Iden i ica ion o he Rep esso
Subdomain wi hin he Signal
Recep ion Module o he
P oka yo ic Enhance -binding
P o ein XylR o Pseudomonas
pu ida*
(Recei ed o publica ion, Oc obe 25, 1995, and in e ised o m,
Janua y 16, 1996)
Jose´Pe´ ez-Ma ı´n and Vı´c o de Lo enzo‡
F om he Cen o de In es igaciones Biolo´gicas,
Consejo Supe io de In es igaciones Cien ı´ icas,
Vela´zquez 144, 28006 Mad id, Spain
In he p esence o m-xylene, he p o ein XylR encoded
by he TOL plasmid o Pseudomonas pu ida, ac i a es
he
s
54
-dependen p omo e Pu. Ea ly ac i a ion s ages
in ol e he elease o he in amolecula ep ession
caused by he signal ecep ion N- e minal (A domain) o
XylR on he cen al module o he p o ein. A gene ic
app oach has been ollowed o loca e he speci ic seg-
men wi hin A domain o XylR ha is di ec ly esponsi-
ble o i s down- egula ion in he absence o induce , as
compa ed o ha in ol ed in e ec o (m-xylene) bind-
ing. Fo his, a epo e Esche ichia coli s ain ca ying
a monocopy ansc ip ional usion o Pu o lacZ was
ans o med wi h a collec ion o plasmids encoding
equi alen unca ed a ie ies o XylR, consis ing o
nes ed and in e nal dele ions h oughou he en i e A
domain. Examina ion o he esul ing pheno ypes al-
lowed he assignmen o he A domain egion nea he
cen al ac i a ion domain, as he po ion o he p o ein
esponsible o he speci ic ep ession o XylR ac i i y
in he absence o m-xylene.
S ains o he genus Pseudomonas ha bo ing he TOL plas-
mid pWW0 can g ow on oluene, m-xylene, and p-xylene as
he only ca bon sou ce owing o he ac i i y o a complex
ca abolic pa hway (summa ized in Fig. 1) ha p oceeds in
wo majo biochemical s eps (Nakazawa e al., 1990). These
a e de e mined by wo independen ope ons ha become
coo dina ely ansc ibed when bac e ia ace pa hway sub-
s a es such as m-xylene (see Ma que´s and Ramos (1993) o
a e iew). The main egula o o he sys em is he so-called
XylR p o ein, a membe o he amily o p oka yo ic enhanc-
e -binding egula o s ha ac in conce wi h he al e na i e
s
ac o
s
54
(Mo e and Sego ia, 1993; No h e al., 1993). In
he p esence o xylenes, XylR bound o ups eam sequences
ac i a es he Pu p omo e o he uppe -TOL ope on, hus
igge ing exp ession o he co esponding ca abolic genes (de
Lo enzo e al., 1991; Ab il e al., 1991). The e y ea ly chain
o e en s ha ansla es he p esence o xylenes in o ac i a-
ion o Pu, in ol e e ec o -media ed con e sion o XylR in o
a ansc ip ionally compe en o m. Fo his, he induce
binds di ec ly o he N- e minal, signal ecep ion module o
XylR e med he A domain (Delgado and Ramos, 1994; Fe -
na´ndez e al., 1995; see Fig. 1) and igge s he elease o he
ep ession caused by his domain on he cen al po ion o he
p o ein ha is in ol ed in he con ac and ac i a ion o he
s
54
-con aining RNA polyme ase (Pe´ ez-Ma ı´n and de
Lo enzo, 1995). This no ion is based on he obse a ion ha
dele ing he en i e A domain o XylR gi es ise o a unca ed
p o ein ha ac i a es cons i u i ely Pu in he absence o
a oma ic e ec o s, bo h in i o (Fe na´ndez e al., 1995) and
in i o (Pe´ ez-Ma ı´n and de Lo enzo, 1996). I seems, he e-
o e, ha he A domain o XylR has a leas wo unc ions: (a)
ecogni ion o he a oma ic induce s and (b) in amolecula
ep ession. Since hese wo a e ob iously connec ed, he
ques ion a ises as whe he disc e e subdomains wi hin he
N- e minal module can be assigned o each o hem.
To explo e he p esence and he loca ion o speci ic po ions
wi hin he A module o XylR ha a e accoun able o he
cen al domain ep ession, we chose a epo e sys em in which
he e ec o changes in he egula o could be ela ed immedi-
a ely o a dis inc pheno ype in ac i a ion o Pu. Since all
ansc ip ional con ol elemen s can be ai h ully ep oduced in
Esche ichia coli, his epo e sys em employs a de i a i e o E.
coli YMC10 ha had been lysogenized wi h an specialized
l
phage con aining a ansc ip ional Pu-lacZ usion (Pe´ ez-
Ma ı´n and de Lo enzo, 1995). This s ain (E. coli
l
RSPu) was
ans o med independen ly wi h each one o a collec ion o
plasmids bea ing unca ed xylR alleles ha di e ed only in A
domain sequences (Fig. 1). These we e gene a ed wi h a polym-
e ase chain eac ion-based s a egy (PCR)
1
desc ibed in he
legends o Figs. 2 and 3. The ac i i y o he unca ed p oduc s
exp essed in ans was measu ed as he accumula ion in i o o
b
-galac osidase in he p esence o absence o he XylR e ec o
m-xylene. Simul aneously o each ac i i y assay, we examined
he le el o exp ession o each XylR-de i ed p o ein h ough
Wes e n blo assays (Fe na´ndez e al., 1995) o ensu e ha he
p o eins we e p oduced a simila le els (no shown).
To ha e a p elimina y indica ion on he po ion o he A
domain o XylR in ol ed in in amolecula ep ession, we
sough o di ide he A domain (211 amino acids, Inouye e al.
(1988) and Shingle e al. (1993); see Fig. 2) in 6 la ge seg-
men s, ha we e p og essi ely dele ed om he N e minus
(Fig. 2). These dele ions we e gene a ed by ampli ying wi h
PCR he sequences o in e es wi h an adequa e collec ion o
p ime s, so ha he esul ing p oduc s we e lanked by EcoRI
and BamHI si es as speci ied in he legend o Fig. 1. T ans e
o he esul ing DNA agmen s o he specialized exp ession
ec o pP (Pe´ ez-Ma ı´n and de Lo enzo (1995); see legend o
Fig. 2) p o ided a ansla ion ini ia ion sequence and a leading
ATG o exp ession o he six xylR dele ion alleles shown in Fig.
2. These we e named, espec i ely, D30, D120, D150, D180,
D210, and D226. The co esponding p o eins we e p oduced in
i o a le els compa able o hose o he wild- ype XylR wi h
* This wo k was suppo ed by G an BIO95–0788 o he Spanish
Comisio´n In e minis e ial de Ciencia y Tecnologı´a (CICYT) and Con-
ac BIOTECH BIO2-CT92–0084 o he Eu opean Union. The cos s o
publica ion o his a icle we e de ayed in pa by he paymen o page
cha ges. This a icle mus he e o e be he eby ma ked “ad e isemen ”
in acco dance wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac .
‡ To whom co espondence should be add essed. P esen add ess:
Cen o Nacional de Bio ecnologı´a-CSIC, Campus de Can oblanco,
28049 Mad id, Spain. E-mail: [email p o ec ed].
1
The abb e ia ion used is: PCR, polyme ase chain eac ion.
Communica ion THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 271, No. 14, Issue o Ap il 5, pp. 7899–7902, 1996
© 1996 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc.
P in ed in U.S.A.
7899
This is an Open Access a icle unde he CC BY license.
he same exp ession sys em, as assessed by Wes e n blo , and
hey we e equally able o shu down exp ession o he P
p omo e o he xylR gene in an in i o au o ep ession assay
(Fe na´ndez e al. (1995); no shown). These expe imen s e i-
ied ha he p o eins we e exp essed a simila le els and ha
hey we e e icien ly able o bind DNA. In e media e dele ions
be ween posi ions 30 and 120 nei he p oduced any de ec able
p o ein no ga e posi i e in an au o ep ession assay, and, hus,
hey we e no u he conside ed (no shown). The six p oduc-
i e dele ions displayed e y dis inc pheno ypes when he
co esponding plasmids we e ans o med in he Pu-lacZ E.
coli epo e s ain (Fig. 2). While unca ion o he 30 leading
amino acids (XylRD30) ga e ise o a p o ein wi h only a esid-
ual esponsi eness o he XylR induce , m-xylene (Fig. 2), sub-
sequen emo al o he p o ein segmen up o posi ion 120
(XylRD120) and beyond (XylRD150) ga e ise o p o eins un-
able o ac i a e he Pu p omo e o any signi ican ex en .
Howe e , dele ion o 30 addi ional amino acids (XylRD180)
pa ially es o ed he abili y o he p o ein o p omo e an-
sc ip ion (Fig. 2), al hough esponsi eness o m-xylene was los
and he ac i i y became cons i u i e. Dele ion o 30 addi ional
amino acids (XylRD210), which emo ed he A domain en i ely,
inc eased
b
-galac osidase accumula ion o i s highes le el.
Fu he dele ions en e ing he Q-linke (XylRD226) did no
esul in an addi ional inc ease o p omo e ac i i y, while
dele ions beyond posi ion 233, en e ing in o he cen al C do-
main (Fig. 1), abolished any ansc ip ional ac i i y o he
esul ing unca ed p o eins (no shown).
The esul s abo e con i med he exis ence o a speci ic egion
wi hin he A domain o XylR di ec ly in ol ed in in amolecu-
la ep ession loca ed be ween amino acid posi ions 150 and
210. To na ow down he loca ion o he bounda y, we made wo
addi ional dele ions in 10-amino acid inc emen s called
XylRD160 and XylRD170. As shown in Fig. 2, while D160 e-
mained inac i e, D170 ga e a subs an ial, bu no ull, cons i-
u i e ac i a ion o Pu, as compa ed o he maximum p omo e
ac i a ion caused by he dele ion o he en i e A domain
(XylRD210, XylRD226). These esul s loca ed he le wa d limi
o he ep esso subdomain a a ound amino acid posi ion 170,
and, he e o e, he whole unc ional module may span no mo e
han 40 esidues (o 60, i we also include some amino acids
p esen a he hinge B domain, Fig. 1).
To e i y he loca ion o he ep esso segmen o he A
domain sugges ed by he pheno ypes caused by he N- e minal
dele ions, we cons uc ed addi ional xylR alleles bea ing se-
quen ial unca ions o he A domain s a ing in posi ion 210
(i.e. a he C- e minal end o he module) and spanning inc eas-
ingly la ge segmen s owa d he N- e minal end (see legend o
Fig. 3 o he p ocedu e employed o hei cons uc ion). As
be o e, p oduc ion in i o o he p edic ed mu an p o eins was
e i ied h ough au o ep ession assays and Wes e n blo o he
co esponding cell ex ac s (no shown). The pheno ypes en-
dowed by each o he cons uc ions lis ed in Fig. 3 we e exam-
ined as be o e wi h he esul s summa ized in Fig. 3. As ex-
pec ed, dele ions spanning la ge po ions o he A domain (D60/
210, D120/210, and D150/210) o igina ed unca ed p o eins
ha we e ully cons i u i e. I was mos ema kable, howe e ,
ha dele ions D160/210, D180/210, and D190/210 ( ha a e
o ally o pa ially dele ed o he ep esso subdomain), al-
hough capable o ac i a ing ansc ip ion in he absence o
induce , main ained a signi ican deg ee o esponsi eness o
m-xylene. Such esponsi eness was los when he leading N
e minus was dele ed also ( unca ion D30-D190/210). This sug-
FIG.1.The TOL sys em o plasmid pWW0 and domain o gani-
za ion o XylR. The TOL sys em o deg ada ion o oluene and m-/p-
xylene includes wo gene clus e s, he uppe -ope on and he me a-
ope on, as well as wo egula o y genes, xylS and xylR, downs eam o
he me a-ope on. The
s
54
-dependen p omo e s Pu and Ps (unde lined)
a e ac i a ed by he cogna e ac i a o XylR in he p esence o m-xylene,
while he Pm p omo e is ac i a ed by XylS in he p esence o benzoa e
o olua es. Func ional domains o XylR a e shown expanded, wi h an
indica ion o he amino acid posi ions co esponding o he bounda ies
o each domain (Inouye e al., 1988; Shingle , 1996). These include a
signal ecep ion N- e minal module (A domain), he cen al (C) ac i a-
ion domain, and he C- e minal segmen (D domain) con aining a
helix- u n-helix (HTH) mo i o DNA binding. The lowe pa o he
igu e ske ches he s a egy used o ampli y wi h he polyme ase chain
eac ion (PCR) speci ic DNA segmen s co esponding o di e en po -
ions o he XylR p o ein o exp ession o unca ed a ian s o he A
domain. These include N- e minal dele ed o in e nally unca ed p o-
eins (see legends o Figs. 2 and 3).
FIG.2.E ec o sequen ial N- e minal dele ions h ough he A
domain in he ansc ip ional ac i i y o XylR. The ac i i y o he
N- e minal unca ed de i a i es o he A module o XylR (211 amino
acids, Fig. 1) indica ed in he igu e was moni o ed as accumula ion o
b
-galac osidase in he E. coli s ain
l
RSPu, ha ca ied a ch omosomal
Pu-lacZ usion. Fo cons uc ion and exp ession o each o he unca ed
p o eins, he ollowing s a egy was pu sued. A DNA agmen con ain-
ing he wild- ype xylR sequence was subjec ed o a PCR eac ion using
as di ec p ime s (Fig. 1) a ious oligonucleo ides (33-me s) ha a -
ached an EcoRI si e o he le o he si e o dele ion desi ed. Fo he
e e se p iming o he eac ion, he same oligonucleo ide was used in all
cases, ha gene a ed a BamHI si e ollowing he STOP codon o he
xylR sequence, TAG. The ampli ied p oduc s we e cloned as EcoRI-
BamHI agmen s o di e en sizes a he same si es o ec o pP . This
is a de i a i e o pCG1 (Mye s e al., 1987) in which he na i e P
p omo e o xylR wi hin he TOL plasmid (Fig. 1) has been enginee ed
in on o he same polylinke as pT c99A (Amman e al., 1988), ha is
led by an NcoI si e o e lapping a i s s uc u al ATG. This allowed all
unca ed p o eins o be exp essed h ough he e y same na i e p o-
mo e and ansla ion ini ia ion egions as he wild- ype xylR. The use
o he di ec EcoRI p ime s in he PCR eac ion in oduced in all cases
amino acid esidues EF (co esponding o 59-GAA TTC-39), nex o he
leading me hionine o he unca ed p oduc s. Replacemen o he sec-
ond (Se ) and hi d (Leu) amino acid esidue o he wild- ype XylR
p o ein by EF had no e ec on p o ein ac i i y (no shown). Fo he
expe imen o he igu e, each o he E. coli
l
RSPu ans o man s we e
g own in LB medium (Mille , 1972) a 30 °C up o an A
600
50.5, a e
which hey we e exposed, as indica ed, o sa u a ing apo s o m-
xylene. Accumula ion o
b
-galac osidase (Mille , 1972) was hen meas-
u ed a e 5ho induc ion. The igu es o he epo e p oduc a e
indica ed wi h espec o he si e o he dele ions co esponding o each
unca ed egula o . The alues shown a e he a e age o 3 independen
expe imen s ca ied ou wi h duplica e samples. The app oxima e lo-
ca ion o he ep ession subdomain sugges ed by he esul s is indica ed
on op.
Func ional Subdomains o he N Te minus o XylR7900
ges ed ha such a leading egion is in ol ed in e ec o ecog-
ni ion, li ing o in amolecula ep ession, o bo h. Since dele-
ions D160/210, D180/210, and D190/210 a e p edic ed o o se
conside ably he ela i e posi ioning o he emaining A se-
quence wi h espec o he cen al domain o he p o ein, he
egula ion by m-xylene e ained by hese p o eins indica ed
ha e ec o binding and associa ed changes in p o ein s uc-
u e do no in ol e pe se speci ic in e domain in e ac ions. In
addi ion, simul aneous dele ion o he segmen 190–210 and
he leading 30 amino acids ga e ise o a cons i u i e low
ac i i y egula o (Fig. 3), ha may e lec he need o an in ac
N e minus o any esponsi eness o he e ec o . Speci ic
ep ession seems, he e o e, o be due exclusi ely o he po ion
o he A domain encompassing posi ions 160 o 226. Fu he -
mo e, he ac ha he in e nal dele ion D160/185 ( ha lacks
he le mos bounda y o he ep esso subdomain wi h he es
o he p o ein) is i ually inac i e (Fig. 3) sugges ed ha he
p o ein segmen s de e mining he esponse o e ec o ecogni-
ion may be connec ed o each o he close o posi ions 160–170.
In ac , dele ion o he zone a ound 170 seems o o igina e a
p o ein ha is inhibi ed unspeci ically by he emainde o he
A domain (see below). In e es ingly, Delgado and Ramos (1994)
ound ha a mu a ion in esidue 172 made he p o ein o
espond o a new e ec o (m-ni o oluene), hus indica ing ha
some di ec o indi ec de e minan s o ligand speci ici y may
lie also a ound posi ion 170.
The semicons i u i e pheno ype o dele ions D160/210, D180/
210, and D190/210 (Fig. 3) indica ed also ha elimina ion o he
ep esso subdomain s ill a o ds a deg ee o down- egula ion
o XylR by he emainde o he A module. This phenomenon
can be easily unde s ood in ligh o he obse a ions epo ed
elsewhe e (Fe na´ndez e al., 1995) on he inac i a ion o XylR
h ough subs i u ion o i s A domain by a bulky he e ologous
p o ein module. On his basis, i is e y likely ha al hough
dele ions D160/210, D180/210, and D190/210 ha e los he spe-
ci ic egion in ol ed in in amolecula ep ession, hey s ill
e ain a po ion o he p o ein ha inhibi s i s ull ac i i y in
he absence o induce by a me e physical hind ance o an
ac i a ion su ace and no because o an speci ic in e domain
in e ac ion. The same a gumen explains he pheno ype o he
dele ion D160/185 (Fig. 3), ha is i ually inac i e in i o in
spi e o ha ing los a p o ein segmen ha en e s a egion
in ol ed in speci ic in amolecula ep ession (see abo e). In
his case, he lowe ac i i y o D160/185 (Fig. 3) as compa ed o
D180 (Fig. 2) is he likely esul o he s e ic hind ance caused
by he emainde o he A domain p esen in he unca ed
p o ein.
Taken oge he , hese esul s led o he conclusion ha a
p o ein segmen as sho a 40–50 amino acids ully accoun s
o he ep ession caused by he whole A domain on XylR in he
absence o m-xylene. Ou da a a e consis en also wi h he
no ion ha only ha po ion o he A module main ains speci ic
in e ac ions wi h he cen al domain o he p o ein. In addi ion,
he pheno ypes o igina ed by he in e nally unca ed p o eins
(Fig. 3) sugges ha he XylR po ion in ol ed in e ec o ec-
ogni ion may exis as a sepa a e subdomain wi hin he N-
e minal module. P edic ion o seconda y s uc u e wi hin he
egion ca ied ou wi h he PHD p o ile ne wo k me hod
(Bu kha d and Sande , 1993) indica ed ha amino acids 219 o
221 could be o ganized as a somewha long (21 esidues)
a
-he-
lix. In e es ingly, when compa ed o he lib a y o p o ein
c ys al coo dina es o he B ookha en da a bank, pa o ha
a
-helix and a ew p eceding esidues (po ion 204–222 o he A
domain) bea signi ican esemblance o he egion o he eu-
ka yo ic p o ein c-Fos ( esidues 168 o 186) ha is in ol ed in
p o ein-p o ein in e ac ions wi hin a c-Fos/c-Jun/DNA co-c ys-
al (B ookha en ID code: 1FOS). This s uc u al esemblance
migh be ela ed o he speci ic in e ac ions be ween he A
domain and he cen al domain o XylR p oposed o con ol he
ac i i y o he egula o (Pe´ ez-Ma ı´n and de Lo enzo, 1995).
On he con a y, no po ion o he 160–220 egion o XylR
showed any s uc u al simila i y o he N- e minal domain o
Dc D, ano he membe o he amily o
s
54
-dependen egula-
o s whose ac i i y is egula ed h ough in amolecula ep es-
sion as well (Gu e al., 1994).
The A domain o XylR is he key componen o his ac i a o
ha endows speci ici y in he esponse o m-xylene (Delgado
and Ramos, 1994; Shingle and Moo e, 1994), so ha di ec
e ec o binding is ansla ed in o elease o in amolecula
ep ession (Delgado e al., 1995; Pe´ ez-Ma ı´n and de Lo enzo,
1995). Al hough he p ecise mechanism by which his happens
emains unsol ed, he da a p esen ed in his wo k sugges ha
di e en po ions o he A domain ha e speci ic oles in he
p ocess. I is possible ha , simila ly o wha may happen o
N C (Fiedle and Weiss, 1995), de ep ession could in ol e he
dime iza ion o he N- e minal module in esponse o he e-
cep ion o he ac i a ing signal. The di e en p edic ions aised
by his hypo hesis and o he al e na i es a e cu en ly unde
s udy in ou labo a o y.
REFERENCES
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Labo a o y P ess, Cold Sp ing Ha bo , NY
FIG.3.Pheno ypes endowed by in e nal unca ions o he A
domain o XylR. The abili y o xylR alleles ca ying he in e nal
dele ions wi hin he A domain indica ed in he igu e was examined as
o he sequen ial N- e minal dele ions (Fig. 2). The s a egy o gene -
a e he in e nally unca ed p o eins in ol ed he p oduc ion by PCR o
wo es ic ion agmen s ha we e sequen ially assembled in ec o
pP (see legend o Fig. 2). The “le ” es ic ion agmen s ( lanked by
EcoRI-XbaI si es) we e p oduced by ampli ying he desi ed pa o he
A domain sequence wi h a di ec EcoRI p ime and a e e se XbaI
p ime (Fig. 1). The “ igh ” es ic ion agmen s we e simila ly p o-
duced wi h a di ec XbaI p ime , a ge ed o he si e o in e es wi hin
he sequence o he A domain, and a e e se BamHI p ime a he end
o he xylR sequence (Fig. 1). Thei assembly downs eam o he P
p omo e in pP ga e ise o he unca ed p o eins unde examina ion.
Accumula ion o
b
-galac osidase by each o he epo e s ains, ans-
o med wi h he co esponding plasmids, was examined as desc ibed in
he legend o Fig. 2. The ha ched po ion o he A module shows he
posi ion o he ep ession subdomain.
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