Identification of the repressor subdomain within the signal reception module of the prokaryotic enhancer-binding protein XyIR of Pseudomonas putida

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 Ab il, M. A., Buck, M., and Ramos, J. L. (1991) J. Biol. Chem. 266, 15832–15838 Amann, E., Ochs, B., and Abel, K. J. (1988) Gene (Ams .) 69, 301–315 Bu kha d, R., and Sande , C. (1993) J. Mol. Biol. 232, 584–599 Delgado, A., and Ramos, J. L. (1994) J. Biol. Chem. 269, 8059–8062 Delgado, A., Sal o, R., Ma que´s, S., and Ramos, J. L. (1995) J. Biol. Chem. 270, 5144–5150 de Lo enzo, V., He e o, M., Me zke, M., and Timmis, K. N. (1991) EMBO J. 10, 1159–1167 Fe na´ndez, S., de Lo enzo, V., and Pe´ ez-Ma ı´n, J. (1995) Mol. Mic obiol. 16, 205–213 Fiedle , U., and Weiss, V. (1995) EMBO J. 14, 3696–3705 Gu, B., Lee, J. H., Hoo e , T. R., Scholl, D., and Nixon, B. T. (1994) Mol. Mic obiol. 13, 51–56 Inouye, S., Nakazawa, A., and Nakazawa, T. (1988) Gene (Ams .) 66, 301–306 Ma que´s, S., and Ramos, J. L. (1993) Mol. Mic obiol. 9, 923–929 Mille , J. H. (1972) Expe imen s in Molecula Gene ics, Cold Sp ing Ha bo 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. Func ional Subdomains o he N Te minus o XylR 7901 Mo e , E., and Sego ia, L. (1993) J. Bac e iol. 175, 6067–6074 Mye s, R., Mania is, T., and Le man, L. (1987) Me hods Enzymol. 155, 501–527 Nakazawa, T., Inouye, S., and Nakazawa, A. (1990) in Pseudomonas: Bio ans o - ma ions, Pa hogenesis and E ol ing Bio/Technology (Sil e , S., Chak aba y, A., Iglewski, B., and Kaplan, S., eds) pp. 133–140, Ame ican Socie y o Mic o- biology, Washing on, D. C. No h, A. K., Klose, K. E., S edman, K. M., and Kus u, S. (1993) J. Bac e iol. 175, 4267–4273 Pe´ ez-Ma ı´n, J., and de Lo enzo, V. (1995) P oc. Na l. Acad. Sci. U. S. A. 92, 9392–9396 Pe´ ez-Ma ı´n, J., and de Lo enzo, V. (1996) J. Mol. Biol., in p ess Shingle , V. (1996) Mol. Mic obiol. 19, 409–416 Shingle , V., and Moo e, T. (1994) J. Bac e iol. 176, 1555–1560 Shingle , V., Ba ilson, M., and Moo e, T. (1993) J. Bac e iol. 175, 1596–1604 Func ional Subdomains o he N Te minus o XylR7902