nucleo ide-p o ein complexes. Nucleic Acids
Res 39(19):8628-8637.
225. Fon eca e M, Choudens SO, Py B, & Ba as F (2005) Mechanisms o i on-sul u
clus e assembly: he SUF machine y. J Biol Ino g Chem 10(7):713-721.
226. Takahashi Y & Nakamu a M (1999) Func ional assignmen o he ORF2-iscS-
iscU-iscA-hscB-hscA- dx-ORF3 gene clus e in ol ed in he assembly o Fe-S
clus e s in Esche ichia coli. J Biochem 126(5):917-926.
227. Riboldi GP, de Oli ei a JS, & F azzon J (2011) En e ococcus aecalis Su U
sca old p o ein enhances Su S desul u ase ac i i y by acqui ing sul u om i s
cys eine-153. Biochim Biophys Ac a 1814(12):1910-1918.
228. By ne-Bailey KG, W igh on KC, Melnyk RA, Agbo P, Hazen TC, & Coa es JD
(2010) Comple e genome sequence o he elec ici y-p oducing "The mincola
po ens" s ain JR. J Bac e iol 192(15):4078-4079.
229. Ca lson HK, Ia a one AT, Go u A, Yeo BS, T an R, Melnyk RA, Ma hies RA,
Aue M, & Coa es JD (2012) Su ace mul iheme c- ype cy och omes om
The mincola po ens and implica ions o espi a o y me al educ ion by G am-
posi i e bac e ia. P oc Na l Acad Sci U S A 109(5):1702-1707.
230. Yu aniyama P, Aga JN, Cash VL, Johnson MK, & Dean DR (2000) Ni S-di ec ed
assembly o a ansien [2Fe-2S] clus e wi hin he Ni U p o ein. P oc Na l Acad
Sci U S A 97(2):599-604.
231. T o e V, Vinella D, Loiseau L, Ollagnie de Choudens S, Fon eca e M, & Ba as
F (2009) The CsdA cys eine desulphu ase p omo es Fe/S biogenesis by ec ui ing
Su componen s and pa icipa es o a new sulphu ans e pa hway by ec ui ing
CsdL (ex-YgdL), a ubiqui in-modi ying-like p o ein. Mol Mic obiol 74(6):1527-
1542.
232. Ho KG, Ta DT, Tapley TL, Silbe g JJ, & Vicke y LE (2002) Hsc66 subs a e
speci ici y is di ec ed owa d a disc e e egion o he i on-sul u clus e empla e
p o ein IscU. J Biol Chem 277(30):27353-27359.
233. And e G, Haudecoeu E, Mono M, Oh ani K, Shimizu T, Dupuy B, & Ma in-
Ve s ae e I (2010) Global egula ion o gene exp ession in esponse o cys eine
a ailabili y in Clos idium pe ingens. BMC Mic obiol 10:234.
234. Shen G, Balasub amanian R, Wang T, Wu Y, Ho a LM, K ebs C, B yan DA, &
Golbeck JH (2007) Su R coo dina es wo [4Fe-4S]2+, 1+ clus e s and unc ions as
a ansc ip ional ep esso o he su BCDS ope on and an au o egula o o su R in
cyanobac e ia. J Biol Chem 282(44):31909-31919.
235. Mansy SS, Wu G, Su e us KK, & Cowan JA (2002) I on-sul u clus e
biosyn hesis. The ma oga ma i ima IscU is a s uc u ed i on-sul u clus e assembly
p o ein. J Biol Chem 277(24):21397-21404.
236. La kin MA, Blackshields G, B own NP, Chenna R, McGe igan PA, McWilliam H,
Valen in F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, & Higgins
DG (2007) Clus al W and Clus al X e sion 2.0. Bioin o ma ics 23(21):2947-2948.
237. Reboi o-Ja o D, Reboi o-Ja o M, Fdez-Ri e ola F, Viei a CP, Fonseca NA, &
Viei a J (2012) ADOPS--Au oma ic De ec ion O Posi i ely Selec ed Si es. J
In eg Bioin o m 9(3):200.

136 Bibliog aphy
238. Choi YS, Shin DH, Chung IY, Kim SH, Heo YJ, & Cho YH (2007) Iden i ica ion
o Pseudomonas ae uginosa genes c ucial o hyd ogen pe oxide esis ance. J
Mic obiol Bio echnol 17(8):1344-1352.
239. Zeng J, Zhang K, Liu J, & Qiu G (2008) Exp ession, pu i ica ion, and
cha ac e iza ion o i on-sul u clus e assembly egula o IscR om
Acidi hiobacillus e ooxidans. J Mic obiol Bio echnol 18(10):1672-1677.
240. Fuen es-P io P & Sal esen GS (2004) The p o ein s uc u es ha shape caspase
ac i i y, speci ici y, ac i a ion and inhibi ion. Biochem J 384(P 2):201-232.
241. G ego e LM, Rade SD, Fle e ick RJ, & Cohen FE (1991) Hyd ogen bonds
in ol ing sul u a oms in p o eins. P o eins 9(2):99-107.
242. Tu k V, S oka V, Vasilje a O, Renko M, Sun T, Tu k B, & Tu k D (2012) Cys eine
ca hepsins: om s uc u e, unc ion and egula ion o new on ie s. Biochim
Biophys Ac a 1824(1):68-88.
243. Zhou P, Tian F, L F, & Shang Z (2009) Geome ic cha ac e is ics o hyd ogen
bonds in ol ing sul u a oms in p o eins. P o eins 76(1):151-163.
244. Tsane a IR & Weiss B (1990) soxR, a locus go e ning a supe oxide esponse
egulon in Esche ichia coli K-12. J Bac e iol 172(8):4197-4205.
245. W igh on KC, Th ash JC, Melnyk RA, Bigi JP, By ne-Bailey KG, Remis JP,
Schichnes D, Aue M, Chang CJ, & Coa es JD (2011) E idence o di ec elec on
ans e by a g am-posi i e bac e ium isola ed om a mic obial uel cell. Appl
En i on Mic obiol 77(21):7633-7639.
246. Alb ach SP & Sub amanian J (1977) The numbe o Fe a oms in he i on-sulphu
cen e s o he espi a o y chain. Biochim Biophys Ac a 462(1):36-48.
247. Lanciano P, Ve gnes A, G imaldi S, Guiglia elli B, & Magalon A (2007)
Biogenesis o a espi a o y complex is o ches a ed by a single accesso y p o ein. J
Biol Chem 282(24):17468-17474.
248. Lumppio HL, Shen i NV, Summe s AO, Voo douw G, & Ku z DM, J . (2001)
Rub e y h in and ub edoxin oxido educ ase in Desul o ib io ulga is: a no el
oxida i e s ess p o ec ion sys em. J Bac e iol 183(1):101-108.
249. W igh on KC, Agbo P, Wa necke F, Webe KA, B odie EL, DeSan is TZ,
Hugenhol z P, Ande sen GL, & Coa es JD (2008) A no el ecological ole o he
Fi micu es iden i ied in he mophilic mic obial uel cells. ISME J 2(11):1146-1156.
250. Balsalob e LC, D opa M, & Ma e MH (2014) An o e iew o an imic obial
esis ance and i s public heal h signi icance. B az J Mic obiol 45(1):1-5.
251. D inkwa e N & McGowan S (2014) F om c ys al o compound: s uc u e-based
an imala ial d ug disco e y. Biochem J 461(3):349-369.
137
The unique egula ion o i on-sul u clus e biogenesis
in a G am-posi i e bac e ium
Joana A. San os
a,b
, Noelia Alonso-Ga cía
a
, Sand a Macedo-Ribei o
a,1
, and Ped o José Ba bosa Pe ei a
a,1
a
Ins i u o de Biologia Molecula e Celula (IBMC), Uni e sidade do Po o, 4150-180 Po o, Po ugal; and
b
Ins i u o de Ciências Biomédicas de Abel Salaza
(ICBAS), Uni e sidade do Po o, 4050-313 Po o, Po ugal
Edi ed by G ego y A. Pe sko, Weill Co nell Medical College, New Yo k, NY, and app o ed Ap il 28, 2014 ( ecei ed o e iew Decembe 6, 2013)
I on-sul u clus e s unc ion as co ac o s o a wide ange o p o eins,
wi h di e se molecula oles in bo h p oka yo ic and euka yo ic
cells. Dedica ed machine ies assemble he clus e s and deli e hem
o he inal accep o molecules in a igh ly egula ed p ocess. In he
p o o ypical G am-nega i e bac e ium Esche ichia coli, he wo
exis ing i on-sul u clus e assembly sys ems, i on-sul u clus e
(ISC) and sul u assimila ion (SUF) pa hways, a e closely in e con-
nec ed. The ISC pa hway egula o , IscR, is a ansc ip ion ac o o
he helix- u n-helix ype ha can coo dina e a [2Fe-2S] clus e .
Redox condi ions and i on o sul u a ailabili y modula e he liga-
ion s a us o he labile IscR clus e , which in u n de e mines
a swi ch in DNA sequence speci ici y o he egula o : clus e -con-
aining IscR can bind o a amily o gene p omo e s ( ype-1)
whe eas he clus e less o m ecognizes only a second g oup o
sequences ( ype-2). Howe e , i on-sul u clus e biogenesis in
G am-posi i e bac e ia is no so well cha ac e ized, and mos
o ganisms o his g oup display only one o he i on-sul u clus e
assembly sys ems. A no able excep ion is he unique G am-posi-
i e dissimila o y me al educing bac e ium The mincola po ens,
whe e genes om bo h sys ems could be iden i ied, albei wi h
a di e ging o ganiza ion om ha o G am-nega i e bac e ia.
We demons a ed ha one o hese genes encodes a unc ional
IscR homolog and is likely in ol ed in he egula ion o i on-sul u
clus e biogenesis in T. po ens. S uc u al and biochemical cha ac e -
iza ion o T. po ens and E. coli IscR e ealed a s ikingly simila
a chi ec u e and un eiled an un o eseen conse a ion o he unique
mechanism o sequence disc imina ion cha ac e is ic o his dis inc-
i e g oup o ansc ip ion egula o s.
R 2-like egula o
|
ansc ip ion egula ion
|
helix- u n-helix mo i
|
DNA ecogni ion
|
speci ici y modula ion
I on-sul u (Fe/S) p o eins play c ucial oles o he unc ioning
o bo h p oka yo ic and euka yo ic cells, being equi ed o
biological unc ions anging om elec on anspo o edox and
non edox ca alysis, and om DNA syn hesis and epai o sens-
ing in egula o y p ocesses (1). The main ole o he Fe/S clus e
assembly machine ies is o mobilize i on and sul u a oms om
hei s o age sou ces, assemble he wo componen s in o an Fe/S
clus e , and hen ans e he newly o med clus e o he inal
p o ein accep o s (2). In Esche ichia coli, he e a e wo o hese
Fe/S clus e ‘‘ ac o ies,’’ he ISC (i on-sul u clus e ) and SUF
(sul u assimila ion) sys ems, whose co esponding genes a e
o ganized in wo ope ons, iscSUA-hscBA- dx and su ABCDSE,
espec i ely (2, 3). Dele ion mu an s o he ISC sys em display
a a ie y o g ow h de ec s due o loss o Fe/S clus e -con aining
enzyme ac i i y and dis up ion o sul u me abolism whe eas
ailu e o bo h he ISC and SUF sys ems leads o syn he ic
le hali y (4, 5).
In E. coli, he ISC machine y is conside ed he housekeeping
sys em esponsible o he ma u a ion o a la ge a ie y o Fe/S
p o eins whe eas he SUF sys em is igge ed unde s ess con-
di ions, such as oxida i e s ess o i on s a a ion (6). ISC
pa hway egula o (IscR) is a [2Fe-2S] clus e -con aining an-
sc ip ion ac o wi h a single p edic ed helix- u n-helix mo i ,
i s iden i ied o i s ole in egula ing exp ession o he ISC
biogenesis pa hway (7) and subsequen ly ound o con ol he
exp ession o mo e han 40 genes in E. coli (7, 8). Acco ding o
he cu en ly accep ed model o Fe/S clus e biogenesis, unde
condi ions un a o able o Fe/S clus e o ma ion, he labile IscR
clus e is los , and IscR-media ed ep ession o he isc (i on-
sul u clus e ) ope on is alle ia ed. A he same ime, apo-IscR
ac i a es he su (sul u assimila ion) ope on o u he com-
pensa e o damage o loss o Fe/S clus e s (9, 10). Once he
demand o Fe/S biogenesis is me , highe le els o clus e -con-
aining holo-IscR exis , causing an inc eased ep ession o he
ISC pa hway. Mo eo e , unde i on limi a ion, he ISC and SUF
machine ies a e unable o main ain he le els o holo-IscR, and
he e o e his eedback mechanism allows IscR o sense Fe/S
demand and enables E. coli o espond app op ia ely o s ess
condi ions (11).
The e a e wo classes o IscR binding si es in he E. coli ge-
nome: a ype-1 si e deduced om iscR,yadR, and yhgI p omo e
egions and a ype-2 si e compiled om he IscR si es ups eam
o he hyaA,ydiU, and su A p omo e s (8). In e es ingly, IscR
binds ype-1 p omo e s solely in i s holo- o m whe eas binding o
ype-2 p omo e s was shown o be independen o he p esence
o he Fe/S clus e (12). In E. coli, IscR mu a ion E43A enabled
speci ic ecogni ion o ype-1 p omo e s by apo-IscR, likely
mimicking he in e ac ion mode o he clus e -bound o m o he
p o ein (13).
Signi icance
I on-sul u clus e s a e ubiqui ous co ac o s o p o eins in-
e ening in dispa a e biological p ocesses. I on-sul u clus e
biosyn hesis pa hways a e igh ly egula ed in G am-nega i e
bac e ia. One o he pa icipa ing ansc ip ion ac o s, i on-
sul u clus e pa hway (ISC) egula o (IscR), can i sel bind an
i on-sul u clus e . Depending on i s liga ion s a us, IscR
ecognizes and binds o dis inc p omo e s, he e o e modu-
la ing clus e biosyn hesis. This unique p o ein a he c oss oad
be ween he ISC and sul u assimila ion (SUF) i on-sul u clus-
e biosyn he ic pa hways was hough o be es ic ed o G am-
nega i e bac e ia. We demons a ed he exis ence o a unc-
ional IscR in he unique G am-posi i e bac e ium The mincola
po ens. S uc u al and unc ional analysis o T. po ens and
Esche ichia coli IscR un eiled a conse ed mechanism o p o-
mo e disc imina ion, along wi h sub le s uc u al di e ences
ha explain hei dis inc DNA sequence ecogni ion speci ici y.
Au ho con ibu ions: J.A.S., S.M.-R., and P.J.B.P. designed esea ch; J.A.S., N.A.-G., and
P.J.B.P. pe o med esea ch; J.A.S., N.A.-G., S.M.-R., and P.J.B.P. analyzed da a; and J.A.S.,
S.M.-R., and P.J.B.P. w o e he pape .
The au ho s decla e no con lic o in e es .
This a icle is a PNAS Di ec Submission.
Da a deposi ion: The a omic coo dina es and s uc u e ac o s ha e been deposi ed in he
P o ein Da a Bank, www.pdb.o g (PDB ID codes 4CHU and 4CIC).
1
To whom co espondence may be add essed. E-mail: [email p o ec ed] o s ibei o@
ibmc.up.p .
This a icle con ains suppo ing in o ma ion online a www.pnas.o g/lookup/suppl/doi:10.
1073/pnas.1322728111/-/DCSupplemen al.
www.pnas.o g/cgi/doi/10.1073/pnas.1322728111 PNAS Ea ly Edi ion
|
1o 10
BIOCHEMISTRY PNAS PLUS
Al hough a molecula -le el unde s anding o he complex
p ocesses o Fe/S clus e biosyn hesis in se e al o ganisms is now
eme ging om he combina ion o in i o and in i o app oaches,
hese machine ies a e s ill poo ly unde s ood in G am-posi i e
bac e ia. Al hough homologs o he E. coli ISC o SUF sys ems
a e p esen in se e al o ganisms, some species exhibi unusual
Fe/S clus e biosyn he ic machine ies. Mos G am-posi i e bac-
e ia ca y only a su ope on, con aining genes coding o Su U
and he Su BCD complex (14, 15), bu no su E o su A- ela ed
genes, e en i in some cases su A can be ound elsewhe e in he
genome (14, 16).
The mincola po ens (s ain JR) is an anae obic, he mophilic,
G am-posi i e dissimila o y me al- educing bac e ium (DMRB),
isola ed om a he mophilic mic obial uel cell (17). I is o he
i s G am-posi i e DMRB o which he e is a comple e genome
sequence, which e ealed an unusual abundance o mul iheme
c- ype cy och omes (17, 18). Using homology sea ches, we iden-
i ied a se ies o genes wi h sequence simila i y o bo h E. coli
SUF and ISC machine ies in he T. po ens genome, including a
gene locus coding o a pu a i e IscR p o ein. Taken oge he ,
ou esul s bo h iden i y and cha ac e ize a unique Fe/S bio-
genesis egula o in G am-posi i e bac e ia. Th ough s uc u al
and biochemical analysis o bo h T. po ens and E. coli apo-IscR
p o eins and hei E43A mu an s, we we e able o un eil sub le
s uc u al ea u es impo an o DNA ecogni ion and binding
speci ici y.
Resul s
Unique Fe/S Clus e Biogenesis in T. po ens.In G am-posi i e bac-
e ia, he e is conse a ion o he su ope on, o en p esen as
su CDSUB, which is he only machine y o Fe/S clus e bio-
syn hesis in he majo i y o hese o ganisms (14, 19). Su p is-
ingly, homology sea ches on he G am-posi i e DMRB T. po ens
JR genome (17) allowed iden i ying wo gene loci wi h sequence
simila i y o E. coli SUF and ISC machine ies (Fig. 1A). In
T. po ens, he e a e ORFs coding o homologs o he an-
sc ip ion ac o IscR (The JR_1914, 37% iden ical o he E. coli
p o ein) (7), he cys eine desul u ase IscS (The JR_1913) (4, 20),
and he sca old IscU (The JR_1912) (21) om he ISC pa hway.
An addi ional su -like ope on in T. po ens comp ises homologs o
su C (The JR_0923), su B, and su D (The JR_0924) om he
E. coli SUF pa hway, and he chape ones hcsA (The JR_0925)
and hcsB (The JR_0926) om he E. coli isc ope on (22–24).
Compa ed wi h o he G am-posi i e bac e ia, namely om he
Fi micu es phylum, some unique ea u es o he T. po ens su
ope on become e iden . In T. po ens, he su ope on does no
code o cys eine desul u ase (Su S) homologs al hough he e
a e elsewhe e in he T. po ens genome wo addi ional genes
coding o pu a i e cys eine desul u ases (The JR_0460 and
The JR_3003) homologous o CsdA/Su S, which can unc ion
as complemen a y sul u sou ces o Fe/S clus e biogenesis,
possibly h ough he ec ui men o he SUF machine y (25).
The T. po ens su ope on is also de oid o homologs o Su U,
ecen ly epo ed o be a zinc-dependen sul u ans e ase in
Bacillus sub ilis (26), bu encodes a su BD p o ein, which o-
ge he wi h su C was shown o ac as sca old in G am-nega-
i e bac e ia (27, 28). Fu he mo e, he su ope on in T. po ens
includes he hscA and hscB genes coding o he chape ones
esponsible o ans e ing p e o med clus e s om he sca -
old IscU o inal accep o s and ha , in E. coli,a eco an-
sc ibed wi h he isc and no wi h he su ope on (4). Addi-
ionally, genes coding o A- ype ca ie s a e absen om he
T. po ens genome.
IscU is a highly conse ed p o ein ha unc ions as sca old o
clus e assembly and subsequen ans e . P ese ed ea u es
include he clus e ligands ( h ee cys eines and one his idine), an
aspa a e esidue ha plays a c i ical ole in clus e ans e o
apo-p o eins and he LPPVK mo i ecognized by he chape one
HscA (29) (Fig. S1). Some G am-posi i e bac e ia (e.g., En-
e ococcus aecalis) we e shown o possess an IscU homolog,
Su U, which does no con ain he HscA ecogni ion si e and has
a 19- esidue inse ion be ween he i s wo conse ed cys eines
(14). The T. po ens sca old p o ein displays conse a ion o he
cha ac e is ic IscU LPPVK mo i and does no con ain he
inse ion signa u e speci ic o Su U- ype p o eins. Acco dingly,
phylogene ic analysis o IscU and Su U p o ein sequences places
he p o ein encoded by he The JR_1912 gene be ween he
G am-nega i e IscU- ype and he Su U-like p o eins om
G am-posi i e bac e ia (Fig. 1B).
As p e iously epo ed o Clos idium pe ingens (30),
sea ches o Fe/S clus e biogenesis ope ons in G am-posi i e
bac e ia wi h comple ely sequenced genomes, namely he DMRBs
Desul i obac e ium ha niense and Desul o omaculum educens,
e ealed ha hey possess a single ISC gene locus (iscRSU) bu
no SUF appa a us. In con as , in o he G am-posi i e bac e ia
(e.g., E. aecalis), only he SUF pa hway can be ound. The e o e,
con a y o o he G am-posi i e bac e ia desc ibed so a , T. po ens
no only has wo gene loci coding o he wo Fe/S clus e bio-
syn hesis machine ies p esen in E. coli (ISC and SUF), bu hese
sys ems display a unique o ganiza ion.
The T. po ens The JR_1914 Gene Codes o IscR. IscR is a [2Fe-2S]
clus e -con aining ansc ip ional egula o encoded by he i s
gene o he iscRSUA-hscBA- dx ope on ha egula es bo h ISC
and SUF sys ems in E. coli and o he G am-nega i e bac e ia
(10, 11). Apa om he sequence-un ela ed Su R ound in cya-
nobac e ia (31, 32), no IscR homolog was desc ibed in G am-
posi i e bac e ia, wi h he possible excep ion o some species o
he Clos idium genus o which unc ional da a a e s ill lacking
(30). In T. po ens, he The JR_1914 gene encodes a p o ein o he
R 2 amily o ansc ip ional egula o s, sha ing only 37% iden i y
wi h E. coli IscR bu wi h ull conse a ion o he cys eine esidues
known o coo dina e he [2Fe-2S] clus e (Cys
92, 98, 104
,E. coli
numbe ing) (Fig. 1C)(10).
Clus e less (apo) IscR om E. coli was shown o ac i a e su
ope on exp ession du ing s ess condi ions, such as i on s a a-
ion (6). The as-pu i ied apo o m o he p o ein encoded by
T. po ens gene The JR_1914 (apo-IscR
Tp
-w ) was ound o bind
o he ups eam egion o he pu a i e su ope on, be ween genes
The JR_0922 and The JR_0923 (Fig. 1D). A simila beha io
was obse ed o a iple mu an (C92/101/107S) o IscR
Tp
(apo-
IscR
Tp
)(Fig.1D) whe e all pu a i e clus e -binding cys eine
esidues (Fig. 1C) we e eplaced by se ine. Gi en he s uc u al
simila i y be ween cys eine and se ine and he equi emen o
homogeneous sample o downs eam unc ional and s uc u -
al assays, his a ian was used in all expe imen s whe e he apo
o m o IscR
Tp
was equi ed. The abili y o apo-IscR
Tp
-w
and apo-IscR
Tp
o bind he p omo e egion o he su ope on
sugges s ha IscR
Tp
can unc ionasanFe/Sclus e egula o in
his o ganism, wi h he apo o m in ol ed in he egula ion o
he su ope on exp ession, as obse ed o E. coli. Al hough
such egula o s ha e been ound and cha ac e ized in a numbe
o G am-nega i e bac e ia (7, 8, 33–35), he cha ac e iza ion o
o hologous p o eins om G am-posi i e species has no ye
been epo ed. The e o e, T. po ens has a unique o ganiza ion
and egula ion o Fe/S clus e assembly genes, among G am-
posi i e bac e ia.
O e all S uc u e o T. po ens IscR. The 3D s uc u e o ee apo
T. po ens IscR, wi h he pu a i e clus e -binding cys eines mu-
a ed o se ine ( he clus e less IscR iple-mu an s C92/101/107S
o T. po ens o C92/98/104S o E. coli a e he eby e med apo-
IscR
Tp
and apo-IscR
Ec
, espec i ely) was de e mined by X- ay
c ys allog aphy om e agonal (P4
1
) c ys als di ac ing o
1.6-Å esolu ion. The c ys allog aphic asymme ic uni con ains he
unc ional IscR homodime (Fig. 2A). Apo-IscR
Tp
monome s
2o 10
|
www.pnas.o g/cgi/doi/10.1073/pnas.1322728111 San os e al.
20]. Ligand dilu ions we e p epa ed in assay bu e wi hou Tween 20 and
mixed wi h each p o ein sample a a olume a io o 1:1. Measu emen s
wi h apo-IscR
Tp
, apo-IscR
Tp
P40S, and apo-IscR
Tp
E43A we e pe o med in
s anda d capilla ies whe eas hyd ophilic capilla ies we e used o measu e-
men s wi h apo-IscR
Ec
E43A. Fo each in e ac ion, da a om a leas wo
independen uns we e a e aged, and he a e age cu e was i ed wi h
NTAnalysis so wa e (NanoTempe Technologies).
C ys alliza ion o apo-IscR
Tp
and apo-IscR
Ec
:hya Complex. Ini ial c ys alliza ion
condi ions o apo-IscR
Tp
we e sc eened a 20 °C using he si ing-d op
me hod wi h comme cial spa se-ma ix c ys alliza ion sc eens. D ops con-
sis ing o equal olumes (1 μL) o p o ein (a 20 mg/mL) and p ecipi an
solu ion we e equilib a ed agains a 300-μL ese oi . C ys als we e ob ained
a e 2 d using 0.1 M Bis-T is (pH 6.5) and 3 M NaCl as p ecipi an . Be o e
da a collec ion, c ys als we e c yop o ec ed by imme sing hem b ie ly in
a 1:1 mix u e o p ecipi an solu ion and 40% ( ol/ ol) o 2 mg/mL NDSB-201
(3-(1-py idino)-1-p opane sul ona e) solu ion in e hylene glycol and lash-
cooled in liquid ni ogen (46). Selenome hionyl apo-IscR
Tp
c ys allized in
he same condi ions and was c yop o ec ed ollowing he p ocedu e
desc ibed abo e.
A 3.8- old mola excess o apo-IscR
Ec
was mixed wi h double-s anded
oligonucleo ide (p epa ed as desc ibed in Elec opho e ic Mobili y-Shi
Assay) comp ising egion −30 o −55 o he E. coli hya p omo e sequence
wi h a single-base 5′o e hang (hya_26_OH)(Table S1) and incuba ed a
oom empe a u e o 30 min. The complex was ei he used immedia ely o
lash ozen in liquid ni ogen and s o ed a −80 °C. Ini ial c ys alliza ion
condi ions we e es ablished a he High Th oughpu C ys alliza ion Labo-
a o y o he Eu opean Molecula Biology Labo a o y, using he si ing-d op
me hod. C ys als we e ob ained a 20 °C, om 0.2-μL d ops composed o
iden ical olumes o complex solu ion [350 μM p o ein and 92 μM oligonu-
cleo ide in 40 mM T is·HCl (pH 8.0), 150 mM KCl, 10% ( ol/ ol) glyce ol,
1 mM DTT] and o p ecipi an [0.1 M ci ic acid (pH 4.0 o 6.0), 1 M li hium
chlo ide, 20% (w / ol) PEG 6000]. Be e and la ge c ys als could be ob ained
om he condi ion a pH 4.0 using he hanging-d op apo di usion me hod.
The op imized c ys als we e c yop o ec ed in he same condi ions as he apo-
IscR
Tp
c ys als.
Da a Collec ion and P ocessing. X- ay di ac ion da a we e collec ed om
cooled (100 K) single c ys als a synch o on beam lines ID29 (apo-IscR
Tp
and
Se-Me apo-IscR
Tp
) (47) and ID23-EH2 (apo-IscR
Ec
:hya complex) (48) o he
Eu opean Synch o on Radia ion Facili y. The apo-IscR
Tp
da a we e eco ded
on a Pila us 6M de ec o (Dec is) using a wa eleng h o 0.9763 Å (na i e
da ase ) o 0.9792 Å (Se-Me da ase ). Fo he na i e da a, 1,200 images
we e collec ed in 0.1° oscilla ion s eps wi h 0.1-s exposu e pe ame
whe eas, o he Se-Me da a, 3,600 images we e eco ded in 0.1° oscilla ion
s eps wi h 0.037-s exposu e pe ame. The apo-IscR
Ec
:hya complex da a
we e eco ded on a MX-225 de ec o (Ma esea ch) using a wa eleng h o
0.8726 Å. One hund ed images we e collec ed in 0.95° oscilla ion s eps wi h
5.43-s exposu e pe ame. Di ac ion da a we e in eg a ed wi h XDS (49),
scaled wi h XSCALE (50), and educed wi h u ili ies om he CCP4 p og am
sui e (51). Da a collec ion s a is ics a e summa ized in Table 2.
S uc u e Solu ion and Re inemen . The s uc u e o apo-IscR
Tp
was sol ed by
single-wa eleng h anomalous di ac ion using he anomalous signal o se-
lenium-subs i u ed c ys als wi h he SHELXC/SHELXD/SHELXE pipeline (52)
and he HKL2MAP GUI (53). The esul ing elec on densi y maps we e eadily
in e p e able. The s uc u e o he apo-IscR
Ec
:hya complex was sol ed by
molecula eplacemen wi h PHASER (54) using a unca ed e sion o he
e ined apo-IscR
Tp
s uc u e as sea ch model. Fo bo h s uc u es, al e na ing
cycles o model building wi h COOT (55) and o e inemen wi h PHENIX (56)
we e pe o med un il model comple ion. Fo he apo-IscR
Tp
s uc u e, he
inal model comp ises esidues Gly-3 o Gly85 and Se 101 o Ile149 o subuni
Table 2. S a is ics o da a collec ion, p ocessing, and e inemen
Da ase T. po ens IscR* (na i e) T. po ens IscR* (Se-Me ) E. coli IscR-DNA complex*
C ys allog aphic analysis
Wa eleng h, Å 0.9763 0.9792 0.8726
Space g oup P4
1
P4
1
P2
1
2
1
2
1
Uni cell dimensions, Å a =b=53.6; c =118.4 a =b=53.4; c =118.7 a =49.0; b =75.8; c =173.4
Resolu ion ange, Å 53.6–1.60 (1.69–1.60) 48.7–2.47 (2.61–2.47) 46.0–2.49 (2.62–2.49)
Re lec ions (measu ed/unique) 196,159/43,750 (28,394/6,325) 111,297/11,861 (13,614/1,647) 87,182/23,387 (12,463/3,244)
Comple eness, % 99.7 (98.7) 99.1 (94.1) 99.3 (96.3)
Mul iplici y 4.5 (4.5) 9.4 (8.3) 3.7 (3.8)
R
me ge†
0.046 (1.299) 0.190 (1.573) 0.103 (0.911)
R
pim‡
0.024 (0.688) 0.064 (0.555) 0.061 (0.531)
〈I/σ(I)〉14.3 (1.6) 7.3 (1.4) 8.6 (1.5)
Monome s pe asymme ic uni 2 2 2
Ma hews coe icien , Å
3
·Da
−1
2.53 2.52 3.13
Sol en con en , % 51.4 51.2 60.7
S uc u e e inemen
Resolu ion ange, Å 48.8–1.60 —46.0–2.49
R
ac o §
/F ee R
ac o ¶
0.202/0.220 —0.207/0.251
Unique e lec ions (wo k/ es se ) 41,642/1,973 —22,057/1,193
Wa e molecules 156 —15
To al no. o a oms 2,363 —2,999
No. o mac omolecule a oms 2,205 —2,984
msd bond leng hs, Å 0.011 —0.008
msd bond angles, ° 1.09 —1.38
A e age o e all B ac o , Å
2
38.1 —77.7
Ramachand an a o ed, % 97.5 —96.0
Ramachand an ou lie s, % 0.0 —0.4
PDB en y 4cic —4chu
*Values in pa en heses co espond o he ou e mos esolu ion shell. Each da ase was eco ded om a single c ys al.
†
R
me ge
=P
hkl
P
i
jI
i
(hkl)–〈I(hkl)〉j/P
hkl
P
i
I
i
(hkl), whe e I
i
(hkl) is he obse ed in ensi y and〈I(hkl)〉is he a e age in ensi y o mul iple obse a ions o
symme y- ela ed e lec ions.
‡
R
pim
=P
hkl
[1/(N–1)]
1/2
P
i
jI
i
(hkl)–〈I(hkl)〉j/P
hkl
P
i
I
i
(hkl), whe e I
i
(hkl) is he obse ed in ensi y and〈I(hkl)〉is he a e age in ensi y o mul iple obse a ions
o symme y- ela ed e lec ions.
§
R
ac o
=PjjF
o
j−jF
c
jj/PjF
o
j, whe e jF
o
jand jF
c
ja e obse ed and calcula ed s uc u e ac o ampli udes, espec i ely.
¶
F ee R
ac o
is he c oss- alida ion R
ac o
compu ed o a andomly chosen subse o 5% o he o al numbe o e lec ions, which we e no used du ing
e inemen .
San os e al. PNAS Ea ly Edi ion
|
9o 10
BIOCHEMISTRY PNAS PLUS

A and Gly-3 o Gly85 and Se 101 o Ty 148 o subuni B whe eas he apo-
IscR
Ec
:hya complex comp ises esidues Me 0 o Asp88 and Lys103 o Se 139 o
subuni A, and Gly1 o Asp84 and Gln93 o Val135 o subuni B. Model e-
inemen s a is ics a e summa ized in Table 2.
ACKNOWLEDGMENTS. We hank Jo ge Viei a o help wi h Au oma ic
De ec ion o Posi i ely Selec ed Si es. We acknowledge he Eu opean
Synch o on Radia ion Facili y (ESRF) o p o ision o synch o on adia ion
acili ies and hank he ESRF s a o help wi h da a collec ion. Mic oscale
he mopho esis da a collec ion was ca ied ou a he Campus Science
Suppo Facili ies P o ein Technologies Facili y (www.cs .ac.a ). This wo k
was unded by Fundo Eu opeu de Desen ol imen o Regional h ough he
Ope a ional Compe i i eness P og amme-COMPETE and by na ional unds
h ough Fundação pa a a Ciência e a Tecnologia unde p ojec FCOMP-01-
0124-FEDER-028116 (PTDC/BBB ‐BEP/2127/2012) and PhD Fellowship SFRH/
BD/66461/2009 ( o J.A.S.). The esea ch leading o hese esul s has ecei ed
unding om he Eu opean Communi y’s Se en h F amewo k P og amme
(FP7/2007-2013) unde BioS uc -X (G an Ag eemen 283570).
1. Beine H, Holm RH, Münck E (1997) I on-sul u clus e s: Na u e’s modula , mul i-
pu pose s uc u es. Science 277(5326):653–659.
2. Ayala-Cas o C, Saini A, Ou en FW (2008) Fe-S clus e assembly pa hways in bac e ia.
Mic obiol Mol Biol Re 72(1):110–125.
3. Fon eca e M, Choudens SO, Py B, Ba as F (2005) Mechanisms o i on-sul u clus e
assembly: The SUF machine y. J Biol Ino g Chem 10(7):713–721.
4. Takahashi Y, Nakamu a M (1999) Func ional assignmen o he ORF2-iscS-iscU-iscA-
hscB-hscA- dx-ORF3 gene clus e in ol ed in he assembly o Fe-S clus e s in Esche ichia
coli.JBiochem126(5):917–926.
5. Takahashi Y, Tokumo o U (2002) A hi d bac e ial sys em o he assembly o i on-
sul u clus e s wi h homologs in a chaea and plas ids. J Biol Chem 277(32):
28380–28383.
6. Ou en FW, Djaman O, S o z G (2004) A su ope on equi emen o Fe-S clus e as-
sembly du ing i on s a a ion in Esche ichia coli.Mol Mic obiol 52(3):861–872.
7. Schwa z CJ, e al. (2001) IscR, an Fe-S clus e -con aining ansc ip ion ac o , ep esses
exp ession o Esche ichia coli genes encoding Fe-S clus e assembly p o eins. P oc Na l
Acad Sci USA 98(26):14895–14900.
8. Giel JL, Rodiono D, Liu M, Bla ne FR, Kiley PJ (2006) IscR-dependen gene exp es-
sion links i on-sulphu clus e assembly o he con ol o O2- egula ed genes in
Esche ichia coli.Mol Mic obiol 60(4):1058–1075.
9. Lee KC, Yeo WS, Roe JH (2008) Oxidan - esponsi e induc ion o he su ope on, en-
coding a Fe-S assembly sys em, h ough Fu and IscR in Esche ichia coli.J Bac e iol
190(24):8244–8247.
10. Yeo WS, Lee JH, Lee KC, Roe JH (2006) IscR ac s as an ac i a o in esponse o oxida i e
s ess o he su ope on encoding Fe-S assembly p o eins. Mol Mic obiol 61(1):206–218.
11. Giel JL, e al. (2013) Regula ion o i on-sulphu clus e homeos asis h ough an-
sc ip ional con ol o he Isc pa hway by [2Fe-2S]-IscR in Esche ichia coli.Mol Mic o-
biol 87(3):478–492.
12. Nesbi AD, Giel JL, Rose JC, Kiley PJ (2009) Sequence-speci ic binding o a subse o
IscR- egula ed p omo e s does no equi e IscR Fe-S clus e liga ion. J Mol Biol 387(1):
28–41.
13. Rajagopalan S, e al. (2013) S udies o IscR e eal a unique mechanism o me al-
dependen egula ion o DNA binding speci ici y. Na S uc Mol Biol 20(6):740–747.
14. Riboldi GP, Ve li H, F azzon J (2009) S uc u al s udies o he En e ococcus aecalis
Su U [Fe-S] clus e p o ein. BMC Biochem 10:3.
15. Riboldi GP, de Oli ei a JS, F azzon J (2011) En e ococcus aecalis Su U sca old p o ein
enhances Su S desul u ase ac i i y by acqui ing sul u om i s cys eine-153. Biochim
Biophys Ac a 1814(12):1910–1918.
16. Alb ech AG, e al. (2010) Su U is an essen ial i on-sul u clus e sca old p o ein in
Bacillus sub ilis.J Bac e iol 192(6):1643–1651.
17. By ne-Bailey KG, e al. (2010) Comple e genome sequence o he elec ici y-p oducing
“The mincola po ens”s ain JR. J Bac e iol 192(15):4078–4079.
18. Ca lson HK, e al. (2012) Su ace mul iheme c- ype cy och omes om The mincola
po ens and implica ions o espi a o y me al educ ion by G am-posi i e bac e ia.
P oc Na l Acad Sci USA 109(5):1702–1707.
19. Riboldi GP, de Ma os EP, F azzon J (2013) Biogenesis o [Fe-S] clus e in Fi micu es:An
unexploi ed ield o in es iga ion. An onie an Leeuwenhoek 104(3):283–300.
20. Zheng L, Cash VL, Flin DH, Dean DR (1998) Assembly o i on-sul u clus e s. Iden i-
ica ion o an iscSUA-hscBA- dx gene clus e om Azo obac e inelandii.J Biol Chem
273(21):13264–13272.
21. Yu aniyama P, Aga JN, Cash VL, Johnson MK, Dean DR (2000) Ni S-di ec ed assembly o
a ansien [2Fe-2S] clus e wi hin he Ni U p o ein. P oc Na l Acad Sci USA 97(2):599–604.
22. Rangacha i K, e al. (2002) Su C hyd olyzes ATP and in e ac s wi h Su B om The -
mo oga ma i ima.FEBS Le 514(2-3):225–228.
23. Saini A, Mapolelo DT, Chahal HK, Johnson MK, Ou en FW (2010) Su D and Su C
ATPase ac i i y a e equi ed o i on acquisi ion du ing in i o Fe-S clus e o ma ion
on Su B. Biochemis y 49(43):9402–9412.
24. Silbe g JJ, Tapley TL, Ho KG, Vicke y LE (2004) Regula ion o he HscA ATPase e-
ac ion cycle by he co-chape one HscB and he i on-sul u clus e assembly p o ein
IscU. J Biol Chem 279(52):53924–53931.
25. T o e V, e al. (2009) The CsdA cys eine desulphu ase p omo es Fe/S biogenesis by
ec ui ing Su componen s and pa icipa es o a new sulphu ans e pa hway by
ec ui ing CsdL (ex-YgdL), a ubiqui in-modi ying-like p o ein. Mol Mic obiol 74(6):
1527–1542.
26. Selbach BP, e al. (2014) Fe-S clus e biogenesis in G am-posi i e bac e ia: Su U is
a zinc-dependen sul u ans e p o ein. Biochemis y 53(1):152–160.
27. Chahal HK, Dai Y, Saini A, Ayala-Cas o C, Ou en FW (2009) The Su BCD Fe-S sca old
complex in e ac s wi h Su A o Fe-S clus e ans e . Biochemis y 48(44):
10644–10653.
28. Tian T, He H, Liu XQ (2014) The Su BCD p o ein complex is he sca old o i on-sul u
clus e assembly in The mus he mophiles HB8. Biochem Biophys Res Commun 443(2):
376–381.
29. Ho KG, Ta DT, Tapley TL, Silbe g JJ, Vicke y LE (2002) Hsc66 subs a e speci ici y is
di ec ed owa d a disc e e egion o he i on-sul u clus e empla e p o ein IscU.
J Biol Chem 277(30):27353–27359.
30. And é G, e al. (2010) Global egula ion o gene exp ession in esponse o cys eine
a ailabili y in Clos idium pe ingens.BMC Mic obiol 10:234.
31. Shen G, e al. (2007) Su R coo dina es wo [4Fe-4S]2+,1+clus e s and unc ions as
a ansc ip ional ep esso o he su BCDS ope on and an au o egula o o su R in
cyanobac e ia. J Biol Chem 282(44):31909–31919.
32. Wang T, e al. (2004) The su R gene (sll0088 in Synechocys is sp. s ain PCC 6803)
unc ions as a ep esso o he su BCDS ope on in i on-sul u clus e biogenesis in
cyanobac e ia. J Bac e iol 186(4):956–967.
33. Choi YS, e al. (2007) Iden i ica ion o Pseudomonas ae uginosa genes c ucial o
hyd ogen pe oxide esis ance. J Mic obiol Bio echnol 17(8):1344–1352.
34. Runyen-Janecky L, e al. (2008) Role and egula ion o i on-sul u clus e biosyn hesis
genes in Shigella lexne i i ulence. In ec Immun 76(3):1083–1092.
35. Zeng J, Zhang K, Liu J, Qiu G (2008) Exp ession, pu i ica ion, and cha ac e iza ion
o i on-sul u clus e assembly egula o IscR om Acidi hiobacillus e ooxidans.
J Mic obiol Bio echnol 18(10):1672–1677.
36. Shepa d W, e al. (2011) Insigh s in o he R 2 ep esso amily— he s uc u e o
CymR, he global cys eine egula o o Bacillus sub ilis.FEBS J 278(15):2689–2701.
37. Ji Q, e al. (2012) S aphylococcus au eus CymR is a new hiol-based oxida ion-sensing
egula o o s ess esis ance and oxida i e esponse. JBiolChem287(25):
21102–21109.
38. G ego e LM, Rade SD, Fle e ick RJ, Cohen FE (1991) Hyd ogen bonds in ol ing
sul u a oms in p o eins. P o eins 9(2):99–107.
39. Zhou P, Tian F, L F, Shang Z (2009) Geome ic cha ac e is ics o hyd ogen bonds
in ol ing sul u a oms in p o eins. P o eins 76(1):151–163.
40. Fuen es-P io P, Sal esen GS (2004) The p o ein s uc u es ha shape caspase ac i i y,
speci ici y, ac i a ion and inhibi ion. Biochem J 384(P 2):201–232.
41. Tu k V, e al. (2012) Cys eine ca hepsins: F om s uc u e, unc ion and egula ion o
new on ie s. Biochim Biophys Ac a 1824(1):68–88.
42. P ischi F, e al. (2010) O he ulne abili y o o phan complex p o eins: The case s udy
o he E. coli IscU and IscS p o eins. P o ein Exp Pu i 73(2):161–166.
43. Olichon A, Su ey T (2007) Selec ion o gene ically encoded luo escen single domain
an ibodies enginee ed o e icien exp ession in Esche ichia coli.J Biol Chem 282(50):
36314–36320.
44. Ca pen e ML, Kneale GG (1994) Ci cula dich oism o he analysis o p o ein-DNA
in e ac ions. Me hods Mol Biol 30:339–345.
45. Je abek-Willemsen M, Wienken CJ, B aun D, Baaske P, Duh S (2011) Molecula
in e ac ion s udies using mic oscale he mopho esis. Assay D ug De Technol 9(4):
342–353.
46. Pe ei a PJ, e al. (2008) Mycobac e ium ube culosis glucosyl-3-phosphoglyce a e
syn hase: S uc u e o a key enzyme in me hylglucose lipopolysaccha ide biosyn hesis.
PLoS ONE 3(11):e3748.
47. de Sanc is D, e al. (2012) ID29: A high-in ensi y highly au oma ed ESRF beamline o
mac omolecula c ys allog aphy expe imen s exploi ing anomalous sca e ing.
J Synch o on Radia 19(P 3):455–461.
48. Flo D, e al. (2010) The ID23-2 s uc u al biology mic o ocus beamline a he ESRF.
J Synch o on Radia 17(1):107–118.
49. Kabsch W (2010) XDS. Ac a C ys allog D Biol C ys allog 66(P 2):125–132.
50. Kabsch W (2010) In eg a ion, scaling, space-g oup assignmen and pos - e inemen .
Ac a C ys allog D Biol C ys allog 66(P 2):133–144.
51. Collabo a i e Compu a ional P ojec , Numbe 4 (1994) The CCP4 sui e: P og ams o
p o ein c ys allog aphy. Ac a C ys allog D Biol C ys allog 50(P 5):760–763.
52. Sheld ick GM (2010) Expe imen al phasing wi h SHELXC/D/E: Combining chain acing
wi h densi y modi ica ion. Ac a C ys allog D Biol C ys allog 66(P 4):479–485.
53. Pape T, Schneide TR (2004) HKL2MAP: A g aphical use in e ace o mac omolecula
phasing wi h SHELX p og ams. J Appl C ys 37:843–844.
54. McCoy AJ, e al. (2007) Phase c ys allog aphic so wa e. J Appl C ys 40(P 4):658–674.
55. Emsley P, Cow an K (2004) Coo : Model-building ools o molecula g aphics. Ac a
C ys allog D Biol C ys allog 60(P 12 P 1):2126–2132.
56. Adams PD, e al. (2010) PHENIX: A comp ehensi e Py hon-based sys em o mac o-
molecula s uc u e solu ion. Ac a C ys allog D Biol C ys allog 66(P 2):213–221.
57. Reboi o-Ja o D, e al. (2012) ADOPS—Au oma ic De ec ion O Posi i ely Selec ed
Si es. J In eg Bioin o m 9(3):200.
58. Thompson JD, Gibson TJ, Higgins DG (2002) Mul iple sequence alignmen using
Clus alW and Clus alX. Cu P o oc Bioin o ma ics Chap e 2: Uni 2 3.
59. Bond CS, Schü elkop AW (2009) ALINE: A WYSIWYG p o ein-sequence alignmen
edi o o publica ion-quali y alignmen s. Ac a C ys allog D Biol C ys allog 65(P 5):
510–512.
60. Holm L, Rosens öm P (2010) Dali se e : Conse a ion mapping in 3D. Nucleic Acids
Res 38(Web Se e issue):W545–W549.
10 o 10
|
www.pnas.o g/cgi/doi/10.1073/pnas.1322728111 San os e al.
Suppo ing In o ma ion
San os e al. 10.1073/pnas.1322728111
Fig. S1. The mincola po ens Fe/S clus e sca old p o ein displays he conse ed ea u es o IscU- ype sca olds om G am-nega i e bac e ia. Amino acid
sequence alignmen o he T. po ens The JR_1912 gene p oduc and homologous p o eins om bo h G am-posi i e (En e ococcus aecalis,S ep ococcus
pyogenes,Bacillus sub ilis) and G am-nega i e bac e ia (Esche ichia coli). S ic ly conse ed amino acids a e highligh ed in ed and inc easing esidue con-
se a ion is ep esen ed by a colo g adien om g een o ed. Cys eine esidues known o coo dina e he Fe/S clus e a e deno ed by a s a (1), he “LPPVK”
HscA-binding mo i is ep esen ed in bold (2), and he cha ac e is ic G am-posi i e inse ion is unde lined (3). Numbe s abo e he alignmen e e o he
T. po ens amino acid sequence numbe ing. Alignmen p epa ed wi h Clus alW (4) and colo ed wi h Aline (5).
1. Mansy SS, Wu G, Su e us KK, Cowan JA (2002) I on-sul u clus e biosyn hesis. The ma oga ma i ima IscU is a s uc u ed i on-sul u clus e assembly p o ein. J Biol Chem 277(24):21397–
21404.
2. Ho KG, Silbe g JJ, Vicke y LE (2000) In e ac ion o he i on-sul u clus e assembly p o ein IscU wi h he Hsc66/Hsc20 molecula chape one sys em o Esche ichia coli.P oc Na l Acad Sci
USA 97(14):7790–7795.
3. Riboldi GP, Ve li H, F azzon J (2009) S uc u al s udies o he En e ococcus aecalis Su U [Fe-S] clus e p o ein. BMC Biochem 10:3.
4. La kin MA, e al. (2007) Clus al W and Clus al X e sion 2.0. Bioin o ma ics 23(21):2947–2948.
5. Bond CS, Schü elkop AW (2009) ALINE: A WYSIWYG p o ein-sequence alignmen edi o o publica ion-quali y alignmen s. Ac a C ys allog D Biol C ys allog 65(P 5):510–512.
Fig. S2. The o e all s uc u e o he biologically ac i e i on-sul u clus e (ISC) pa hway egula o (IscR) dime is mos ly unchanged upon DNA binding. (A)
Supe posi ion o he 3D s uc u es o ee apo-IscR
Tp
(g een), ee E. coli apo-IscR (magen a; PDB ID code 4HF0), and hya p omo e -bound apo-IscR
Ec
(blue). (B)
Supe posi ion o he 3D s uc u es o ee apo-IscR
Tp
and hya p omo e -bound apo-IscR
Ec
(colo s as in A). The molecules we e o a ed ∼90° a ound y, ela i e o
he iew in A.(C) An asymme ic elec os a ic su ace o ien s he unc ional apo-IscR
Ec
dime owa d i s hya DNA a ge . Posi i e su ace elec os a ic po en ial
is shown in blue and nega i e in ed. The DNA molecule backbone is depic ed as a whi e ibbon wi h bases in g een.
San os e al. www.pnas.o g/cgi/con en /sho /1322728111 1o 3
Fig. S3. The T. po ens isc p omo e egion con ains wo binding si es o IscR
Tp
.(A) DNA ecogni ion was assessed by elec opho e ic mobili y-shi assay o he
complexes o med be ween apo-IscR
Tp
o apo-IscR
Tp
E43A and ei he he ull (iscTp_1) o immed (iscTp_2)isc p omo e sequence. DNA band-shi s a e de-
no ed by a ows. (B,Uppe ) Time cou se o Fe/S clus e assembly on apo-IscR
Tp
-w . The e is a ime-dependen inc ease o he cha ac e is ic Fe/S clus e ab-
so p ion peak a 420 nm o econs i u ed apo-IscR
Tp
-w (R-IscR
Tp
-w ; blue ci cles) whe eas no no iceable a ia ion could be obse ed o he assay pe o med in
he absence o cys eine (NR-IscR
Tp
-w , black ci cles). A he end o he assay, he eac ion con aining R-IscR
Tp
-w displayed a cha ac e is ic b own colo (uppe
cu e e; R), which was essen ially absen in he con ol eac ion (lowe cu e e; NR). (Lowe ) UV/Visible abso p ion spec a o R-IscR
Tp
-w . The spec um o
pu i ied R-IscR
Tp
-w (blue cu e) displays local maxima a 420 nm and 320 nm ha a e cha ac e is ic o Fe/S clus e s, which disappea ed upon educ ion wi h
2 mM di hioni e ( ed cu e). (C) Fe/S clus e binding modula es ecogni ion o ype-1 p omo e DNA sequences by IscR. Ci cula dich oism spec a o iscTp_1
DNA sequence (Table S1) wi h inc easing concen a ions o R-IscR
Tp
-w (Uppe ) o NR-IscR
Tp
-w (Lowe ). The spec a we e eco ded upon successi e addi ions o
each pu i ied p o ein o an iscTp_1 solu ion (2 μM). A signi ican change in ellip ici y a he cha ac e is ic B-DNA peak a 285 nm (1, 2) can be obse ed only
upon R-IscR
Tp
-w addi ion, indica ing ha only he Fe/S clus e -con aining o m o IscR
Tp
-w is able o ecognize he T. po ens isc p omo e sequence and induce
local DNA s uc u al changes. Expe imen s we e ca ied ou a 20 °C in 40 mM T is (pH 8), 150 mM KCl, 5% ( ol/ ol) glyce ol, 1 mM DTT.
1. Ca pen e ML, Kneale GG (1994) Ci cula dich oism o he analysis o p o ein-DNA in e ac ions. Me hods Mol Biol 30:339–345.
2. Ca pen e ML, Kneale GG (1991) Ci cula dich oism and luo escence analysis o he in e ac ion o P 1 gene 5 p o ein wi h poly(dT). J Mol Biol 217(4):681–689.
Table S1. Oligonucleo ides used in binding and c ys alliza ion assays
Name Sequence
hyaEc 5′-AAATCCACAC AGTTTGTATT GTTTTG-3′
iscbEc 5′-TAAATAGTTG ACCAATTTAC TCGGGAATGT CAGACT-3′
iscTp_1 5′-TAAAAAATCT TAGTATTTTA GTTGGAATTT TTCTTGACCA GAAAATAACT GTATGC-3′
iscTp_2 5′-TAAAAAATCT TAGTATTTTA GTTGGAATTT TTCTTGACC-3′
iscTp_3 5′-TAAAAAATCT TAGTATTTTA GTTGGAATT-3′
iscTp_4 5′-TTTTTCTTGA CCAGAAAATA ACTGTATGC-3′
iscTp_5 5′-TAAAAAATCT TAGTATTTTA GTTGGAA-3′
hya_26_OH 5′-GAAATCCACA CAGTTTGTAT TGTTTTG-3′
su 5′-CGCTTTATAT TTAGGAAAGA TGCAGCGCCG GCTATAAAAT AGCCGGCTTT TTCTAAGCTC
TTAATCAATA GCCGGATCCA ATTATTGTAG AATCCTGCCC AAAATTATTT GTACTTTTTT
ATCCCGGCAG GAGGATAAAT GTGGGTGGAC AAAGAAAAAT ACCCCTGTAG TTTTTGGATT
TAAACCAGGA ATCCGTCAGG AGGGAATATT GCGCTTTATA TTTAGGAAAG ATGCAGCGCC
GGCTATAAAA TAGCCGGCTT TTTCTAAGCT CTTAATCAAT AGCCGGATCC AATTATTGTA
GAATCCTGCC CAAAATTATT TGTACTTTTT TATCCCGGCA GGAGGATAAA TGTGGGTGGA
CAAAGAAAAA TACCCCTGTA GTTTTTGGAT TTAAACCAGG AATCCGTCAG GAGGGAATAT TG-3′
San os e al. www.pnas.o g/cgi/con en /sho /1322728111 2o 3
Table S2. Binding a ini ies be ween IscR and ype-1 and ype-2
p omo e sequences de e mined by mic oscale he mopho esis
DNA sequence IscR a ian Dissocia ion cons an K
d
,nM
hyaEc Apo-IscR
Ec
E43A 180 ±18
Apo-IscR
Tp
E43A 340 ±52
Apo-IscR
Tp
n.d.
Apo-IscR
Tp
P40S 11,900 ±3,340
iscbEc Apo-IscR
Ec
E43A 154 ±7
Apo-IscR
Tp
E43A 97 ±6
Apo-IscR
Tp
n.d.
iscTp_3 Apo-IscR
Tp
E43A 320 ±19
Apo-IscR
Tp
n.d.
iscTp_4 Apo-IscR
Tp
E43A 905 ±87*
Apo-IscR
Tp
n.d.
iscTp_5 Apo-IscR
Tp
E43A n.d.
n.d., binding no de ec ed.
*The K
d
alue migh be o e es ima ed because sa u a ion was no eached.
San os e al. www.pnas.o g/cgi/con en /sho /1322728111 3o 3