Re iew
Analysis o sol en ole ance in Pseudomonas pu ida DOT-T1E based on i s
genome sequence and a collec ion o mu an s
Zulema Udaondo
a,1
, Es ella Duque
a,1
, Ma ilde Fe nández
b
, Láza o Molina
c
, Jesús de la To e
a
,
Pa icia Be nal
a
, José-Luis Niqui
b
, Cecilia Pini
a
, Amalia Roca
b
, Miguel A. Ma illa
b
,
M. An onia Molina-Hena es
a
, Ho encia Sil a-Jiménez
a
, Glo ia Na a o-A ilés
a
, And eas Busch
a
,
Jesús Lacal
a
, Tino K ell
a
, Ana Segu a
a
, Juan-Luis Ramos
a,
⇑
a
Consejo Supe io de In es igaciones Cien íficas, EEZ, Depa men o En i onmen al P o ec ion, 18008 G anada, Spain
b
Bio-Ilibe is R&D, Polígono Junca il, Calle Capilei a 7, 18210 G anada, Spain
c
CIDERTA-Uni e sidad de Huel a, Huel a, Spain
a icle in o
A icle his o y:
Recei ed 4 July 2012
Re ised 9 July 2012
Accep ed 9 July 2012
A ailable online 20 July 2012
Edi ed by Miguel De la Rosa, Felix Wieland
and Wilhelm Jus
Keywo ds:
Gene egula ion
Pseudomonas
E flux pumps
Ene gy gene a ion
abs ac
Pseudomonas pu ida s ains a e p e alen in a a ie y o p is ine and pollu ed en i onmen s. The
genome o he sol en - ole an P. pu ida s ain DOT-T1E which h i es in he p esence o high con-
cen a ions o monoa oma ic hyd oca bons, con ains a ci cula 6.3 Mbp ch omosome and a 133 kbp
plasmid. Omics in o ma ion has been used o iden i y he genes and p o eins in ol ed in sol en ol-
e ance in his bac e ium. This s ain uses a mul i ac o ial esponse ha in ol es fine- uning o lipid
fluidi y, ac i a ion o a gene al s ess- esponse sys em, enhanced ene gy gene a ion, and induc ion
o specific e flux pumps ha ex ude sol en s o he medium. Local and global ansc ip ional eg-
ula o s pa icipa e in a complex ne wo k o me abolic unc ions, ac ing as he decision make s in
he esponse o sol en s.
1. In oduc ion
S ains o he species Pseudomonas pu ida a e ubiqui ous, me a-
bolically e y e sa ile and adap ed o p ospe in di e se habi a s.
These s ains can use a wide ange o compounds as ca bon, ni o-
gen, phospha e and sul u sou ces [1–4]. They exhibi an unusual
weal h o de e minan s o high a fini y nu ien anspo sys-
ems; and a wide a ie y o mono- and di-oxygenases, which a e
use ul o deg ada ion o na u al p oduc s and xenobio ics, e flux
pumps ha comba an imic obial compounds o di e en o igin,
an ex ensi e se o ex acy oplasma ic unc ion (ECF) sigma ac-
o s; and a wide ange o egula o s and s ess esponse sys ems
ha pe mi hese mic obes o apidly espond o en i onmen al
changes and challenges [5]. The me abolic flexibili y o Pseudomo-
nas de i es no only om an a ay o gene ic de e minan s, bu also
om he igh egula ion o he exp ession o he di e en me a-
bolic pa hways ha allow me abolism o a wide a ie y o chemi-
cals [6,7]. In addi ion, as o he ee-li ing mic oo ganisms, s ains
o he genus Pseudomonas ha e genomes ha con inuously acqui e
new DNA and unde go expansion a he han educ ion [3,4,8]. The
subjec o his s udy is he DOT-T1E (T1E) P. pu ida s ain ha was
isola ed om a was ewa e ea men plan as an e ficien deg a-
de o benzene, e hylbenzene, oluene and o he a oma ic hyd o-
ca bons [9]. These a oma ic hyd oca bons a e oxidized o hei
co esponding ca echols and upon me a clea age, he esul ing
alkyl muconic acid semialdehydes a e di ec ed owa ds he K ebs
cycle [10], whe e gene a ion o NADH akes places and elec ons
a e channeled o a weal h o espi a o y chains [11]. In addi ion,
his s ain is unusually highly esis an o o ganic sol en s such
as oc anol, decanol, benzene, oluene and o he s [9,12], which
has led he scien ific communi y o conside his mic oo ganism
as a model sys em o he s udy o he esponse o g am nega i e
bac e ia o oxic o ganic chemicals. I should be no ed ha
u iliza ion o sol en s as C-sou ces and ole ance o sol en s a e
independen e en s [10]. S ain T1E has been also used o he
de elopmen o bio echnological bio ans o ma ion p ocesses
wi hin wo-phase sys ems o he p oduc ion o p-hyd oxybenzo-
a e, alkylca echols and o he chemical p oduc s [12,13]. T1E has
also been used o he p oduc ion o y osine-de i ed chemicals
h p://dx.doi.o g/10.1016/j. ebsle .2012.07.031
⇑
Co esponding au ho . Add ess: EEZ-CSIC, C/P o . Alba eda, 1, E-18008 G anada,
Spain.
E-mail add ess: [email p o ec ed] (J.-L. Ramos).
1
These wo co-au ho s con ibu e equally o his s udy.
FEBS Le e s 586 (2012) 2932–2938
jou nal homepage: www.FEBSLe e s.o g
Ó2012 Fede a ion o Eu opean Biochemical Socie ies. Published by Else ie B.V.
Open access unde CC BY-NC-ND license.
0014-5793 Ó2012 Fede a ion o Eu opean Biochemical Socie ies. Published by Else ie B.V.
Open access unde CC BY-NC-ND license.
such as p-hyd oxyphenylpy u a e,
L
-DOPA and phenol (Udaondo
e al., unpublished esul s). In his e iew we used he genomics
da a and a ailable ansc ip omic, p o eomic and me abolomic
da a o e eal a pic u e o he asso men o genes in ol ed in sol-
en ole ance.
2. The sequence o he P. pu ida T1E genome
The genomic sequence o T1E was de e mined o iden i y po-
en ial sol en ole ance clus e s (o islands) esponsible o he
enhanced sol en - ole ance o his s ain o e o he P. pu ida
s ains. To his end he T1E genome was sequenced using he 454
echnology. Sange sequencing was used o confi m ameshi s,
po en ial sho dele ions and closing gaps. The esul ing anno a ed
sequence has been submi ed o GenBank (numbe GDSUB19779).
The 6.39 Mbp genome o P. pu ida s ain T1E comp ises wo ci cu-
la eplicons: a single ch omosome o 6,260,702 bp (GC con en o
63%) and a 133,451 bp sel - ansmissible plasmid named pGRT1.
Based on sequence co e age i is mos likely ha pGRT1 is p es-
en in cells a a copy numbe o one plasmid pe ch omosome. The
pGRT1 plasmid encodes one hund ed and 26 p o eins, which ep-
esen less han 2% o he o al numbe o encoded p o eins [14],
and has a sligh ly lowe G + C con en (58%) han he ch omosome,
bu a simila coding densi y [14].
Global alignmen o he T1E genome wi h hose o o he s ains
o his species was ca ied ou using BioEdi so wa e package o
iden i y s ain-specific egions [8,15]. Whole genome alignmen
wi h BioEdi o bo h nucleo ide and deduced p o ein sequences
o GB1, W619, KT2440, F1 and o he s ains e ealed a high ch o-
mosomal gene syn eny wi h some genome ea angemen s and
conside able in e ed alignmen s on bo h sides o he ch omo-
somal eplica ion o igin as compa ed o he o he P. pu ida s ains.
O he 5756 (open eading ames) iden ified in he genome o T1E,
a ound 84% a e sha ed wi h he genomes o he o he ou P. pu ida
s ains. The T1E s ain has 82, 47, 45 and 108 CDS ha a e uniquely
sha ed wi h he s ains KT2440, GB-1, W619 and F1, espec i ely. I
should also be no ed ha T1E con ains 170 unique CDS ha sha e
no simila i y (E alue o < 10
5
) wi h CDS p esen among he se-
quenced Pseudomonas genomes, which sugges s ha hese genes
may ha e o igina ed om mic oo ganisms ou side o he genus
Pseudomonas.
We ha e p e iously gene a ed a genome-wide mu an collec-
ion o T1E and ha e sc eened he collec ion o sol en -sensi i e
s ains. This collec ion comp ises mo e han 30000 mini-Tn5inse -
ions. Sequencing o he inse ion si es e ealed ha hey a e an-
domly dis ibu ed in he genome, wi h an es ima ed a e age
inse ion e e y 200 bp. No mu an exhibi ing sol en -sensi i i y
was associa ed wi h inse ion in any o he 170 unique CDS. The e-
o e, gene iden i y alone could no indica e whe he sol en - e-
la ed genes we e en iched in T1E-specific egions. Below we
p o ide an o e iew o he gene al ea u es o oluene u iliza ion
and sol en - ole ance ai s o T1E, while highligh ing genomic
de e minan s ha a e ele an o hese ai s.
3. The oluene ca abolic pa hway and i s egula ion
To be e unde s and he gene ic con ex o he oluene deg a-
da ion gene clus e in s ain T1E, we examined he ch omosomal
egion in de ail. Homologous sequences nea he 5
0
and 3
0
ends
o a conspicuous 96 kb egion a e con inuous in s ain KT2440
bu in e up ed in s ain T1E. Toluene deg ada ion ( od) akes
places h ough he oluene dioxygenase pa hway encoded by he
odFC1C2BADE ope on [16,17], ollowed in 5
0
by he genes encoding
he co esponding wo-componen egula o y sys em, odST
[18,19]. In addi ion o i s abili y o use oluene, e hylbenzene and
benzene as a sole ca bon sou ce, T1E is able o g ow on p-cymene
(p-isop opyl oluene) and i s acid de i a i e, p-cuma e simila ly o
F1 s ain [20,21]. In T1E as in F1, he cym/cm and he od pa hways
a e loca ed less han 3 kbp apa on a pu a i e genomic island,
which exhibi s lack o syn eny wi h o he P. pu ida s ains. Taken
oge he his da a and conside ing ha he genes a e su ounded
by phage- ela ed genes, sugges s ha his egion may ha e been
acqui ed ia ansduc ion.
Molina-Hena es and Ramos (unpublished esul s) isola ed mu-
an s o he T1E s ain capable o g owing on n-p opylbenzene,
n-bu ylbenzene and isop opylbenzene as a sole ca bon sou ce.
These s ains we e de i ed om he accumula ion o mu a ions
in he genes coding o oluene dioxygenase and ca echol 2,3-diox-
ygenase. Simila ly o he F1 s ain, he wild- ype b oad subs a e
oluene dioxygenase o he T1E s ain can oxidize ichlo oe hylene
(TCE), indole and o he subs i u ed a oma ic compounds [22–24].
The TodS and TodT p o eins o m a highly specific wo-compo-
nen egula o y sys em ha egula es he exp ession o he genes
in ol ed in he deg ada ion o oluene, benzene, and e hylbenzene
h ough he oluene dioxygenase pa hway. TodS is a senso p o ein
ha con ains wo inpu domains, each o which a e ollowed in se-
quence by a his idine kinase domain [19]. TodS has basal au o-
phospho yla ion ac i i y, which is enhanced by he p esence o
e ec o s. We used iso he mal i a ion calo ime y o s udy he
binding o di e en e ec o molecules o TodS, and ela ed hese
findings o hei capaci y o induce gene exp ession in i o. Tolu-
ene was ound o bind o TodS wi h high a fini y (K
d
a ound
700 nM) and a 1:1 s oichiome y, which in u n inc eased he le el
o au ophospho yla ion o TodS. The analysis o he unca ed a i-
an s o TodS e ealed ha oluene binds o he N- e minal inpu
domain (K
d
a ound 2
l
M) bu no o he C- e minal hal . o ho-Sub-
s i u ions o oluene educed o abolished in i o esponses, as
exemplified by o-xylene which is ecognized by TodS wi h high
a fini y, bu does no enhance au ophospho yla ion. Compounds
ha bind TodS bu do no p omo e au ophospho yla ion- egula-
ion we e conside ed an agonis s, in con as wi h agonis s ha
bind and s imula e phospho yla ion. An agonis s and agonis s
compe e o binding o TodS bo h in i o and in i o.
We p opose in amolecula TodS signal ansmission, no
molecula ecogni ion o compounds by TodS, o be he phenome-
non ha de e mines whe he a gi en compound will lead o ac i-
a ion o exp ession o he od genes. Molecula modeling
iden ified esidues F46, I74, F79 and I114 as po en ially in ol ed
in he binding o e ec o molecules. Alanine subs i u ion mu an s
o hese esidues showed educed a fini ies (2- o 345- old) o
bo h agonis ic and an agonis ic compounds [25]. In esponse o
oluene TodS ansphospho yla es TodT, which binds o a ge
DNA binding si es. Lacal e al. [19] epo ed ha in addi ion o TodT
phospho yla ion, in eg a ion hos ac o (IHF) also plays a ele an
ole in he p ocess o ansc ip ional ac i a ion, since exp ession
om he od p omo e was ound o be eigh - old lowe in an
IHF-deficien backg ound. IHF binds be ween he TodT boxes and
he -10 hexame egion, and a unc ional model was p oposed
[19] in which IHF a o s he con ac be ween he TodT ac i a o
and he
a
-subuni o RNA polyme ase (while bound o he down-
s eam p omo e elemen ), leading o e ficien ansc ip ion om
he od ope on.
4. Sol en ole ance de e minan s
The genome o he T1E s ain was also examined o ea u es e-
la ed o adap a ion o he p esence o sol en s, combined wi h
in o ma ion ga he ed h ough a ange o physiological, biochemi-
cal and gene ic analyses ca ied ou in ou labo a o y and o he
labo a o ies ha ha e wo ked wi h he sol en - ole an S12 and
Z. Udaondo e al. / FEBS Le e s 586 (2012) 2932–2938 2933
18733468, 2012, 18, Downloaded om h ps:// ebs.onlinelib a y.wiley.com/doi/10.1016/j. ebsle .2012.07.031 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [09/04/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
he Idaho s ains [26–29]. Sol en ole ance is an ene gy in ensi e
p ocess, and i has been ound ha g ow h yields o Pseudomonas
in he p esence o suble hal oluene concen a ions can be educed
by up o 50% o d y cell weigh . Physiological and biochemical
analyses, oge he wi h he p ofile o p o eins and ansc ip s in
P. pu ida T1E and P. pu ida S12 upon exposu e o suble hal oluene
concen a ions was analyzed e sus cells g owing wi hou oluene.
Wi je e al. [28], an de We e al. [29], Segu a e al. [30] and o h-
e s iden ified almos 90 p o eins ha we e up egula ed as a esul
o an exposu e o he S12 o T1E s ains o oluene. These s udies
showed ha T1E and S12 espond o oluene by: al e ing lipid
composi ion o adap memb ane fluidi y o he p esence o sol-
en s [31,32]; inducing e flux pumps such as gDEF and gGHI
o s pABC o emo e hese oxic chemicals om cell memb anes
[12,33,34]; ac i a ing he ROS de ence sys em o emo e eac i e
oxygen species; and by syn hesizing a numbe o chape ones o e-
old p o eins dena u ed by oluene (Fig. 1)[28,30,35]. Induc ion o
K ebs cycle enzymes and ano he se o enzymes ela ed o ene gy
p oduc ion indica es a equi emen o enhanced me abolism in
o de o powe e flux pumps ha emo e sol en s om he cell
memb anes a p ocess ha seems o be he mos impo an de e -
minan in sol en ole ance. These sol en ole ance esponses a e
bo h mul i ac o ial and combina o ial, and he syne gy be ween
hese elemen s, which include a pool o e flux pump genes and
s ess de ence sys ems, con ibu e o sol en - ole ance.
4.1. Modifica ions a he memb ane lipid le el
O ganic sol en s accumula e in bac e ial memb anes inc easing
memb ane fluidi y [32,36] and many mic oo ganisms espond o
sol en s a he memb ane le el by coun e ac ing he inc ease in
fluidi y. In he sho - e m Pseudomonas esponds by implemen ing
isome isa ion o he cis unsa u a ed a y acids o ans unsa u a ed
a y acids, a eac ion media ed by he cis– ans isome ase [37–39].
The isome isa ion is a quick esponse ha p o ides he bac e ia
wi h dense memb anes and a selec i e ad an age. The isome iza-
ion occu s ia he CTI isome ase, which is encoded by he c i gene.
Be nal e al. [32] used fluo escence pola iza ion assays o show
ha mu an s deficien in he c i gene exhibi ed less igid mem-
b anes han he wild ype s ain. The c i gene was also ound o
be monocis onic and exp essed cons i u i ely a low basal le els
in he log and s a iona y phase. The CTI enzyme is ca aly ically
inac i e in he absence o sol en s, bu is apidly ac i a ed in he
p esence o sol en s. A mu an deficien in CTI exhibi ed e a ded
g ow h wi h espec o he pa en al s ain when exposed o olu-
ene [37].
The cyclop opane a y acids (CFAs) ha e long been ecognized
as an impo an de e minan o acid and alcohol esis ance in Esch-
e ichia coli [40]. Exp ession o he c a syn hase gene in Pseudomo-
nas is also dependen on he RpoS sigma ac o , and exp ession
o he gene occu s when cells en e he s a iona y phase [40,41].
We iden ified a ole o CFAs in sol en ole ance due o he ac
ha a P. pu ida T1E c aB mu an was mo e sensi i e o oluene
shock han he pa en al s ain [42], al hough, c aB exp ession is
no enhanced in esponse o oluene in P. pu ida [43].
In G am-nega i e bac e ia, cell memb ane fluidi y is also influ-
enced by phospholipid head g oup composi ion and he leng h o
linked a y acids [44]. Changes in phospholipid head g oups ha e
also been shown o be in ol ed in sol en ole ance [36,45,46].
Howe e , i is no clea i he changes in ela i e phospholipid con-
Fig. 1. Schema ic ep esen a ion o he main mechanisms in ol ed in he mul i ac o ial sol en ole ance p ocess in P. pu ida s ains mic oo ganisms.
2934 Z. Udaondo e al. / FEBS Le e s 586 (2012) 2932–2938
18733468, 2012, 18, Downloaded om h ps:// ebs.onlinelib a y.wiley.com/doi/10.1016/j. ebsle .2012.07.031 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [09/04/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
en co esponded wi h an inc eased packing o de in he mem-
b ane. Based on da a on a ca diolipin-deficien mu an o P. pu ida
T1E i has been specula ed ha changes in he memb ane a chi ec-
u e, as a consequence o low ca diolipin con en , p o okes a de-
c ease in he e ficiency o he e flux pumps which is ansla ed
in o educed sol en ole ance [32].
The ca diolipin syn hase (cls) genes in he sol en - ole an P.
pu ida T1E s ain is loca ed on a monocis onic ope on and is ex-
p essed om sigma-70 p omo e s. Exp ession om he cls p o-
mo e is six- old highe in he s a iona y phase han in he log
phase, and exp ession o he cls gene is no influenced by sol en s.
Mu an s wi h a knockou in he cls gene exhibi inc eased mem-
b ane igidi y.
4.2. Chape ones and oxida i e s ess esponse
The educed pe meabili y o cells induced ia changes in lipid
composi ion is only pa ial and sol en s ha en e he pe iplasmic
space and cy oplasm dena u e p o eins. This esul s in damage o
which he cell eac s by e olding p o eins and by an ac i a ion
o he ROS de ence sys em o educe ROS-media ed damage.
P o eomic and ansc ip omic assays iden ified genes anno a ed
as in ol ed in ‘hea s ess esponse’ ha we e o e exp essed in
he p esence o sol en s such as e hanol, bu anol, oluene and
xylenes. In ac , i has been well es ablished ha he poH egulon
is up egula ed in he p esence o se e al alcohols [47,48]. The p es-
ence o o ganic sol en s in he cy oplasm and pe iplasm al e p o-
ein olding; hus i is no su p ising ha p oduc ion o di e se
chape ones is necessa y o cope wi h he p esence o he sol en .
A se ies o assays in s ains KT2440, S12 and T1E ha e shown ha
in esponse o oluene a numbe o chape ones a e induced. Ou
bioin o ma ics analysis e ealed ha T1E encodes a leas 41 chap-
e ones, wi hin his se o chape ones only G oEL, G oS, G pE, IbpA
a e exp essed a highe le els in he p esence o oluene. I should
be no ed ha KT2440, a sol en -sensi i e s ain, ac i a es mo e
chape ones han T1E and S12 in esponse o oluene and his
may be ela ed o he ac ha T1E and S12 a e mo e e ficien han
KT2440 in oluene emo al (See e flux pumps below).
S ain T1E was isola ed om a was ewa e ea men plan , a
habi a likely o fluc ua e be ween ae obic, anae obic and mic o-
ae obic condi ions. In expe imen s designed o es he p e e ence
o s ain T1E o oxygen ension, he as es g ow h o s ain T1E in
so aga (0.2%) was obse ed abou 2 mm below he aga su ace,
sugges i e o a p e e ence o a mic oae ophilic li es yle. P. pu ida
T1E is ac i e a low dissol ed O
2
le els in bio eac o s. A mic oae -
ophilic o igin o P. pu ida T1E would be consis en wi h he p es-
ence o he TCA cycle and he anae obic cobalamin biosyn hesis
pa hway. Compa a i e genomics shows ha many o he enzymes
in ol ed in he oxida i e s ess esponse a e common among di -
e en s ains o P. pu ida. Se e al s udies ha e demons a ed ha
alcohols and a oma ic compounds ac i a e he esponse agains
oxida i e agen s and e en p o oke ypical oxida i e damage in
Pseudomonas and o he bac e ia. This is mos likely due o he
in e e ence o sol en s wi h he elec on anspo sys ems ha
leads o an inc ease in he p oduc ion o hyd ogen pe oxide and
o he eac i e oxygen species [35,47,49]. In esponse o oxida i e
s ess se e al genes o he OxyR egulon a e induced by oluene
in P. pu ida KT2440 [35], and genes egula ed by OxyR o N dR
a e commonly up egula ed in E. coli e hanol- ole an s ains [49].
The T1E genome encodes wo supe oxide dismu ases: SodA, a
Mn supe oxide dismu ase (T1E-5070); SodB, a Fe supe oxide dis-
mu ase (T1E-1925). P. pu ida T1E is also p edic ed o con ain fi e,
pe oxidases, namely: Ka A, a pu a i e ca alase; wo alkyl hyd o-
pe oxide educ ases; a chlo ope oxidase; wo hiol pe oxidases;
and wo pu a i e glu a hione pe oxidases (T1E-1488 and T1E-
3636). Addi ionally we ound se e al glu a edoxin genes and six
hio edoxin genes ha may play a ole in hiol edox con ol in
he T1E s ain [50]. The Xen-like enzymes a e known o be ele an
in he esponse o P. pu ida o agen s ha p o oke oxida i e s ess
[51]. In his ega d i is wo h no ing he p esence o six Xenobi-
o ic-like educ ases.
4.3. Ene gy Me abolism and ope a ion o e flux pumps
As men ioned abo e cul u es o T1E and S12 can exhibi lowe
yields when g owing in he p esence o sol en s, sugges ing ha
high le els o ene gy a e essen ial o sol en ole ance [9,52]. In-
deed, p o eomic analysis e ealed ha a numbe o p o eins e-
la ed o ene gy me abolism we e up egula ed upon oluene
exposu e [28]. As desc ibed below he main sol en de ense mech-
anism is media ed by e flux pumps ha ex ude oxic chemicals,
which is an ene ge ically in ensi e p ocess. In s ain S12 i was
ound ha p o eins associa ed wi h he s o age o suga s, such as
GlgX and GlgP, we e down egula ed in he p esence o oluene,
whe eas p o eins in ol ed in he con e sion o glucose, glucoki-
nase (Glk) and glucose-6-phospha e 1-dehyd ogenase (Zw 1) we e
up egula ed, indica ing inc eased p oduc ion o 6-phosphogluco-
na e – he key in e media e o he En ne –Doudo o pa hway
[53]. Con e sely, in S12 gluconeogenesis appea s o be supp essed
upon oluene exposu e as e idenced by he down egula ion o
bo h Fbp, which con e s uc ose 1,6-biphospha e o uc ose 6-
phospha e; and Pgi2, which con e s uc ose-6-phospha e in o
glucose-6-phospha e and ice e sa. These obse a ions s ongly
sugges an inc eased a e o glucose consump ion and dec eased
me abolic gene a ion and s o age o glucose unde sol en s ess.
The inc eased p oduc ion o 6-phosphoglucona e leads o high-
e py u a e le els, which is con e ed o ace yl-CoA – he p ima y
subs a e o he TCA cycle. Mo eo e , he inc eased ace a e up ake
and con e sion o ace yl-CoA sugges s inc eased ac i a ion o he
TCA cycle by an al e na i e means o glucose con e sion. Se e al
p o eins o he TCA cycle we e also ound o be up egula ed in
he p esence o oluene, including Mqo-1, SdhD and SucD, as well
as se e al NADH dehyd ogenase and ATP syn hase subuni s
[28,30]. The up egula ion o p o eins in ol ed in ene gy p oduc-
ion sugges s ha P. pu ida S12 and T1E ha e he abili y o espond
o high ene gy demands due o oluene exposu e by inc easing
ene gy me abolism, which is in ag eemen wi h a ious fluxomics,
p o eomics and ansc ip omics s udies [20,30].
Se e al e minal oxidase genes we e ound o be up egula ed in
he p esence o sol en s (pu a i e cy och ome aa
3
- ype oxidase,
cy och ome cbb
3
- ype oxidase and cy och ome bd- ype quinol oxi-
dase), sugges ing adap a ion by s ain T1E o a iable ae obic and
mic oae obic condi ions as well as o sol en s, a si ua ion ha de-
mands ene gy consump ion due o he high ac i i y o e flux
pumps [11].InP. pu ida T1E complex I consis s o an NADH ubiqui-
none oxido educ ase (EC 1.6.5.3) wi h 14 subuni s coded o by he
nuo genes (A h ough N) ha a e g ouped in a gene clus e . The
cy och ome bc1 complex, o quinol:cy och ome coxido educ ase
(EC: 1.10.2.2); and a NADH dehyd ogenase (EC: 1.6.99.3), made
o h ee independen subuni s ha a e FAD-dependen and a e un-
linked in he genome (nda, ORF 00102 and ORF 00044 and 1.6.99.5
F2) a e used o op imize he NADH/NAD
+
balance unde changing
en i onmen al condi ions. Complex II is a succina e dehyd ogenase
(EC1.3.99.1) comp ising ou subuni s (SdhA, SdhBCD ha a e clus-
e ed [TIE 00016 h ough 0019]. Complex IV consis s o cy o-
ch ome-coxidase/cy och ome aa3 genes. I is likely ha unde
high oxygen ension he aa3- ype oxidase is used, and ha he
cbb3- ype oxidase is used unde mic oae ophilic condi ions [54].
The quinol oxidase bd is a high-a fini y e minal oxidase o g ow h
a low O
2
ension. S ikingly, he cbb3- ype oxidase genes ha e a
G + C con en o 63%, a alue highe han a e age GC con en o
he T1E and S12 s ains.
Z. Udaondo e al. / FEBS Le e s 586 (2012) 2932–2938 2935
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In he S12 s ain, p o eomics analyses sugges ed di e en ial
exp ession o he cy och ome oubiquinol oxidase complex upon
exposu e o sol en s, and showed ha CyoB and CyoA exhibi a
simila change in exp ession le el in oluene adap ed cells, while
o he NADH dehyd ogenase complex, only ou subuni s o he
se en iden ified subuni s we e di e en ially exp essed when cells
g ew in he p esence o oluene. One o he mos ele an ea u es
obse ed wi h a
D
cyoB mu an o T1E is ha i exhibi ed a ma ked
educ ion in global me abolism sugges ing ha he limi a ion in
ene gy gene a ion associa ed wi h his e minal oxidase has a gen-
e al e ec on cell me abolism in he p esence o oluene. Fo exam-
ple, a y acid biosyn hesis is g ea ly limi ed, which ag ees wi h
p e ious obse a ions ha CyoB mu an cells p oduce memb ane
in agina ions ha a ec cell memb ane s uc u e and conse-
quen ly esul in ex eme sensi i i y o sol en shocks [55].
E flux pumps, especially hose belonging o he RND amily, a e
conside ed o be mos impo an mechanism o sol en ole ance
in G am-nega i e bac e ia [45]. RND anspo e s a e p o on-d i-
en e flux sys ems comp ising h ee p o eins ha o m a mul i-
componen complex ex ending om he inne memb ane o he
ou e memb ane (Fig. 2)[56]. This molecula o ganiza ion pe mi s
bac e ia o ex ude compounds ia wo possible pa hways: om
he pe iplasm o he ex e nal medium o om he cy oplasm o
he ex e nal medium [57]. Membe s o he ABC- anspo e s
(ATP Binding Casse e) ha e also been implica ed in sol en ole -
ance [58].
Mos G am-nega i e bac e ia encode se e al RND e flux pumps
in hei genomes (up o 20 in he highly sol en ole an s ain P.
pu ida T1E). Su i al analysis o P. pu ida T1E cul u es a e oluene
shock (addi ion o a second phase o oluene) e ealed ha h ee
RND e flux pumps, wi h di e en bu o e lapping subs a e speci-
fici y, a e di ec ly in ol ed in oluene esis ance. We ha e gene -
a ed a collec ion o hese mu an s in T1E and ha e es ed hei
ole in oluene ole ance. The p esence o T gGHI, encoded on he
133-kb pGRT1 plasmid, was ound o be absolu ely necessa y o
su i e a 0.3% ( / ) oluene shock [14,62]. Mu an s o wo o he
pumps, namely T gABC [59] and T gDEF [33], we e also isola ed.
The T gABC pump is he main an ibio ic ex usion pump, and is
able o ex ude ampicillin, chlo amphenicol, e acycline and fla o-
noids in addi ion o oluene and o he sol en s [43,60,61]. The T g-
DEF (PP3425–PP3427) e flux pump has been shown o be in ol ed
in a oma ic hyd oca bon de oxifica ion [33].
In he S12 s ain upon oluene exposu e mos RND anspo e
sys ems become down egula ed excep he e flux pumps S pABC
(highly simila o T gGHI) and PP1272. The mos p ominen
up egula ion was obse ed o p o eins o he S pABC e flux sys-
em (>15- old), which can be di ec ly linked o he sol en ole -
ance o P. pu ida S12. The up egula ion o PP1272 in he p esence
o oluene is in e es ing because his obse a ion sugges s ha
P. pu ida S12 also uses addi ional e flux sys ems o o ganic sol en
anspo .
4.4. Ex acy oplasmic sigma ac o s (ECFs) and egula ion o e flux
pump exp ession
P. pu ida encodes 20 ECFs. We ha e shown ha one o hese
ECFs, known as ECF-Pp12 (PP3006), plays a ole in ole ance o ol-
uene and o he o ganic sol en s. Based on his finding, we ha e
called he gene ha encodes his new ECF poT. The poT gene
o ms an ope on wi h he p eceding gene and wi h he gene lo-
ca ed downs eam. The ansla ed gene p oduc o he PP3005
open eading ame is an inne memb ane p o ein, whe eas he
PP3007 p o ein is pe iplasmic (G. Na a o-A ilés and J.L. Ramos,
unpublished). A non-pola
D
poT mu an was gene a ed by homol-
ogous ecombina ion, and su i al o he mu an was es ed unde
a ious s ess condi ions. The mu an s ain was hype sensi i e o
oluene and o he sol en s, bu jus as ole an as he wild ype o
s ess imposed by hea , an ibio ics, NaCl, pa aqua , sodium dodecyl
sul a e, H
2
O
2
and benzoa e. In he
D
poT mu an backg ound,
exp ession o app oxima ely 50 ansc ip ional uni s was a ec ed:
31 cis ons we e up egula ed, and 23 cis ons we e down egu-
la ed. This indica es ha abou 1% o all P. pu ida genes a e unde
Fig. 2. (A) Model o an RND e flux pump based on he s uc u e o i s componen s. This model ep esen s he possible assemblage o he T gGHI p o eins based on he Ac
sys em [34]. (B) Gene o ganiza ion o he e flux pumps in ol ed in oluene ole ance in P. pu ida DOT-T1E (le panel) and esponse o he wild- ype (black) and i s isogenic
mu an s o a sudden sol en shock ( igh panel). Fu he de ails can be ound in [42].
2936 Z. Udaondo e al. / FEBS Le e s 586 (2012) 2932–2938
18733468, 2012, 18, Downloaded om h ps:// ebs.onlinelib a y.wiley.com/doi/10.1016/j. ebsle .2012.07.031 by Csic O ganización Cen al Om (O icialia Mayo ) (U ici), Wiley Online Lib a y on [09/04/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License

he di ec o indi ec influence o RpoT. The poT gene con ols he
exp ession o a numbe o memb ane p o eins, including compo-
nen s o he espi a o y chains, po ins, anspo e s, and mul id ug
e flux pumps. Hype sensi i i y o he P. pu ida RpoT-deficien mu-
an o o ganic sol en s can be a ibu ed o he ac ha in he
D
poT s ain exp ession o he oluene e flux pump gGHI genes
is se e al old lowe han in he pa en al s ain.
Recen ly, ex ensi e analyses o he egula ion o he gABC, g-
DEF and gGHI ope ons in T1E and S pABC in S12 ha e been ca -
ied ou . T gR, which belongs o he Te R amily o egula o s, is
he specific ansc ip ional ep esso o he gABC. The basal
exp ession o his pump is inc eased in he p esence o an ibio ics,
fla onoids and alcohols bu i does no a y in he p esence o a o-
ma ic compounds [1,63,64]. The T gR ope a o is a 36 bp sequence
loca ed in he gR– gA in e genic egion and o e laps wi h he -
10 and -35 egions o he gABC p omo e , and he -10 egion o
he gR p omo e . In he absence o e ec o s, he T gR dime is
bound o i s ope a o si e ep essing i s own exp ession and ha
o he e flux pump. E ec o binding o he p o ein–DNA complex
induces he dissocia ion o T gR, and allows ansc ip ion. The
c ys al s uc u e o he T gR p o ein in complex wi h e ec o s
has been de e mined [65]. T gR has a hyd ophobic binding pocke ,
which explains T gR’s abili y o bind di e en ligands. Wi hin his
pocke wo binding si es we e iden ified: one ha binds ligands
wi h high a fini y and he second ha binds molecules wi h low
a fini y.
T gV belongs o he IclR amily o ansc ip ional egula o s and
is he main egula o in he modula ion o he exp ession o gDEF
and gGHI ( e iewed in [66]). The gDEF ope on is silen in he ab-
sence o e ec o s, while a basal exp ession le el o he gGHI op-
e on has been epo ed [64,67]. Exposu e o P. pu ida T1E o
a oma ic compounds (i.e. 4-ni o oluene, benzoni ile, 1-naph-
hol), inc eased he ansc ip ion a e o gDEF and gGHI [67].
T gV is a e ame in solu ion and also when bound o i s 42-bp a -
ge ope a o in he gDEF and gGHI in e genic egions. The
ep esso binds o and masks he -10 egion o each p omo e
[66,68]. I should be no ed ha he p omo e s o gG and gV pa -
ially o e lap each o he [67].
Each T gV monome has wo domains, one comp ising he HTH
DNA binding domain a he amino e minal end, and an e ec o
binding egion a he cen al egion and C e minus o he p o ein
[69]. The wo domains a e b idged by a linke , whose ole is o
se e as a signal ansmission elemen be ween he wo domains
ha a e physically disconnec ed [70]. Residues R98 and E102 wi h-
in he linke , which b idge bo h domains, a e c i ical o he co ec
ansmission o he signal om he e ec o binding pocke o he
DNA binding domain. Once he ansmission is achie ed T gV mod-
ifies i s s uc u e and is eleased om he DNA, a his poin he
RNA polyme ase is able o bind he p omo e egions and ini ia e
gG and gV ansc ip ion [67,68]. Al hough a T gV/e ec o co-
c ys al s uc u e has no been ob ained, compa ison o he apo-
T gV s uc u e wi h he s uc u e o he T gV–DNA complex
showed a majo e-a angemen in T gV [69,70], which is consis-
en wi h a model p oposed abo e.
Regula ion o he S pABC e flux pump in P. pu ida S12 (homol-
ogous o he T gGHI in T1E) exhibi s g ea complexi y. In his bac-
e ium, wo di e en egula o s, S pS (T gV in T1E) and S pR
pa icipa e in con ol o he exp ession o he e flux pump ope on
[71,72]. In addi ion, wo inse ion elemen s, ISS12 and ISPpu21,
can inse in o s pS o block exp ession, hus de ep essing exp es-
sion o he e flux pump [72]. S pS is a ep esso o he e flux pump
and S pR is an an i ep esso which binds o S pS in such a way ha
i inhibi s he S pS–DNA in e ac ion, he eby acili a ing i s elease
i p ebound o he p omo e egion [72].
In sho , sol en ole an s ains uses a mul i ac o ial esponse
ha in ol es fine- uning o lipid fluidi y, ac i a ion o a gene al
s ess- esponse sys em, enhanced ene gy gene a ion, and induc-
ion o specific e flux pumps ha ex ude sol en s o he medium.
Local and global ansc ip ional egula o s pa icipa e in a complex
ne wo k o me abolic unc ions, ac ing as he decision make s in
he esponse o sol en s.
Acknowledgemen s
Wo k in ou labo a o y was suppo ed by Fondo Social Eu opeo
and Fondos FEDER om he Eu opean Union h ough p ojec
BIO2010-17227 and Jun a de Andalucía P oyec o de Excelencia
CVI-3010.
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