Zaključno poročilo o rezultatih raziskovalnega projekta - 2012 Oznaka poročila: ARRS-RPROJ-ZP-2012/12 ZAKLJUČNO POROČILO O REZULTATIH RAZISKOVALNEGA PROJEKTA A. PODATKI O RAZISKOVALNEM PROJEKTU 1.Osnovni podatki o raziskovalnem projektu Šifra projekta Z1-2142 Naslov projekta Uravnavanje koproteazne aktivnosti proteina RecA v bakterijah Vodja projekta 24290 Matej Butala Tip projekta Zt Podoktorski projekt - temeljni Obseg raziskovalnih ur 3400 Cenovni razred B Trajanje projekta 05.2009 - 04.2011 Nosilna raziskovalna organizacija 481 Univerza v Ljubljani, Biotehniška fakulteta Raziskovalne organizacije -soizvajalke Raziskovalno področje po šifrantu ARRS 1 NARAVOSLOVJE 1.05 Biokemija in molekularna biologija Družbeno-ekonomski cilj .3 0. Naravoslovne vede - RiR financiran iz drugih virov (ne iz 13.01 SUF) 2.Raziskovalno področje po šifrantu FOS1 Šifra 1.05 - Veda 1 Naravoslovne vede - Področje 1.05 Vede o zemlji in okolju B. REZULTATI IN DOSEŽKI RAZISKOVALNEGA PROJEKTA 3.Povzetek projekta2 SLO Odkritje in uporaba antibiotikov je eden največji dosežkov sodobne medicine, kar je omogočilo zdravljenje infektivnih bolezni. Danes je v svetovnem merilu ena največjih groženj zdravju ljudi odpornost patogenih bakterij proti antibiotikom. Potrebujemo nove pristope k zdravljenju infekcij. Novejše raziskave so razkrile, da številni klinično pomembni antibiotiki v nizkih koncentracijah (v našem telesu lahko prisotna v začetku / koncu terapije z antibiotiki; v določenih delih telesa tekom terapije) poškodujejo DNA v bakterijah in posledično aktivirajo bakterijski odziv SOS. Sistem SOS je regulatorno omrežje genov odgovorno za popravilo poškodovane DNA. Bakterije se prilagodijo na stres, ki ga povzročijo antibiotiki, sprožijo odziv SOS, kar vodi v popravljanje DNA, nastanek točkovnih mutacij in prenosa genov med bakterijami. Z oviranjem sprožitve odziva SOS pri bakterijah, znižamo nastanek odpornosti proti nekaterim antibiotikom ter tako podaljšamo njihovo učinkovitost. Ključni proteini odziva SOS so pomembna tarča za izdelavo učinkovin, ki bi podaljšali učinkovitost obstoječih antibiotikov z znižanjem mutageneze in prenosa genov med bakterijami, kar sproži večina v kliniki uporabljenih antibiotikov! Odziv SOS je široko razširjen med bakterijami, preučevali smo odziv pri modelni bakteriji Escherichia coli. Sistem SOS je uravnavan z dvema proteinoma, LexA je dejavnik transkripcije, ki v pogojih normalne bakterijske rasti zmanjša lastno izražanje in v E. coli, izražanje vsaj 43 fizično nepovezanih genov. Protein RecA je induktor, ki se kot odziv na poškodbe DNA veže na enoverižno DNA (ssDNA) in tvori filament. Filament RecA-ssDNA-ATP (RecA*) interagira z LexA in aktivira samocepitveno aktivnost LexA, ianktivacija LexA vodi v sprožitev prepisa genov SOS. V projektu sem poskusal, v sodelovanju z ostalimi raziskovalci, pojasniti ključne mehanizme sprožitve odziva SOS. Razumevanje teh mehanizmov nam je omogočilo začetek razvoja učinkovine, s katero želimo zamrzniti zaznavanje stresa pri bakterijah, njihovo prilagoditev na antibiotike. Za izvedbo projekta sem vzpostavil sodelovanja z raziskovalci iz Biotehniške Fakultete (UL), Fakultete za Farmacijo (UL), Kemijskega inštituta, Univerze v Birminghamu (Anglija) ter Univerze v Osnabrucku (Nemčija). ANG One of the most serious health care problems worldwide is bacterial resistance to antibiotics. Although revolutionizing the treatment of infectious diseases, have antibiotics also rapidly selected for the emergence of resistant pathogens. Today, resistance has rendered most of the original antibiotics obsolete for many infections, typically by acquiring chromosomal mutations. Traditional methods of antibiotic discovery have failed to keep pace with the evolution of the resistance, which suggests that new strategies to combating the emerging threat of antibiotic resistant bacteria are needed. It has recently been shown that numerous clinically significant antibiotics can in bacteria induce the production of single stranded DNA and thus activate the SOS response. The SOS response induces the expression of a set of genes in response to DNA damage, leading to the arrest of cell division and induction of DNA repair and prophages and concommitant mutagenesis. The SOS system is a programmed DNA repair regulatory network, which results in mutations and genetic exchange, presumably to facilitate bacterial evolution in times of stress. Recent studies have shown that the antibiotic induced SOS response can modulate the evolution and spread of drug resistance as well as virulence factors. The SOS response is wide-spread among bacteria. Thus, key regulators of this system are important targets for the development of substances that would prolong the efficiency of the currently used antibiotics and act as antibiotic adjuvants. We studied the molecular mechnaism of the induction of the SOS response in a model bacterial organism, Escherichia coli. The SOS system is controlled by the interplay of 2 key regulatory proteins which alternate between on and off states. These are a repressor, LexA, which, during normal bacterial growth downregulates its own expression and, in E. coli, the expression of at least of 43 unlinked genes. The RecA protein is the inducer, which, in response to DNA damage, binds to single-stranded DNA (ssDNA) to form a filament. The RecA-ssDNA-ATP (RecA*) filament interacts with LexA and activates a self-cleaving activity in LexA, leading to induction of the SOS genes. In collaboration with other researchers, I tried to determine the key steps in the induction of the SOS response. The insights into this mechanism enabled us to set us a system for developing a drug that would disable bacteria to sense the antibiotic stress and adapt to antibiotics. To acomplish this project I continued with the previously establiset, or newly set up a colaboration with researchers from the Biotechnical faculty and the Faculty of Pharmacy, University of Ljubljana, Chemical institute (Slovenia), University of Birmingham (UK) and from the University of Osnabruck (Germany). 4.Poročilo o realizaciji predloženega programa dela na raziskovalnem projektu3 1. Ali aktivni filament RecA sproži cepitev proteina LexA, ko je represor specifično vezan na tarčna mesta DNA? Hipoteza: aktivni filament RecA sproži cepitev na DNA specifično vezanega represorja LexA. Dokazali smo, da aktivni filament RecA (RecA*), bakterij Escherichia coli, ne sproži cepitve transkripcijskega faktorja LexA, ko je represor LexA specifično vezan na tarčna mesta DNA. Prikažemo, da konformacijska sprememba v proteinu LexA omogoči programiran prepis genov bakterijskega stresnega odziva na poškodbe DNA. LexA je homo-dimeren protein, C-terminalna domena (CTD) služi za dimerizacijo, N-terminalna (NTD) za vezavo na DNA. Pripravili smo >95% očiščene proteine bakterije E. colt LexA, necepljivo različico (LexASA119), različico, ki se boljše veže na DNA (LexAEK71), različice z uvedenim aminokislinskim ostankom cistein na NTD ali CTD (LexA54, LexA29, LexA191 ter LexA29-191) ter protein RecA. V sodelovanju s skupino prof. H.J. Steinhoffa, Nemčija, smo z elektronsko paramagnetno resonanco (EPR) dokazali, da sta NTD LexA prosto gibljivi, ko protein ni vezan na DNA, a v specifični konformaciji, ko je protein vezan na tarčno DNA. V nasprotju, ob vezavi na DNA, ni velike konformacijske spremembe v CTD. Dokazali smo, da aktivni filament RecA (RecA*) sproži inaktivacijo ene podenote prostega LexA in ob ponovni interakciji med proteinoma, cepitev še preostale podenote. Razložili smo mehanizem sinhronizirane sprožitve bakterijskega odziva na poškodbe DNA: S površinsko plazmonsko resonanco (SPR) smo razjasnili, da v specifični, na DNA vezani konformaciji LexA ne interagira z RecA*. Nadalje, s SPR smo dokazali različne hitrosti sproščanja represorja iz različnih tarčnih zaporedij DNA E. coli. Posledično: na mestu poškodovane DNA se tvori RecA*, slednji sproži samo-cepitev prostega represorja LexA, znižanje koncentracije na DNA nevezanega/nespecifično vezanega LexA v celici. Slednje povzroči programiran prepis genov SOS, saj imajo zgodnji geni v odzivu (produkti, ki omogočijo natančno popravljanje poškodb) promotorska področja s tarčnim zaporedjem LexA, nizko afiniteto do represorja. Obratno, ob dolgotrajni poškodbi se prepišejo pozni geni SOS, ki imajo visoko afiniteto do LexA (mutageneza, sineteza toksinov). Pridobljeno temeljno zananje sem uporabil v primeru nastanka bakterij tolerantnih na antibiotike (dormantnih, perzisterskih celic). Nastanek slednjih je uravnano v odzivu SOS in predstavlja veliko težavo v zdravstvu. Dokazal sem, da z uravnavanjem funkcij LexA vplivamo na nastanek bakterij tolerantnih na antibiotike. Patentna prijava je v postopku: EPO, #10005558.1-2405, popravilo pomanjkljivosti. Članek je bil sprejet v reviji Nucleic Acids Research (točka 6.1). Raziskava je plod vzpostavitve/nadljevanje sodelovanja raziskovalcev iz Slovenije, Anglije in Nemčije. 2. Preučili smo zakaj LexA v DNA vezani konformaciji ne interagira z RecA*. Strukturni vpogled v interakcijo RecA* ter LexA ni poznan. Iz biokemijskih raziskav se predvideva, da le CTD LexA interagira z RecA*. Izdelali smo mutante proteina LexA v različnih konformacijah (LexAQM - LexA v cepitvi zmožni konformaciji, LexA13-91 - represor v cepitvi nezmožni konformaciji, LexA54 -represor v konformaciji nevezani na DNA, LexA24 - represor v konformaciji vezani na DNA). Pripravili smo tudi CTD ter NTD različic. S SPR smo dokazali, da poleg CTD tudi DNA vezavne domene LexA (NTD) interagirajo direktno z RecA*. Dokazali smo, da RecA* sproži cepitev mutante LexA24 (LexA v na DNA vezani konformaciji). posledično smo dokazali, da specifična DNA sterično ovira interakcijo RecA* z LexA. Iz rezultatov smo izdelali represor LexA v konformaciji, ki stabilno interagira z RecA*, z namenom kristalizacije RecA*-LexA. 3. Hipoteza: Neidentificirani proteini uravnavajo izražanje genov SOS, vplivajo na srostitev represorja LexA iz DNA. Preučilo smo ali obstajajo proteini, ki interagirajo z DNA vezanim represorjem LexA ter vplivajo na pozen prepis nekaterih genov odziva SOS. Kolicini so toksini bakterije E. coli, ki toksično učinkujejo na bakterije iste ali sorodne vrste ter vplivajo na raznovstnost bakterij v prebavilih sesalcev. Kolicini so uravnani z LexA in prepisani zadnji v odzivu SOS, saj se sprostijo ob lizi producentske bakterije. Predvidevali smo, da obstaja protein, ki stabilizira represor LexA na promotorskem področju gena za kolicin K (cka). Izvedli smo in vitro različico nedavno razvite metode "DNA sampling" (Butala et al, 2009, NAR). Z masno spektormetrijo smo prepoznali 6 DNA vezavnih proteinov (H-NS, DeoR, IscR, GlcC, UlrR, MqsA), z morebitnm vplivom na LexA pri prepisu cka. Z določevanjem aktivnosti promotorja smo dokazali, da protein IscR omogoči zakasneli prepis cka (2h lag fazo po nastanku poškodb DNA), najverjetneje stabilizira protein LexA na DNA. S SPR smo dokazali vezavno mesto za IscR na promotorskem področju cka, vezavno mesto prekriva element -35 promotorja. Kot prvi smo dokazali, da je prepis nekaterih genov za kolicine uravnan z dvema transkripcijskima faktorjema in se odzove na dva signala iz okolja. Dokazali smo, da nivo železa in dostopnost hranil vplivata na koncentracijo proteina IscR v celici ter na vezavne lastnosti proteina na tarčno zaporedje količina K. Med ~50 z LexA uravnanimi geni, je to drugi primer, da pri izražanju gena SOS, poleg LexA, sodeluje še dodaten dejavnik transkripcije. Dokazali smo fiziološki pomen tega skrbno uravnanega prepisa, ki privede do lize producentskih celic. V iscR' sevu, se kolicin K prepiše med prvimi geni odziva SOS, posledično bakterije ne morejo vključiti popravljalnih mehanizmov zaradi prezgodnje lize bakterij. Z difuzijskimi antibiogrami smo dokazali, da IscR vpliva na prepis ter posledično sintezo kolicina K in preživetje producentskih bakterij. Rezultati so bili poslani v revijo Molecular Microbiology, pregledani s strani urednika in treh recezentov, popravki bodo poslani v revijo sredi marca, 2012. Raziskava je plod sodelovanja raziskovalcev iz Biotehniške fakultete (UL), Kemijskega inštituta (Ljubljana) ter raziskovalcev iz Univerze v Birminghamu (Anglija). 4. Izdelava učinkovine, ki repreči inaktivacijo LexA, inhibicijo sprožitve odziva SOS. Kot navedeno zgoraj, smo z bazičnimi raziskavami ugotovli v kakšni konformaciji bi bilo najustrezneje zamrzniti protein LexA v celici, da bi preprečili porajanje odpornosti proti antibiotikom med baterijami. Uporabili smo knjižnice peptidnopredstavitvenih fagnih klonov, a neuspešno. Nadajle smo izvedli in silico iskanje učinkovine, ki bi mimikrirala cepitevno regijo LexA ter inhibirala samo-inaktivacijo LexA (preprečila sprožitev odziva SOS) v bakteriji E. coli. Izbrali in pridobili smo 30 učinkovin, izdelali hiter (in vitro) test inhibitornega učinka substanc na LexA. Rezultati raziskave nakazujejo potencial nekaterih učinkovin na inhibicijo inaktivacije represorja LexA. Ob izteku financiranja projekta, nisem uspel pridobit sredstev, ki bi nam omogočale nadaljevanje razvoja učinkovine. Raziskave so bile opravljene v sodelovanju s skupino prof. S. Gobca, Fakulteta za farmacijo, UL. S.Ocena stopnje realizacije programa dela na raziskovalnem in zastavljenih raziskovalnih ciljev4 Projekt je bil uspešno realiziran, kar je razvidno iz objavjenih rezultatov točke 7, 8, 9. Zaradi aktualnosti določenih tem je bilo v primerjavi s prvotno načrtovanim projektom izvedenih nekaj sprememb. Hipotezo 1, da aktivni filament RecA sproži cepitev na DNA specifično vezanega represorja LexA, smo ovrgli, tako razjasnili programiran odziv sos. Naši rezultati razložijo zakaj se nekateri geni odziva SOS prepišejo pred drugimi. Hipoteze 2, da ima protein RecA preferenčna vezavna mesta za vezavo in tvorbo aktivnega filamenta na genomu bakterije E. coli, še proučujemo. Vzrok zakasnitve je zaradi visokih stroškov analize - raziskave smo prilagodili finančnim zmožnostim projekta. Hipotezo 3, da dodatni proteini (poleg osmih poznanih proteinov) interagirajo z RecA* filamentom in uravnavajo sprožitev cepitve represorja LexA ne morem povsem zavreči. Rezultati kažejo na vlogo YdjM proteina pri uravnavanju funkcij RecA*. Hipotezo 4, da obstajajo proteini, ki z vezavo na DNA stabilizirajo interakcijo LexA z DNA in preprečijo prepis genov SOS smo potrdili, ob uporabi "DNA sampling" metode in vitro. IscR je prvi opisani protein, ki vpliva na stabilnost vezave LexA-DNA. V kombinaciji z LexA omogoči pozni prepis gena cka. Hipoteza 5, identificirati peptid, ki se veže na LexA in prepreči cepitveno aktivnost represorja LexA: knjižnice peptidnopredstavitvenih fagnih klonov se niso izkazale za uporabne v primeru LexA. Posledično, z mimikrijo cepitvene regije LexA in silico smo pridobili več kot 30 učinkovin. Uporabili smo znanje pridobljeno tekom izvedbe projekta, uporabili kot protimikrobno tarčo protein LexA v konformaciji vezani na specifično DNA. Analizo delovanja učinkovin na preprečitev z RecA* sprožene inaktivacije LexA smo torej uspešno začeli, izdelali hiter test za identifikacijo učinkovine in upam, da bomo ustrezno učinkovino tudi izdelali. 6.Utemeljitev morebitnih sprememb programa raziskovalnega projekta oziroma sprememb, povečanja ali zmanjšanja sestave projektne skupine5 Dokazali smo da RecA* (aktivator odziva SOS) ne interagira z LexA (represorjem odziva) vezanim na DNA. Slednji rezultati nakazujejo, da lokacija nastanka poškodb DNA v genomu bakterij ni ključna za uravnavanje sinhronizirane sprožitve prepisa genov SOS. Torej ni nujno potrebna za izdelavo inhibitorja odziva SOS. Posledično smo analizo mesta tvorbe filamenta RecA* v genomu bakterije E. coli tekom normalne rasti bakterij oziroma, tekom z antibiotiki sproženega odziva SOS začeli izvjati v zaključnih mesecih raziskovalnega projekta, ki pa je še nismo uspeli zaključiti. Sodelujemo s skupino dr. David C. Graingerja, Univerza v Birminghamu. Obratno, ob pisanju predloga projekta nisem predvidel poglobljene študije dinamike strukture represorja LexA, izdelava mutant LexA, študije EPR/SPR, v kar je privedla aktualnost naših rezultatov iz prvega sklopa. Poznavanje pridobljenih rezultatov je bil predpogoj za snovanje načina inhibicije inaktivacije LexA. Sprememba načina iskanja inhbitorja LexA (in silico mimikrija) je bila razumna, saj uporaba knjižnice peptidnopredstavitvenih fagnih klonov ni bila uspešna. 7.Najpomembnejši znanstveni rezultati projektne skupine6 Znanstveni dosežek 1. COBISS ID 2368847 Vir: COBISS.SI Naslov SLO Pretvorba LexA iz DNA nevezane v DNA vezano konformacijo orkestrira bakterijski odziv SOS. ANG Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response Opis SLO RecA* ne sproži inaktivacije LexA ko je ta specifično vezan na DNA. Z meritvami EPR dokažemo, da so DNA vezavne domene nevezanega LexA gibljive, a protein v specifični konformaciji ko je vezan na DNA. V slednji konformaciji je interakcija RecA* z LexA-DNA preprečena. Disociacija LexA iz različnih operatorjev poteka z različno hitrostjo, kar sinhronizira prepis genov SOS. S spreminjanjem aktivnoti LexA smo uravnali nastanek perzisterskih celic v bakterijski populaciji. ANG We show that self cleavage of LexA repressor is prevented by binding to specific DNA operator targets, depends on LexA dissociation from the targets and, hence, this controls the SOS response. Distance measurements using EPR spectroscopy reveal that in unbound LexA the DNA binding domains sample different conformations, one of which is captured when bound to operator targets, precluding RecA interaction. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations. Objavljeno v Oxford University Press; Nucleic acids research; 2011; Vol. 39, issue 15; str. 6546-6557; Impact Factor: 7.836;Srednja vrednost revije / Medium Category Impact Factor: 3.787; A': 1; WoS: CQ; Avtorji / Authors: Butala Matej*, Klose Daniel, Hodnik Vesna, Rems Ana, Podlesek Zdravko, Klare Johann P., Anderluh Gregor, Busby Steve J. W., Steinhoff Heinz-Jurgen, Žgur-Bertok Darja Tipologija 1.01 Izvirni znanstveni članek 2. COBISS ID 1 Vir: vpis v poročilo Naslov SLO Regulatorni sistem LexA ANG The LexA regulatory system Opis SLO Na povabilo dr. Nancy L. Craig (Howard Hughes Medical Institute) smo za drugo izdajo "Encyclopedia of Biological Chemistry" spisali poglavje o stresnem odzivu bakterij na poškodovano DNA, izdaja Elsevier. ANG The chapter named The LexA regulatory system for the second edition of "Encyclopedia of Biological Chemistry" published by Elsevier. An invitation from Nancy L. Craig (Howard Hughes Medical Institute). Objavljeno v Butala, M.*, Zgur-Bertok, D., and Busby, S.J.W. (2012) The LexA Regulatory System. In Lennarz, W.J., and Lane, M.D. (eds.), Encyclopedia of Biological Chemistry, 2nd Edition, Elsevier, in press. Tipologija 1.16 Samostojni znanstveni sestavek ali poglavje v monografski publikaciji 3. COBISS ID 2 Vir: vpis v poročilo Naslov SLO Dvojno zklenjen promotor gena za kolicin K, z dvema represorjema, prepreči prezgodnjo lizo bakterij po poškodbi DNA ANG Double-locking of the Escherichia coli colicin K gene promoter by two repressors prevents premature cell lysis after DNA damage Opis SLO Sinteza kolicinov bakterije E. coli je letalna za producentsko bakterijo. Izražanje kolicinov je zato tekom normalne bakterijske rasti močno utišano z represorjem LexA. Ob poškodbi DNA, se prvi prepišejo geni za popravilo DNA in z zamikom geni za kolcine. Ni bilo poznano, kaj omogoči zakasnjen prepis genov za kolicine. Dokazali smo, da globalni dejavnik transkripcije IscR, omogoči zakasneli prepis nekaterih genov za kolicine, tekom sproženega odziva SOS. Idfentificirali smo DNA vezavno mesto za IscR. Razložimo molekularni mehanizem, kako lahko bakterije omogočijo prepis gena za kolicine le ko so bakterije močno poškodovane in ne morejo vzdrževati integritete DNA. ANG The synthesis of Eschericha coli colicins is lethal to the producing cell and is repressed during normal growth by the LexA transcription factor, which is the master repressor of the SOS system for repair of DNA damage. Following DNA damage, LexA is inactivated and SOS repair genes are induced immediately, but colicin production is delayed and induced only in terminally damaged cells. The cause of this delay is unknown. Here we identify the global transcription repressor, IscR, as being directly responsible for the delay in colicin K expression during the SOS response and identify the DNA target for IscR at the colicin K operon promoter. Hence, this promoter is 'double locked' to ensure that suicidal colicin K production is switched on only as a last resort. Objavljeno v Revision, due March 2012. Matej Butala*, Douglas F. Browning, Silva Sonjak, Milan Hodošček, Darja Žgur Bertok, Stephen J. W. Busby. Molecular Microbiology; Impact Factor: 4.819; Srednja vrednost revije / Medium Category Impact Factor: 3.787; A': 1; WoS: CQ; Tipologija 1.01 Izvirni znanstveni članek S.Najpomembnejši družbeno-ekonomsko relevantni rezultati projektne skupine7 Družbenoekonomsko relevantni dosežki 1. COBISS ID 3639160 Vir: COBISS.SI Naslov SLO Interakcija represorja LexA in rekombinaze RecA ANG Interaction of repressor LexA with recombinase RecA Opis SLO Komentor pri diplomskem delu ANG Co-menthor, graduation thesis Šifra D.10 Pedagoško delo Objavljeno v [A. Rems]; 2009; X, 47 f.; Avtorji / Authors: Rems Ana Tipologija 2.11 Diplomsko delo 2. COBISS ID 3782008 Vir: COBISS.SI Naslov SLO Identifikacija nepoznanih proteinov, ki uravnavajo odziv SOS bakterije Escherichia coli ANG Identification of the unknown proteins taht regulate the induction of the bacterial SOS response Opis SLO Komentor pri diplomskem delu ANG Co-menthor, graduation thesis Šifra D.10 Pedagoško delo Objavljeno v [T. Đapa]; 2010; X, 66 f.; Avtorji / Authors: Đapa Tanja 2.11 Tipologija Diplomsko delo 3. COBISS ID Vir: vpis v poročilo Naslov SLO PathoGenoMics PhD award 2009 ANG PathoGenoMics PhD award 2009 Opis SLO Doktorat dr. Mateja Butale je bil ocenjen kot eden izmed treh najboljših doktoratov s področja genetike človeku patogenih mikroorganizmov. Doktorsko delo je bilo predstavljeno v obliki kratkega predavanja na tretjem evropskem kongresu mikrobiologov: 3rd FEMS Congress of European Microbiologists 2009, Goteborg, Švedska. ANG Matej Btala's PhD thesis were selected as one of teh best three thesis from the field of genetics on the research on disease-causing microorganisms by a review board of internationally renowned experts in the field of microbial research. Work was presented in a short lecture at 3rd FEMS Congress of European Microbiologists 2009, Goteborg, Sweden. Šifra E.02 Mednarodne nagrade Objavljeno v http://www.pathogenomics-era.net/index.php?index=322 Tipologija 1.08 Objavljeni znanstveni prispevek na konferenci 9.Drugi pomembni rezultati projetne skupine8 Vložena je patentna prijava: Controlling antibiotic tolerance, persister formation in a bacterial cell population by modulating LexA repressor functions (5/2010, patentna prijava, številka: 10005558.1, European Patent Office, Munchen, Germany). 2.05 Drugo učno gradivo AMBROŽIČ, Jerneja, BUTALA, Matej, STARČIČ ERJAVEC, Marjanca. Učno gradivo za program iz biologije genov : laboratorijske vaje in delavnice. Ljubljana, 2010: [S.n.]. 47 f., ilustr., graf.prikazi. [COBISS.SI-ID 26962393] lO.Pomen raziskovalnih rezultatov projektne skupine9 10.1.Pomen za razvoj znanosti10 SLO Bakterijski odziv SOS je ključen za vzdrževanje integritete genoma, a tudi za porajanje odpornosti proti antibiotikom. Rezultati raziskave so pomembni za razumevanje kompleksnega bakterijskega odziva na poškodbe DNA kot je odziv SOS, tvorijo temelj za nadaljne raziskave oziroma, izhodišče za razvoj učinkovin ali ko-učinkovin katere bomo lahko uporabljali skupaj z že obstoječimi antibiotiki. Za izvedbo projekta sem vzpostavil mednarodno sodelovanje s skupino prof. Heinz-Juergen Steinhoffa v Nemčiji. Z raziskovalcema Danielom Klose ter dr. Johannom Klare smo preučili konformacijske spremembe proteina LexA. Nadaljevali smo sodelovanje s skupino prof. Steva Busby-a ter se povezali tudi s skupino dr. Davida Graingerja v Veliki Britaniji. Vpetost projekta v Sloveniji: meritve izvedene v infrastrukturnem centru SPR v sodelovanju s prof. Gregorjem Anderluhom ter Vesno Hodnik; razvoj anti-LexA učinkovine, sodelovanje z dr. Mojco Lunder in s skupino prof. Stanislava Gobca, Fakulteta za Farmacijo. Nadaljevali smo s sodelovanjem s dr. Milanom Hodoščkom, Kemijski inštitut, Ljubljana. Povezave so razvidne iz skupnih publikacij. S projektom smo poglobili razumevanje kako bakterije uvnavajo izražanja genov v stresnih razmerah, kot je poškodba DNA, kar je lahko povod za razvoj odpornost proti antibiotikom. Poglavitna dodana vrednost rezultatov projekta je, povezovanje Evropskih inšitutov pri preučevanju teh pomembnih vprašanj. Področje raziskav molekularnih mehanizmov porajanja odpornosti proti protimikrobnim učinkovinam je visoko kompetitativno po svetu. Centri raziskav s tega področja so v ZDA, Japonskem in na Kitajskem. Posledično so vzpostavitev sodelovanj tekom tega projekta in pridobljena dognanja pomembna za odličnost Evropskih raziskav na področju odziva SOS ter porajanja odpornosti. Z zgoraj omenjenimi raziskovalci nadaljujemo z raziskavami na odzivu SOS. ANG The bacterial SOS response is essential for the maintenance of genomes, but also modulates antibiotic resistance. Our results provide insights into the mechanisms underlying SOS response and are prerequisite to understand the mechanism behind programmed expression of the LexA regulon genes. Hence, this work sets a novel platform for drug discovery to treat bacterial pathogens and offers an approach to control bacterial survival of antibiotic therapy. I have established international colaborations in order to carry out this project. I have collaborated with prof. Steinhoff's group from Germany. We have applied EPR methods to LexA. We continued collaboration with the group of prof. Steve Busby and established collaboration with dr. David Grainger from the UK. Collaborations established in Slovenia: Infrastractural centre for surface plasmon resonance, measurements performed in collaboration with prof. Gregor Anderluh and Vesna Hodnik; for development of anti-LexA compounds I collaborated with dr. Mojca Lunder and the group of prof. Stanislav Gobec, Faculty of Pharmacy, UL. We continued collaboration with dr. Milan Hodošček, Chemical institute, Ljubljana. International colaboration in this project can be observed from the joint publications. This research project focused on deepening and broadening the understanding of bacterial gene regulation due to stress response in bacteria and their influence on phenomena of antibiotic resistance. One of the main added values to the European research community lies in increasing the potential of Slovenia as a centre for fundamental research in molecular microbiology. The area of antimicrobial stress response is highly competitive internationally. There are rapidly developing centres of excellence in this research area within Japan, China and US. This project established the international community in this field and promoted its general ability to make high impact research contributions to further European Excellence. Thus, after this project is finished we will continue collaborating on the SOS response with the above mentioned research groups. 10.2.Pomen za razvoj Slovenije11 SLO Rezultati projekta prispevajo k razumevanju molekulskega mehanizma, ki omogoči bakterijam da se odzovejo na stres in predstavlja učno gradivo za študente. Naši rezulati pripomorejo k prepoznavnosti Slovenske zananosti v svetu, saj so/bodo rezultati projekta objavljeni v revijah z visokim faktojem citiranosti ter v encilopediji. Iz vsebine projekta sta diplomirali Ana Rems uni. dipl. mikrobiol., ki nadaljuje s podiplomskim študijem na Danskem ter Tanje Đapa uni. dipl. mikrobiol., ki je trenutno doktorantka v Novartisu, Siena. Projekt je torej omogočil razvoj dveh odličnih mladih Slovenskih znanstvenic. Potreben je nov pristop k zdravljenju bakterijskih okužb. Rezultati projekta so ogrodje za nadaljne raziskave v tej smeri. Odkritje in uporaba spojin, ki inhibirajo mehanizme razvoja odpornosti proti antibiotikom, kot je odziv SOS, bo omogočilo učinkovitejše zdravljenje z že obstoječimi antibiotiki. Vložena je patentna prijava (Točka 9). Razvoj učinkovitega inhibitorja odziva SOS, bo lahko omogočil farmacevtskim družbam ohranitev proizvodnje obstoječih klinično pomembnih antibiotikov, kar je izjemnega pomena za Slovensko gospodarstvo. Sredstva, ki so bila vložena v projekt so bila ustrezno porabljena! Verajmem, da je projekt Uravnavanje koproteazne aktivnosti proteina RecA v bakterijah, le eden od mnogih projektov mlajših raziskovalcev, ki so bili uspešno ralizirani. Pomen izvedenega projekta za Slovenijo je torej tudi, da se zavedamo, da je koristno (in nujno) omogočiti čim večjemu številu mlajšim raziskovalcem sredstva za izvedbo/razjasnitev svojih idej in razvoja lastnega potenciala! ANG Results obtained from this project elucidate how bacteria respond to the environmental stress, promote bacterial evolution, which is important for further studies on the SOS response and presents a model for textbooks for the students. The results from this project will benifit to the recognition of Slovenian science abroad as the results are /will be published in a high impact journals and in the encyclopedia. Part of this project was performed by Ana Rems uni. dipl. microbiol. (currently a PhD student at Technical University of Denmark), Tanje Đapa uni. dipl. microbiol. (currently a PhD student in Novartis, Siena), results from this project were used for their graduation thesis. Thus, this project established two talented Slovanian young scientist. As the treatments to treat bacterial pathogens are narrowing, new methods are needed. The set up collaborations and the obtained results enabled us to elucidate the important insights into the molecular mechanism of the bacterial response to antibiotics. Thus, development of an efficient inhibitor that will block SOS response and prevent development and spread of antibiotic resistance genes among bacteria, will hopefuly allow pharmaceutical companies to maintain production of clinically significant antibiotics, which is of great importance for the Slovenian economy. ll.Samo za aplikativne projekte! Označite, katerega od navedenih ciljev ste si zastavili pri aplikativnem projektu, katere konkretne rezultate ste dosegli in v kakšni meri so doseženi rezultati uporabljeni Cilj F.01 Pridobitev novih praktičnih znanj, informacij in veščin Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.02 Pridobitev novih znanstvenih spoznanj Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.03 Večja usposobljenost raziskovalno-razvojnega osebja Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.04 Dvig tehnološke ravni Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.05 Sposobnost za začetek novega tehnološkega razvoja Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.06 Razvoj novega izdelka Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.07 Izboljšanje obstoječega izdelka Zastavljen cilj Oda One Rezultat 1 d Uporaba rezultatov 1 d F.08 Razvoj in izdelava prototipa Zastavljen cilj Oda One Rezultat d Uporaba rezultatov 1 d F.09 Razvoj novega tehnološkega procesa oz. tehnologije Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.10 Izboljšanje obstoječega tehnološkega procesa oz. tehnologije Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.11 Razvoj nove storitve Zastavljen cilj Oda One Rezultat d Uporaba rezultatov 1 d F.12 Izboljšanje obstoječe storitve Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov d F.13 Razvoj novih proizvodnih metod in instrumentov oz. proizvodnih procesov Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.14 Izboljšanje obstoječih proizvodnih metod in instrumentov oz. proizvodnih procesov Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.15 Razvoj novega informacijskega sistema/podatkovnih baz Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.16 Izboljšanje obstoječega informacijskega sistema/podatkovnih baz Zastavljen cilj Oda One Rezultat d Uporaba rezultatov 1 d F.17 Prenos obstoječih tehnologij, znanj, metod in postopkov v prakso Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.1S Posredovanje novih znanj neposrednim uporabnikom (seminarji, forumi, konference) Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.19 Znanje, ki vodi k ustanovitvi novega podjetja ("spin off") Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.20 Ustanovitev novega podjetja ("spin off") Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.21 Razvoj novih zdravstvenih/diagnostičnih metod/postopkov Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.22 Izboljšanje obstoječih zdravstvenih/diagnostičnih metod/postopkov Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.23 Razvoj novih sistemskih, normativnih, programskih in metodoloških rešitev Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.24 Izboljšanje obstoječih sistemskih, normativnih, programskih in metodoloških rešitev Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.25 Razvoj novih organizacijskih in upravljavskih rešitev Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov d F.26 Izboljšanje obstoječih organizacijskih in upravljavskih rešitev Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.27 Prispevek k ohranjanju/varovanje naravne in kulturne dediščine Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.28 Priprava/organizacija razstave Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.29 Prispevek k razvoju nacionalne kulturne identitete Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.30 Strokovna ocena stanja Zastavljen cilj Oda One Rezultat d Uporaba rezultatov d F.31 Razvoj standardov Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.32 Mednarodni patent Zastavljen cilj Oda One Rezultat d Uporaba rezultatov 1 d F.33 Patent v Sloveniji Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov d F.34 Svetovalna dejavnost Zastavljen cilj Oda One Rezultat I d Uporaba rezultatov 1 d F.3S Drugo Zastavljen cilj Oda One Rezultat d Uporaba rezultatov H Komentar 12.Samo za aplikativne projekte! Označite potencialne vplive oziroma učinke vaših rezultatov na navedena področja Vpliv Ni vpliva Majhen vpliv Srednji vpliv Velik vpliv G.01 Razvoj visoko-šolskega izobraževanja G.01.01. Razvoj dodiplomskega izobraževanja O o o o G.01.02. Razvoj podiplomskega izobraževanja o o o o G.01.03. Drugo: o o o o G.02 Gospodarski razvoj G.02.01 Razširitev ponudbe novih izdelkov/storitev na trgu o o o o G.02.02. Širitev obstoječih trgov o o o o G.02.03. Znižanje stroškov proizvodnje o o o o G.02.04. Zmanjšanje porabe materialov in energije o o o o G.02.05. Razširitev področja dejavnosti o o o o G.02.06. Večja konkurenčna sposobnost o o o o G.02.07. Večji delež izvoza o o o o G.02.08. Povečanje dobička o o o o G.02.09. Nova delovna mesta o o o o G.02.10. Dvig izobrazbene strukture zaposlenih o o o o G.02.11. Nov investicijski zagon o o o o G.02.12. Drugo: o o o o G.03 Tehnološki razvoj G.03.01. Tehnološka razširitev/posodobitev dejavnosti o o o o G.03.02. Tehnološko prestrukturiranje dejavnosti o o o o G.03.03. Uvajanje novih tehnologij o o o o G.03.04. Drugo: o o o o G.04 Družbeni razvoj G.04.01 Dvig kvalitete življenja o o o o G.04.02. Izboljšanje vodenja in upravljanja o o o o G.04.03. Izboljšanje delovanja administracije in javne uprave o o o o G.04.04. Razvoj socialnih dejavnosti o o o o G.04.05. Razvoj civilne družbe o o o o G.04.06. Drugo: o o o o Ohranjanje in razvoj nacionalne G.05. naravne in kulturne dediščine in identitete O o o o G.06. Varovanje okolja in trajnostni razvoj o o o o G.07 Razvoj družbene infrastrukture G.07.01. Informacijsko-komunikacijska infrastruktura o o o o G.07.02. Prometna infrastruktura o o o o G.07.03. Energetska infrastruktura o o o o G.07.04. Drugo: o o o o G.08. Varovanje zdravja in razvoj zdravstvenega varstva o o o o G.09. Drugo: o o o o Komentar 13.Pomen raziskovanja za sofinancerje12 Sofinancer 1. Naziv Naslov Vrednost sofinanciranja za celotno obdobje trajanja projekta je znašala: EUR Odstotek od utemeljenih stroškov projekta: % Najpomembnejši rezultati raziskovanja za sofinancerja Šifra 1. 2. 3. 4. 5. Komentar Ocena C. IZJAVE Podpisani izjavljam/o, da: • so vsi podatki, ki jih navajamo v poročilu, resnični in točni • se strinjamo z obdelavo podatkov v skladu z zakonodajo o varstvu osebnih podatkov za potrebe ocenjevanja ter obdelavo teh podatkov za evidence ARRS • so vsi podatki v obrazcu v elektronski obliki identični podatkom v obrazcu v pisni obliki • so z vsebino zaključnega poročila seznanjeni in se strinjajo vsi soizvajalci projekta Podpisi: zastopnik oz. pooblaščena oseba raziskovalne organizacije: in vodja raziskovalnega projekta: Univerza v Ljubljani, Biotehniška Matej Butala fakulteta ŽIG Kraj in datum: Ljubljana [5.3.2012 Oznaka prijave:ARRS-RPROJ-ZP-2012/12 1 Zaradi spremembe klasifikacije je potrebno v poročilu opredeliti raziskovalno področje po novi klasifikaciji FOS 2007 (Fields of Science). Prevajalna tabela med raziskovalnimi področji po klasifikaciji ARRS ter po klasifikaciji FOS 2007 (Fields of Science) s kategorijami WOS (Web of Science) kot podpodročji je dostopna na spletni strani agencije (http://www.arrs.gov.si/sl/gradivo/sifranti/preslik-vpp-fos-wos.asp). Nazaj 2 Napišite povzetek raziskovalnega projekta (največ 3.000 znakov v slovenskem in angleškem jeziku) Nazaj 3 Napišite kratko vsebinsko poročilo, kjer boste predstavili raziskovalno hipotezo in opis raziskovanja. Navedite ključne ugotovitve, znanstvena spoznanja, rezultate in učinke raziskovalnega projekta in njihovo uporabo ter sodelovanje s tujimi partnerji. Največ 12.000 znakov vključno s presledki (približno dve strani, velikosti pisave 11). Nazaj 4 Realizacija raziskovalne hipoteze. Največ 3.000 znakov vključno s presledki (približno pol strani, velikosti pisave 11) Nazaj 5 V primeru bistvenih odstopanj in sprememb od predvidenega programa raziskovalnega projekta, kot je bil zapisan v predlogu raziskovalnega projekta oziroma v primeru sprememb, povečanja ali zmanjšanja sestave projektne skupine v zadnjem letu izvajanja projekta (obrazložitev). V primeru, da sprememb ni bilo, to navedite. Največ 6.000 znakov vključno s presledki (približno ena stran, velikosti pisave 11). Nazaj 6 Znanstveni in družbeno-ekonomski dosežki v programu in projektu so lahko enaki, saj se projekna vsebina praviloma nanaša na širšo problematiko raziskovalnega programa, zato pričakujemo, da bo večina izjemnih dosežkov raziskovalnih programov dokumentirana tudi med izjemnimi dosežki različnih raziskovalnih projektov. Raziskovalni dosežek iz obdobja izvajanja projekta (do oddaje zaključnega poročila) vpišete tako, da izpolnite COBISS kodo dosežka - sistem nato sam izpolni naslov objave, naziv, IF in srednjo vrednost revije, naziv FOS področja ter podatek, ali je dosežek uvrščen v A'' ali A'. Nazaj 7 Znanstveni in družbeno-ekonomski dosežki v programu in projektu so lahko enaki, saj se projekna vsebina praviloma nanaša na širšo problematiko raziskovalnega programa, zato pričakujemo, da bo večina izjemnih dosežkov raziskovalnih programov dokumentirana tudi med izjemnimi dosežki različnih raziskovalnih projektov. Družbeno-ekonomski rezultat iz obdobja izvajanja projekta (do oddaje zaključnega poročila) vpišete tako, da izpolnite COBISS kodo dosežka - sistem nato sam izpolni naslov objave, naziv, IF in srednjo vrednost revije, naziv FOS področja ter podatek, ali je dosežek uvrščen v A'' ali A'. Družbenoekonomski dosežek je po svoji strukturi drugačen, kot znanstveni dosežek. Povzetek znanstvenega dosežka je praviloma povzetek bibliografske enote (članka, knjige), v kateri je dosežek objavljen. Povzetek družbeno ekonomsko relevantnega dosežka praviloma ni povzetek bibliografske enote, ki ta dosežek dokumentira, ker je dosežek sklop več rezultatov raziskovanja, ki je lahko dokumentiran v različnih bibliografskih enotah. COBISS ID zato ni enoznačen izjemoma pa ga lahko tudi ni (npr. v preteklem letu vodja meni, da je izjemen dosežek to, da sta se dva mlajša sodelavca zaposlila v gospodarstvu na pomembnih raziskovalnih nalogah, ali ustanovila svoje podjetje, ki je rezultat prejšnjega dela _ - v obeh primerih ni COBISS ID). Nazaj 8 Navedite rezultate raziskovalnega projekta iz obdobja izvajanja projekta (do oddaje zaključnega poročila) v primeru, da katerega od rezultatov ni mogoče navesti v točkah 7 in 8 (npr. ker se ga v sistemu COBISS ne vodi). Največ 2.000 znakov vključno s presledki. Nazaj 9 Pomen raziskovalnih rezultatov za razvoj znanosti in za razvoj Slovenije bo objavljen na spletni strani: http://sicris.izum.si/ za posamezen projekt, ki je predmet poročanja Nazaj 10 Največ 4.000 znakov vključno s presledki Nazaj 11 Največ 4.000 znakov vključno s presledki Nazaj 12 Rubrike izpolnite / prepišite skladno z obrazcem "izjava sofinancerja" http://www.arrs.gov.si/sl/progproj/rproj/gradivo/, ki ga mora izpolniti sofinancer. Podpisan obrazec "Izjava sofinancerja" pridobi in hrani nosilna raziskovalna organizacija - izvajalka projekta. Nazaj Obrazec: ARRS-RPROJ-ZP/2012 v1.00 89-EB-82-9E-E5-84-81-CD-EE-98-E1-0B-13-D8-90-5B-C9-C4-31-BF 6546-6557 Nucleic Acids Research, 2011, Vol. 39, No. 15 doi:10.1093/nar/gkr265 Published online 16 May 2011 Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response Matej Butala1,*, Daniel Klose2, Vesna Hodnik1, Ana Rems1, Zdravko Podlesek1, Johann P. Klare2, Gregor Anderluh1, Stephen J. W. Busby3, Heinz-Jiirgen Steinhoff2 and Darja zZgur-Bertok1 ^Department of Biology, Biotechnical Faculty, University of Ljubljana, VecCna pot 111, 1000 Ljubljana, Slovenia, ^Department of Physics, University of Osnabrujck, Barbarastrasse 7, D-49076 Osnabrujck, Germany and 3School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK Received November 23, 2010; Revised and Accepted April 6, 2011 ABSTRACT The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional re-pressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations. INTRODUCTION In unstressed, growing Escherichia coli cells, the SOS system is shut off due to repression by LexA of ~50 promoters that control expression of the SOS regulon (1,2). Under these conditions, E. coli is thought to contain ~1300 molecules of LexA (3). Most LexA is DNA bound, but ~20% is thought to be free. LexA is a homodimeric protein (4) that likely locates its target sites by multiple dissociation-reassociation events within the same DNA molecule (5). Around each landing site, the repressor is thought to diffuse along non-specific DNA and to undergo rotation-coupled sliding to facilitate the search for specific binding sites (6). The majority of E. coli SOS promoters are regulated by LexA alone (7). LexA activity is modulated by the active form of RecA (RecA*), that stimulates self-cleavage of a scissile peptide bond between Ala84 and Gly85, thereby de-activating LexA (8), lowering LexA's affinity for the DNA and exposing residues that target LexA for ClpXP and Lon protease degradation (9). As a result, the cellular concentration of LexA drops from ~2 to ~0.2 mM, thereby de-repressing SOS genes (3). A key characteristic of the SOS response is the orchestrated induction of individual SOS genes. Thus, initially, genes with low-affinity SOS boxes are expressed, enabling protection and maintenance of the structural integrity of the replisome, while genes with high-affinity operators are expressed late in the SOS response (1). To circumvent unrepaired DNA damage, even after high-fidelity nucleotide excision, and recombinational repair, low fidelity DNA damage tolerance pathways are induced, presumably to increase bacterial mutation rates c: I M R O •tI L a td L '-i N > u *To whom correspondence should be addressed. Tel: +386 1 320 3405; Fax: +386 1 257 3390; Email: matej.butala@bf.uni-lj.si © The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. and survival in times of stress (10). As DNA damage is repaired, LexA accumulates and the system is reset. Alternatively, if cells are severely damaged and may not survive, the sensing of long-lived-inducing signal triggers the synthesis of bacteriocins and prophages, resulting in cell lysis (11). Thus, RecA* also catalyzes self-cleavage of lambdoid phage repressors (12) whose catalytic, carboxy-terminal domains (CTDs) exhibit homology with the LexA CTD (13). Similarly to LexA inactivation, cleavage of phage re-pressors leads to destruction of the protein's abilities to firmly bind DNA, enabling a switch from the latent or lysogenic to replicative and lytic phase. Interestingly, the À cI repressor is cleaved only when monomeric (14), while the cI repressor of the temperate 434 bacteriophage is inactivated preferably when bound to specific DNA (15). LexA is predominately dimeric in the cell (4) and repressor dimers can undergo RecA*-mediated self-cleavage when off the DNA (16). Therefore, the mechanisms of repressor inactivation among various biological systems related to SOS functions vary from one system to another. Even though many studies have investigated the SOS response, it is still unclear how diversity within SOS boxes co-ordinates temporal induction of the different SOS genes. In addition, it is not known how RecA* induces self-cleavage of LexA and which are the structural determinants required for RecA*-mediated cleavage of LexA (16,17). Here, we present the first report describing LexA repressor with defects in LexA-RecA* interaction. We demonstrate that, the unbound LexA structure is highly flexible in contrast to the rigid DNA-bound state, in which interaction with RecA* is precluded. Thus, we show that RecA* indirectly activates the SOS system, by mediating a decrease in the intracellular pool of unbound LexA provoking dissociation of the operator-bound re-pressor and concomitantly inducing the LexA regulon genes. Our data further imply that two sequential interactions of the unbound LexA with RecA* are required for inactivation of both subunits of the LexA repressor dimer. MATERIALS AND METHODS Cloning, expression and isolation of the proteins The lexA, recA and oxyR genes were amplified by polymerase chain reaction (PCR) from the E. coli K-12 strain RW118 (18) using oligonucleotide primers LexA_u, LexA_d; RecA_u, RecA_d or OxyR_u, OxyR_d, respectively (Supplementary Table S1). The PCR products were subsequently cut with BamHI and Mlul and cloned into an expression vector (19) to prepare plasmids pAna1, pAna2 and pOxyR. The LexA and RecA proteins overexpressed from the pAna1 or pAna2 plasmids, respectively, were constructed as His6 fusion proteins with an N-terminal hexa-histidine tag and a thrombin cleavage site ((H)6SSLVPRGS). A variant of the pAna1 expression plasmids, pLexA29, pLexA54, pLexA71, pLexA119, pLexA71-119 and pLexA191 were constructed employing the QuickChange® Site-directed Mutagenesis kit manual (Stratagene) and pairs of oligonucleotides 29AC_1, 29AC 2 and 54GC 1, 54GC 2; 71EK 1, 71EK 2; 119SA_1, 119SA_2 or 191LC_1, 191LC_2 (Supplementary Tables S1 and S2), respectively. Proteins LexA, LexA29, LexA54, LexA71, LexA119, LexA191 and RecA were expressed with a His-tag present on the N-terminus in the E. coli BL21 (DE3) strain and purified from the bacterial cytoplasm by Ni-chelate chromatog-raphy and gel-filtration chromatography (20). Purified proteins were stored at -80° C in 20 mM NaH2PO4 (pH 7.3), 200 mM NaCl except for LexA, LexA71 and RecA which were stored in buffer containing 20 mM Tris-HCl (pH 7.3), 200 mM NaCl. Protein concentrations were determined using NanoDrop1000 (Thermo SCIENTIFIC) (4). Three LexA cysteine mutants (LexA29, LexA54, LexA191) were used for the electron paramagnetic resonance (EPR) analysis. The LexA71 re-pressor variant exhibits enhanced DNA-binding affinity, but the mechanism for the improved DNA binding is unknown (21). The LexA119 is a non-cleavable repressor derivative with modified Ser119 in the active center to Ala; this mutation does not affect the ability of LexA to bind RecA* (13,16). Thus, the LexA119 variant was used to prevent repressor self-cleavage during the study of the LexA-RecA* interaction. Operator-containing DNA fragments The 88 bp recA and the 114 bp tisB operator-containing DNA fragments were PCR amplified. The colicin K encoding plasmid pKCT1 and its derivatives with altered SOS boxes pKCT3-UP1, pKCT3-UP3 (22) were used to amplify the 121 bp cka, cka-UP1 and the cka-UP3 fragment, respectively. Centered on the generated DNA fragments were none, single or double LexA-binding sites presented in Figure 1. One strand of the amplified PCR products was biotinylated at the 5'-end, and primers RecA_1, RecA_2; TisB_1, TisB_2 were used to amplify DNA fragments with recA or tisB operators and primers Cka_1, Cka_2 to amplify DNA fragments harboring cka, cka-UP1 and cka-UP3 operators, respectively (Supplementary Table S1). The PCR generated fragments were gel purified (QIAquick kit, Qiagen). LexA repressor cleavage assays Activation of the RecA filament (10 mM), carried out on ice for 2h, and the RecA*-induced (2 mM) cleavage of LexA (1.8 mM) at 37°C interacting with specific or non-specific DNA (~1.5 mM) were performed as described previously for the unbound LexA repressor (16). The LexA dimer to operator/modified operator ratio was 1:2. The LexA repressor was preincubated with specific and non-specific DNA or for the titration reactions with increasing concentrations of DNA for 10min at 37°C in a DNA-binding buffer (23). The reaction time course was initiated with the addition of the RecA*. The proteolytic cleavage reactions (20 ml) were stopped by adding 4xNuPAGE LDS sample buffer (Invitrogen). Samples were analyzed on 12% NuPAGE gels (Invitrogen) and stained by Page blue protein stain (Fermentas). The experiments were conducted at least three times and representative gels are shown. The resolved bands were quantified using a G:Box (Syngene). The integrated optical C I M R O •tI L a td L N > u Figure 1. RecA* cannot induce self-cleavage of specifically bound LexA. (A-D) Time course (min) of RecA*-induced LexA proteolysis showing inhibition of cleavage due to operator DNAs compared with non-specific DNA (cka-UP3). Operator sequences used are presented with SOS boxes underlined and mutated nucleotides in bold typeface. (E) Quantitations of the LexA self-cleavage presented are averages with the standard deviation of at least triplicate reactions. (F) LexA was pre-incubated with operators or (G) non-specific DNA in a ratio 1:0.2; 0.7; 1.2; 1.6; 2.1 (mol:mol) for lanes from 2 to 6, or without DNA for lane 1. The RecA*-activated self-cleavage of LexA was stopped after 15 min. RecA protein, LexA repressor and its cleaved products are marked by the CTD or NTD for the dimerization or the DNA-binding domain, respectively. density of the intact LexA monomer was normalized to that determined for the RecA protein to account for lane-dependent artifacts. The ratio of LexA cleavage was calculated as the ratio of the normalized density value for the intact LexA relative to the normalized value of LexA exposed to RecA*. Cross-linking of LexA repressor Glutaraldehyde cross-linking: at the indicated time, RecA*-mediated LexA (both at the final concentration of 5.6 mM) proteolytic cleavage reactions conducted as stated above were stopped with 16 mM glutaraldehyde for 30s before adding glycine to 60mM (16). Covalent cross-linking reactions: the LexA54 variant was reduced with 20 mM dithiothreitol (DTT) or oxidized with a mixture of 0.1 mM CuSO4 and 0.5 mM 1,10-phenantroline for 30 min at room temperature. At the indicated time, RecA*-mediated proteolytic cleavage reactions of the oxidized LexA54 (at the final concentration of 4 and 5.6 mM for the LexA54 and RecA, respectively) conducted as stated above were stopped by adding 4xNuPAGE LDS sample buffer (Invitrogen). Presence of oxidant in the reactions did not affect RecA*-stimulated LexA self-cleavage, as determined by oxidation of wild-type LexA and implementation of self-cleavage reaction (data not shown). Samples were analyzed as described above. We resolved the various repressor forms: dimers, monomers, CTDs, N-terminal domains (NTDs) and combinations of intact LexA protein and its cleavage products, by analysis of protein molar masses in comparison with the PageRuler prestained protein ladder (Fermentas) and by comparing our data with earlier results (16). Spin labeling of LexA mutants For spin labeling, purified single cysteine mutants (~10mg) of E. coli LexA (Supplementary Table S2) were pretreated with DTT at 15 mM final concentration in buffer containing 20 mM NaH2PO4 (pH 7.3), 500 mM NaCl (4h, 4° C). DTT was removed by exchanging the buffer two times with the use of PD-10 desalting column (GE Healthcare) and after removal protein solutions were incubated with 1 mM MTSSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate spin label (Toronto Research, Alexis), for 16 h (8°C). Excess MTSSL was removed by exchanging the buffer two times with 20 mM NaH2PO4 (pH 7.3), 200 mM NaCl with a PD 10 desalting column. The spin-labeled proteins were concentrated to ~100 mM and buffer exchanged by buffer of the same composition containing deuterated water (Acros Organics) by the use of Amicon centrifugal filters (Millipore). Labeling efficiencies have been determined to be -80% for LexA54 and >95% for LexA29 and LexA191. EPR measurements Distance measurements between nitroxide spin labels attached to the LexA variants (-100 mM) were carried out either unbound or bound to the 24 bp tisB operator-containing DNA fragment (50-TTTACTGTAT AAATAAACAGTAAT-30, marked are the SOS boxes) composed of oligonucleotide primers Tis_1b, Tis_2b (Supplementary Table S1). Cw EPR spectra for interspin distance determination in the range from -0.8 to 2.0 nm were obtained on a homebuilt cw X-band EPR spectrometer equipped with a Super High Sensitivity Probehead (Bruker Biospin GmbH, Rheinstetten, Germany). The magnetic field was measured with a B-NM 12 B-field meter (Bruker Biospin). A continuous flow cryostat Oxford ESR9 (Oxford Instruments, Oxfordshire, UK) was used in combination with an Intelligent Temperature Controller (ITC 4; Oxford Instruments) to stabilize the sample temperature to 160 K. The microwave power was C I M R O •tI L a td L N > u set to 0.2 mW and the B-field modulation amplitude to 0.25 mT. EPR quartz capillaries (3 mm inner diameter) were filled with sample volumes of 40 ml. Fitting of simulated dipolar broadened EPR powder spectra to the experimental ones was carried out using the program WinDipFit (24). Double electron-electron resonance (DEER)/PELDOR EPR experiments were performed at X-band frequencies (9.3-9.4 GHz) on a Bruker Elexsys 580 spectrometer equipped with a Bruker Flexline split-ring resonator ER 4118X-MS3. Temperature was stabilized to 50 K using a continuous flow helium cryostat (ESR900; Oxford Instruments) controlled by an Oxford Intelligent Temperature Controller ITC 503 S. EPR quartz capillaries (2.4 mm inner diameter) were filled with sample volumes of 40 ml. All measurements were performed using the four-pulse DEER sequence with two microwave frequencies: ^/2(Uobs) - - n (Uobs) - t' - n (Upump) - (Ti + T2 - f) - ^ (wobs) — t2 - echo (25,26). A two-step phase cycling (+, — ) was performed on n/2(uobs). Time t' is varied, whereas t1 and T2 are kept constant. The dipolar evolution time is given by t = t' - t1. Data were analyzed only for t > 0. The resonator was overcoupled and the pump frequency Upump was set to the center of the resonator dip (coinciding with the maximum of the nitroxide EPR spectrum) whereas the observer frequency ^obs was 65 MHz higher (low-field local maximum of the spectrum). All measurements were performed at a temperature of 50 K with observer pulse lengths of 16 ns for n/2 and 32 ns for n pulses and a pump pulse length of 12 ns. Proton modulation was averaged by adding traces at eight different t1 values, starting at t1,o = 200 ns and incrementing by At1 = 8 ns. For proteins in D2O buffer with deuterated glycerol, used for its effect on the phase relaxation, corresponding values were t1,o = 400 ns and At1 = 56 ns. Data points were collected in 8 ns time steps or, if the absence of fractions in the distance distribution below an appropriate threshold was checked experimentally, in 16 ns time steps. The total measurement time for each sample was 4-24 h. Analysis of the data was performed with DeerAnalysis 2009 (27). Rotamer library analysis The canonical ensemble of spin label side-chain (R1) conformations is modeled by a discrete set of 210 precalculated rotamers (28). From the rotamer library analysis, a conformational distribution of R1 at a specific position in the otherwise fixed protein structure can be determined. Briefly, the superposition of R1's backbone atoms onto the protein backbone at the respective position provides the orientation of R1 with respect to the protein structure. The resulting energy for the R1-protein interaction is then calculated from the Lennard Jones potential using the MD force field CHARMM27 (29). Subsequent Boltzmann weighting and normalization by the partition function gives a probability for each rotamer which is then multiplied by the probability of R1 to exhibit this conformation, resulting in the final rotamer probability distribution at the site of interest. Between two such probability distributions a distance distribution is calculated as the histogram of all pairwise interspin distances weighted by the product of their respective probabilities. Structural aspects of LexA were generated using VMD software (30). Functional properties Of LexA mutants For EPR analysis, we selected LexA residues that are surface exposed and do not impair repressor functions when modified (31). Esherichia coli strain DM936 (lexA41) was transformed with plasmid pLexA29, pLexA54, pLexA191 to complement the temperature-sensitive LexA mutation. As a control strain DM936 expressing the wild-type lexA (pAna1) or expressing the repressor OxyR (pOxyR) was used. To verify the in vivo ability of the LexA mutants to regulate the SOS system and to repress the sulA gene, preventing induction of filamentous growth, strains were grown in Luria-Bertani (LB) ampicillin (Ap, 100 mg/ml) media at 28.0°C or at 42.5°C and in stationary phase cell counts were determined (20). Surface plasmon resonance (SPR) analysis and RecA*-mediated cleavage experiments were conducted as described in this chapter. SPR assays SPR RecA*-LexA interaction measurements were performed on a Biacore X (GE Healthcare) at 25°C. The streptavidin sensor chip was equilibrated with SPR_2 buffer containing 20 mM NaH2PO4 (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 1mM DTT, 1 mM ATP (Sigma-Aldrich), 0.005% surfactant P20 (GE Healthcare). Approximately 200 response units (RU) of 5'-biotinylated 30-mer (32) was immobilized on the flow cell 2. Subsequently, RecA protein (2.1 mM) was passed in the SPR_2 buffer at 2 ml/min to create RecA*. The LexA119 repressor variant interacting with the 24 bp tisB operator (annealed primers Tis_1b, Tis_2b, Supplementary Table S1) or the 24-bp non-specific DNA (annealed primers Tis_1nb, Tis_2nb), free LexA119 or the DNA fragments, were injected across the immobilized RecA* (1000 RU) at 10 ml/min for 60 s, to study the interaction. The sensor chip with bound RecA* was regenerated by injection of 500mM NaCl. A 0.05% SDS was used to additionally regenerate flow cell 1. SPR LexA-operator interaction measurements were performed on a Biacore T100 at 25°C. The 88 bp recA, 114 bp tisB, 121 bp cka operator-containing DNA fragments and the cka-UP3 DNA fragment were PCR amplified and gel purified as described above. The resulting fragments were 50-end biotinylated. The streptavidin sensor chip was equilibrated with SPR_1 buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.005% surfactant P20 (GE Healthcare). The biotinylated DNA in SPR buffer was immobilized to approximately 20 RUs. An empty flow cell was used as a control. The interaction between LexA and chip-immobilized DNA was studied by injecting various concentrations of LexA or LexA71 in SPR buffer. The sensor chip with bound DNA was regenerated by injection of SPR buffer containing 500 mM NaCl. We noted that the interaction of both LexA and LexA71 with DNA was extremely rapid and C I M R O •tI L a td L '-i N > u use of standard assays revealed that it is heavily influenced by the mass transfer effect (33). However, the dissociation of the proteins from the DNA was not influenced by the flow rate of the SPR buffer. For the final determination of dissociation rates, proteins were injected across the surface chip at a saturating concentration (40 nM) for 30 s and dissociation was followed for 20 min at a flow rate of 100 ml/min. The dissociation of LexA71 from cka operator was extremely slow; therefore, we followed dissociation for 40 min. The data were doubly referenced and fitted to a 1:1 binding model to obtain the dissociation rates constants. Three to six independent experiments were performed. Persistence of lexA defective strain complemented by LexA and its variants For the persistence assay, strain RW542 (thr-1 araD139 A(gpt-proA)62 lacY1 tsx-33 supE44 galK2 hisG4 rpsL31 xyl-5 mtl-1 argE3 thi-1 sulA211 lexA51), encoding a defective LexA protein that cannot bind to target DNA sites due to impaired dimerization (18) was used. The ÀDE3 prophage, encoding the T7 RNA polymerase, was integrated into the RW542 chromosome according to instructions (ÀDE3 Lysogenization kit, Novagen). The ÀDE3 lysogenic RW542 strain, designated MB542, exhibited basal-level T7 RNA polymerase expression without addition of isopropyl beta-D-1-thiogalacto-pyranoside as determined according to the manufacturer's instructions. Subsequently, strain MB542 was transformed with plasmid harboring T7 promoter controlled wild-type lexA, mutant lexA119 or the double-mutant lexA71-119. The minimum inhibitory concentration (MIC) for mitomycin C (Sigma) was determined by the broth dilution method (34). The MIC for the strain MB542 lexA (Def) was 3.2 mg/ml, for the strain harboring the plasmid encoding wild-type repressor 4.0 and 1.8 mg/ ml for the strains with lexA119 or the double lexA71-119 mutant. The 2.5 MIC of mitomycin C was used for the persister assay. The isogenic strain RW118 expressing chromosomally encoded lexA exhibited identical mitomycin C MIC as the strain MB542 complemented with the plasmid encoding wild-type repressor. Thus, data indicate that the SOS system of the lexA complemented strain MB542 pAna1 functioned similarly as the wild-type strain. Experiments were conducted at 37°C essentially as described previously (35) except that transformed strains were grown (180 rpm) in 10 ml LB medium supplemented with 100 mg/ml Ap and cell counts determined by plating on LB or LBAp agar plates. No difference in cell count was detected when cells were plated on LB or LBAp media, indicating that plasmid loss did not occur during the experiments (data not shown). The percentage of survival was determined as the ratio of colony forming units (cfu) before to cfu following exposure to mitomycin C and plotted as a function of time. Trypsin cleavage of LexA repressor bound to operator The LexA repressor (2.4 mM) was bound to the recA or cka operator-containing fragments or to the cka variant fragments cka-UP1 or cka-UP3. The LexA dimer to operator/modified operator ratio was 1:2. DPPC-treated Trypsin (Sigma-Aldrich) digestions were conducted at 25°C in DNA-binding buffer at a LexA concentration of 2.4 mM with a protease to repressor ratio of 1:50 (m:m). The reaction time course was initiated with the addition of the protease. Bands were resolved as described above. Western blotting Thrombin (Novagen) digestion of 3.4 mM LexA was carried out at 20°C for 2 h in 20mM Tris (pH 7.3), 200 mM NaCl with a protease to repressor weight ratio of 1:2000. LexA-DNA complex was formed by 10 min incubation of 3.4 mM LexA and DNA fragment-containing recA operator in the LexA dimer toward operator ratio 1:2 at 37°C in DNA-binding buffer prior to trypsin digestion carried out for 30 min as described above. Samples were resolved on a 12% acrylamide gel. Blotting and detection was done as described before (36). Primarily, the proteins were stained with mouse anti-hexa-histidine tag antibody (Quiagen) and secondary antibodies conjugated by horseradish peroxidase. The same membrane was re-stained by primary LexA rabbit poly-clonal antibody (Upstate) and same secondary antibodies. Antibodies were used at a concentration of 0.5 mg/ml. Agarose gel mobility shift assays The LexA repressor was, immediately before use, serially diluted from 2.4 mM to 2.0 nM. The 10 ml reaction mixtures contained ~50mM recA, tisB or ~25mM cka operator-containing DNA or its variants cka-UP1 or cka-UP3, interacting with LexA in the DNA-binding buffer. Protein-DNA complexes were resolved on 2.5% agarose gels (20) after incubation at room temperature for 10min in 20mM Tris (pH 7.5), 200mM NaCl, 1mM EDTA, 12% glycerol. RESULTS AND DISCUSSION DNA is an allosteric effector of bacterial LexA protein It was previously suggested that SOS box-containing DNA fragments can inhibit RecA*-mediated LexA self-cleavage (37). In contrast, recently published LexA-DNA crystal structures indicate that LexA-operator interaction exerts minimal interference with RecA*-induced self-cleavage (38). Most of the E. coli SOS genes possess a single SOS box, but the number of operators can range up to 3 (7). We have measured rates of RecA*-stimulated self-inactivation of purified LexA interacting with either tandem (colicin K gene, cka) or modified, lower LexA affinity tandem operator (cka-UP1) or single (recA) operator-containing DNA fragment in comparison with the non-specific DNA (cka-UP3) (Supplementary Figures S1 and S2). The results shown in Figure 1A-E indicate that RecA* cannot induce self-cleavage in LexA that is bound to target DNA operator sites. This was confirmed by measuring LexA inactivation in reactions with a range of concentrations of specific (cka operators) or non-specific DNA. Non-specific DNA had little inhibitory effect on LexA C I M R O •tI L a td L '-i N > u induced inactivation, in comparison with the operator-containing DNA (Figure 1F and G). It has been suggested that it is not possible for both subunits of a LexA dimer to simultaneously make contact with the deep helical groove of RecA*, and that separate docking events are required to cleave both LexA subunits (38). Thus, we used glutaraldehyde cross-linking to follow the kinetics of RecA*-mediated cleavage of unbound LexA repressor and found that self-cleavage proceeds primarily via one subunit of a dimer (Figure 2A). The reaction reached completion by 20 min (Supplementary Figure S3). Data indicate that RecA* predominately induces self-cleavage in one monomer of the LexA dimer and that the resulting LexA-LexA/CTD heterodimer is an inactive intermediate, exhibiting weaker DNA binding (31). The LexA repressor is mostly dimeric at the concentration used for the glutaraldehyde cross-linking experiment (4); however, complete cross-linking of the dimers could not be achieved. Thus, a cysteine cross-linking experiment was exploited. Structural data of the unbound LexA dimer suggest that residues Gly54 positioned in the DNA-binding NTDs could come in close proximity (13). Data show that the oxidized repressor derivative LexA54, with Gly 54 replaced by Cys, forms covalently bound dimers (Figure 2B). Hence, to complement the glutaraldehyde cross-linking data, RecA*-induced self-cleavage of oxidized LexA54 was determined. The kinetics of appearance of a singly cleaved LexA dimer in the time course of the cleavage reaction indicate that, the LexA heterodimer is an intermediate on the pathway that leads to the fully cleaved dimer (Figure 2). Thus, two successive dockings with RecA* are necessary for the inactivation of both repressor subunits. Intracellularly, almost all LexA is dimeric (4) and preexisting repressors dissociate slowly to monomers (16). Thus, the source of monomers is supposedly newly synthesized LexA. We propose that, following DNA damage repair and disappearance of the SOS-inducing signal, both newly synthesized LexA as well as heterodimers could provide a source of monomers for resetting repression and for fine-tuning of the SOS response. Figure 2. RecA*-induced LexA self-cleavage proceeds primarily by one subunit. (A) Cleavage of unbound LexA was induced by addition of RecA*, and samples were cross-linked by glutaraldehyde at different time points (min) and analyzed by gel electrophoresis. RecA and LexA markers were also cross-linked as indicated. Homodimer (LexA dimer), LexA monomer cross-linked to the C-terminal fragment (LexA-CTD), cross-linked C-terminal fragments (CTD-CTD), monomer (LexA) and cleavage forms of LexA (CTD, NTD) are marked. (B) The LexA54 derivate with residue Gly54 replaced by Cys in the DNA-binding domain was reduced (LexA54 red.) or oxidized (LexA54 ox.) to show that the repressor can be covalently bound at residue 54. Cleavage of oxidized LexA54 was induced by addition of RecA* and samples taken at different time points (min) and analyzed by SDS-PAGE electrophor-esis. Homodimer (LexA dimer), LexA monomer cross-linked to the N-terminal fragment (LexA-NTD), monomer (LexA), cross-linked N-terminal fragments (NTD-NTD), and C-terminal fragment (CTD) are marked. LexA conformational dynamics A recent report of the structure of LexA-operator complexes suggested that flexibility in bound LexA could facilitate interaction with RecA*, leading to LexA self-cleavage, provoking separation of the DNA-binding domain from the rest of the operator-bound dimer and inactivation (38). To test this directly, we used site-directed spin labeling EPR (39) in combination with DEER (25,26) spectroscopy. Interactions between the paramagnetic centers attached to the two subunits of the LexA dimer were measured in order to investigate the mobility of both the N-terminal DNA-binding domain and the C-terminal, regulatory domain, in free and DNA-bound LexA. LexA derivatives with single cysteines substituting residues Ala29 or Gly54 in the DNA-binding domain or residue Leu191 in the dimerization domain were spin labeled (Figure 3A and B, Supplementary Table S2 and Figure S4). Measurements of the interaction between the spin-label side chains (denoted R1) reveal high-conformational flexibility of the DNA-binding domains in the unbound re-pressor (apo), but a defined conformation when bound to a specific DNA target. For spin labels at positions 29 (A29R1) or 54 (G54R1) in the apo state broad, multi-modal interspin distance distributions are revealed ranging from 30 to 65 AA and from 15 to 50 AA, respectively (Figure 3C, solid lines, inset and Supplementary Figures S5 and S6). Remarkably, for A29R1 and G54R1 in the apo state the DEER traces (Supplementary Figure S5) exhibit significantly smaller modulation depths, compared with the DNA bound state. For A29R1, this observation can be explained by the presence of a significant fraction of the protein molecules with interspin distances beyond the range accessible to DEER experiments (>70 For G54R1, the reduced modulation depth in the apo state is caused by the contributions of molecules with interspin distances <15^ which do not contribute to the DEER signal as revealed by cw EPR data. Thus, high C I M R O •tI L a td L '-i N > u Figure 3. Conformational dynamics of the LexA binding to the tisB operator. (A) Structure of unbound LexA dimer [pdb ID:1JHH (13)] with modeled (20) undetermined residues (transparent) and (B), operator-bound LexA [pdb ID:3JSO (38)]. Individual subunits are colored blue and cyan, residues changed to cysteines and spin labeled are presented as yellow beads. Interspin distances were determined for spin-label pairs connected by dashed lines. (C) Experimental interspin distance distributions measured by DEER (solid lines) and simulations based on LexA crystal structures (dashed lines) for the DNA bound (red) and apo states (black). For G54R1 in the apo state, the distribution for interspin distances <2nm (gray) was determined from the dipolar broadened cw EPR spectra (Supplementary Data). Results are shown as normalized probability distributions. flexibility of the DNA-binding domains is obvious as they sample conformations leading to interspin distances ranging from 25 to >70 AA for A29R1 and <15 to 50 AA for G54R1. In contrast, in the operator-bound state both mutants show single population maxima centered at 31 AA (±3 AA) for A29R1, and at 43 AA (±5 AA) for G54R1. Remarkably, the distance distributions of both constructs indicate that the conformations LexA samples in the apo state cover also the DNA bound structure. Measurements with labeled LexA191 (L191R1) revealed that interspin distance distributions were very similar in both the unbound and DN^bound states, with a clear maximum at a distance of 40 ^ (Figure 3C). Hence, the C-terminal regulatory domains of each subunit in the LexA dimer function as a rigid scaffold for the DNA-binding NTDs. In the unbound state, these are flexible and can adapt the conformation in which the RecA*-induced attack of the scissile A84-G85 bond by the active-site Ser119 is facilitated. On the contrary, in the rigid operator-bound state of the LexA dimer, this conformation cannot be accessed and RecA*-induced inactivation of LexA is prevented. Again, an interesting observation concerns the modulation depths of the DEER traces, which is significantly lower for A29R1 and G54R1 in the NTDs compared with L191R1 in the CTD (Supplementary Figure S5). Although a lower labeling efficiency of ~80% has been obtained for G54R1 (A29R1 and L191R1: >95%), this does not explain the observed differences in the modulation depths. Instead, this observation is in line with the fact that unbound LexA has been shown to undergo the process of self-cleavage (13), leading to LexA-LexA/CTD heterodimer formation. Such heterodimers contain two spin labels in the CTD, but only one spin-labeled NTD is present, thus explaining the lower modulation depth for A29R1 and G54R1. A comparison of the experimental interspin distances for LexA-A29R1, G54R1 and L191R1 in the DNA bound state with values predicted from the LexA-DNA crystal structure (pdb ID:3JSO) using the rotamer library approach (Figure 3C, dashed lines) shows reasonable agreement for the two positions located in the NTDs (A29R1 and G54R1) indicating that, the arrangement found in the crystal structures seems to reflect the state in solution well. On the contrary, the data for L191R1 indicate that the conformation of the LexA dimerization domain in solution might slightly differ from that observed in crystals, most probably due to crystal packing effects. Nevertheless, it cannot be excluded that limitations in the accuracy of the rotamer library approach account for the observed differences. Repressor's dissociation from operators orchestrates SOS response SPR analysis was subsequently performed to determine the mechanism of operator-bound repressor interference with RecA*-induced autoproteolysis. Active RecA filament was formed on single-stranded DNA bound to the surface of the sensor chip (Figure 4A). Non-cleavable repressor variant LexA119 (S119A) interacted with chip-immobilized RecA* in a concentration-dependent manner (Figure 4B). The presence of tisB operator interfered with the ability of LexA119 to bind to RecA* (Figure 4C). We show that binding of operator induces LexA in a particular conformation in which interaction with RecA* is precluded (Figure 4D), revealing why RecA*-induced inactivation of specifically bound LexA is unfeasible. The LexA CTD provides the determinants for dimeriza-tion and self-cleavage activity, thus the interface interacting with RecA* (13). In the crystal structure of the unbound LexA mutant dimer (pdb ID: 1JHH) one subunit is well ordered throughout and in a non-cleavable state, whereas the second subunit, while disordered in the NTD, adopts the cleavable state in the CTD (13). The structure of the intact monomer also exhibits LexA intramolecular contacts between the DNA-binding NTD and C I M R O •tI L a td L N > u Figure 4. Interaction of unbound or specifically bound LexA119 with RecA*. (A) SPR sensorgrams of the binding of the 2.1 mM RecA to the flow cell 1 (red) or to the flow cell 2 with immobilized tisB-operator DNA (cyan). (B) Unbound LexA119 repressor in concentration range from 0.7 to 5.2 mM or (C) LexA119 interacting with 24-bp tisB operator DNA in concentration range from 0.3 to 2.7 mM were injected across the chip-immobilized RecA* for 60s at 10ml/min. The used DNA to repressor ratio (mol:mol) was approximately 0.1:1, 0.3:1, 0.5:1, 1:1, 2:1, respectively. (D) Sensorgrams of the 2.6 mM repressor variant LexA119 (black), the 24 bp DNA fragments (2.7 mM) consisting of the tisB operator (violet) or the non-specific DNA (cyan), tisB operator bound LexA119 (red) or LexA119 mixed with the non-specific DNA (green), interacting with the chip-immobilized RecA*. The used DNA to repressor ratio was ~2:1 (mol:mol). the cleavage site loop lying just within the CTD. This is most likely not an artifact due to crystal packing (13) as cleavage site region-NTD interactions were also confirmed by experiments exploiting cysteine cross-linking (20). Thus, orientation of NTDs might affect the position of the cleavage loop containing the scissile peptide bond. Our EPR results indicate that a five residue hydrophilic linker that connects the NTD of LexA to its catalytic core domain does not impede movement of the NTDs, as suggested previously (20). Thus, although LexA is a homodimeric protein, variable positions of its NTDs in the dimer might modulate the position of the cleavage-site regions in the CTDs. The repressor recognizes its targets as a dimer (4) and the dimer does not exert stringency requirement on the binding domain (38). In the operator-bound LexA, an extensive dimer interface is observed between the DNA-binding NTDs, formed of residues which are solvent exposed in the unbound LexA (13). Interactions between the two DNA-binding domains are acting syner-gistic with DNA binding, thus increasing LexA dimer stability by 1000-fold (4,38). In contrast to the alternating conformations of the cleavage loops in the unbound LexA dimers, both scissile peptide bonds in the operator-bound mutant dimers are displaced or docked in the active center (38). The results of this investigation show that the operator is an alosteric effector of the LexA repressor indicating that, a specific orientation of the DNA-binding NTDs sets the repressor in a conformation in which interaction with RecA* and a subsequent self-cleavage reaction is precluded. Interestingly, mutations in LexA that specifically impair RecA*-dependent cleavage, but do not alter catalysis have not been identified (16). Therefore, further studies will be employed to elucidate how diverse positions of the LexA cleavage loop and orientation of the NTDs modulate interaction with the RecA*. Our results imply that LexA dissociation from operators coordinates expression of the SOS genes. This is in agreement with previous reports, showing that the timing of induction of LexA-regulated genes correlates with the binding affinity of the SOS boxes (1). However, previously LexA operator affinity was ranked by quantitative gel retardation and DNase I footprinting experiments and by calculating the relatedness of an operator sequence to that of the consensus sequence derived from the known LexA targets (18,23). To provide further details, we used SPR to measure LexA-operator interactions under near physiological salt and pH conditions in real time. We used DNA fragments that contained recA, tisB, cka operators C I M R O •tI L a td L N > u or non-specific DNA cka-UP3. Binding to operators was concentration dependent (data not shown), but LexA did not bind to the control DNA (Figure 5). The association of LexA with the SOS operators was extremely rapid, and it was therefore not possible to determine accurately the association rate constants due to the mass transfer effect. Control experiments showed that dissociation of LexA from the surface of the chip was not dependent on the flow rate (data not shown), therefore it was possible to determine the rates of dissociation. In spite of rapid LexA association with all the tested operators, the repres-sor exhibited diverse dissociation rates. Dissociation was similar for recA and tisB, but significantly slower from the cka operator. This explains, for example, why recA is one of the first transcribed genes in the SOS response, while expression of the cka gene is delayed, limited to conditions of extensive, long-lived DNA damage (1,11). We conclude that differences between LexA operators affect repressor dissociation and influence the timing of expression of SOS genes. Decreasing persister formation by modulating LexA functions The insights into LexA functions presented here may provide new directions in the battle against the emergence and spread of drug resistance. It has recently been shown that persisters form during the SOS response and depend on the LexA-regulated TisB toxin (40). Hence, bacterial killing by antibiotics can be enhanced by dislabeling the SOS response, either by deleting the recA gene (41) or overexpression of non-cleavable lexA variants (42,43). We used the LexA71 (E71K) repressor variant (21) that exhibits three to nine times slower dissociation from operators compared with wild-type LexA repressor (Figure 5). We then measured persister formation in an E. coli strain defective for lexA, complemented with wild-type LexA or its non-cleavable mutants, exhibiting either normal or enhanced DNA binding, treated with 2.5 times MIC of mitomycin C. Our results (Figure 6) show that the occurrence of persister cells in bacterial populations triggered by DNA damage can be altered by changing LexA activity. Notably, when cells expressed the non-cleavable and enhanced operator-binding LexA re-pressor variant, no persisters were detected 1 h after induced DNA damage. LexA homologs are found in pro-karyotes (31), but to date there are no known orthologs in eukaryotes. Hence, this work sets a novel platform for drug discovery to treat bacterial pathogens and offers an approach to control bacterial survival of antibiotic therapy. CONCLUSIONS In the present paper, we show that RecA*-mediated LexA repressor self-cleavage cannot be induced in LexA specifically bound to target DNA. Our results contradict the observation that the LexA operator bound conformation allows docking to RecA* and subsequent LexA C I M R Figure 5. Interaction of LexA and LexA71 with various promoter regions. SPR was used to assess the interaction of LexA (black) or LexA71 (gray) with various operators as indicated. Biotinylated DNA fragments were immobilized on the surface of the streptavidin sensor chip. Purified protein at saturating concentration was injected across the chip for 30 s and dissociation followed as shown on the graphs. The sensorgrams were doubly referenced and fitted to a 1:1 binding model. Data shown are triplicate injections of the protein and overlaid with fits (red). Calculated dissociation rate constants (average ± standard deviation) are shown for each condition. O •tI L a td L N > u Figure 6. Mitomycin C survival of the E. coli lexA- strain complemented with LexA repressor variants. MB542 (lexA51) strain complemented with wild-type LexA (pLexA) or its non-cleavable mutants exhibiting either normal (pLexA119) or enhanced DNA (pLexA71-119) binding was grown to exponential phase (~108 cfu/ml), when exposed to 2.5 times MIC of mitomycin C. At 0, 0.5, 1, 3, 6h after addition, viable cell number was determined (cfu/ml). As a control, strain MB542 was used. The data points are averages of at least four independent experiments and error bars indicate the standard error. inactivation (38). Thus, diverse LexA conformations enable either repression of SOS genes by specific DNA binding or repressor cleavage in response to DNA damage. Data presented here imply that mobility of the LexA NTDs affects the repressor's interaction with the RecA*. Our results indicate that RecA*-mediated inacti-vation of unbound LexA must decrease the intracellular pool of free LexA which provokes dissociation of the functional repressor from its DNA targets (Figure 7). Taken together, our results indicate how the signal from DNA damage at a particular chromosomal location is transduced into the induction of the SOS genes, co-ordinated by the distinct LexA repressor conformations. In addition, we show that, upon DNA damage, separate interactions between the two key SOS players are required to cleave both subunits of the LexA dimer. Therefore, when the inducing signal disappears, the remaining self-cleavage intermediates, inactive heterodimers, can provide a source of subunits which dimerize into the functional repressor to accelerate resetting of the system. Figure 7. An overview of the SOS response in E. coli. (1) Concentration of LexA monomers increases. (2) LexA monomers in solution form biologically relevant dimers. DNA-binding domains of the unbound LexA are highly mobile and can move freely to one another. (3) Repression of the SOS system occurs when LexA dimers bind specifically to SOS boxes located at the promoter regions of SOS genes and sterically precludes their transcription. (4) The polymerase III holoenzyme (Pol) carries out DNA replication. At the site of DNA damage PolIII arrests, and single-stranded DNA (ssDNA) accumulates. RecA binds to ssDNA in the presence of ATP, forming active RecA-ssDNA-ATP filaments (RecA*). (5) RecA* induces self-cleavage in the unbound LexA but cannot stimulate inactivation of LexA specifically bound to target DNA. (6) In the unbound repressor dimer, one monomer is preferentially inactivated and the uncleaved monomer could affect resetting of the system. Cleaved LexA products are rapidly degraded by the ClpXP and Lon proteases (44). (7) Due to induced unbound LexA self-cleavage, intracellular LexA pool decreases. Specifically bound LexA repressor dissociates from operators, (8) leading to co-ordinated de-repression of SOS genes. (9) The rate of LexA dissociation from target sites is influenced by operator sequences and acts in orchestrating the response. Subsequently, as DNA damage is repaired, SOS induction is reversed. Numbers in red indicate novel insights into the system. C I M R O •tI L a td L '-i N > u SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. FUNDING Slovenian Research Agency (Z1-2142 to M.B., J4-2111 to D.ZZ.B.). 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Read proofs for accuracy and clarity, as well as for typographical errors, but please DO NOT REWRITE. Titles and headings should be checked carefully for spelling and capitalization. Please be sure that the correct typeface and size have been used to indicate the proper level of heading. Review numbered items for proper order - e.g., tables, figures, footnotes, and lists. Proofread the captions and credit lines of illustrations and tables. Ensure that any material requiring permissions has the required credit line and that we have the relevant permission letters. Your name and affiliation will appear at the beginning of the article and also in a List of Contributors. Your full postal address appears on the non-print items page and will be used to keep our records up-to-date (it will not appear in the published work. Please check that they are both correct. Keywords are shown for indexing purposes ONLY and will not appear in the published work. 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The e-mail should state the article code number in the subject line. Corrections should be consecutively numbered and should state the paragraph number, line number within that paragraph, and the correction to be made. 2. If corrections are substantial, send the amended hardcopy by courier to Justin Taylor, Elsevier MRW Production Department, The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK. If it is not possible to courier your corrections, please fax the relevant marked pages to the Elsevier MRW Production Department (fax number: +44 (0)1865 843974) with a covering note clearly stating the article code number and title. Note that a delay in the return of proofs could mean a delay in publication. Should we not receive corrected proofs within 7 days, Elsevier may proceed without your corrections. CHECKLIST Author queries addressed/answered? □ Affiliations, names and addresses checked and verified? □ Permissions details checked and completed? □ Outstanding permissions letters attached/enclosed? □ Figures and tables checked? □ If you have any questions regarding these proofs please contact the Elsevier MRW Production Department at: bch2proofs@elsevier.com Non-Print Items Abstract: Organisms have evolved gene regulatory systems to cope with stress. To maintain the structural and functional integrity of their genomes after damage due to environmental or metabolic assaults, bacteria mount a program of gene expression known as the 'SOS response'. Induction of this response requires a repressor, the LexA protein, and an inducer, the recombinase A (RecA) protein. In Escherichia coli, upon DNA damage, RecA stimulates cleavage of the LexA repressor, inducing expression of approximately 1% of the genes. The coordinated expression of these genes orchestrates a complex program of DNA repair, which can also result in mutations and genetic exchange that facilitate bacterial evolution. In some bacteria, the SOS response also modulates the expression of virulence factor genes and can induce the formation of dormant cells that are highly tolerant to antibiotics. Keywords: Antibiotic resistance;Bacteriophage induction;Cell-cycle control;DNA damage;DNA repair;Gene activation;Induction of gene expression; LexA regulon; Transcription responses; Virulence factor regulation Author and Co-author Contact Information: [aji Matej Butala Department of Biology Biotechnical Faculty University of Ljubljana Vecna pot 111 Ljubljana Slovenia Tel: +368 1 423 3388 Fax: 386 1 257 3390 E-mail: Matej.Butala@bf.uni-lj.si Darja ZZgur-Bertok Department of Biology Biotechnical Faculty University of Ljubljana Vecna pot 111 Ljubljana Slovenia Tel: +368 1 423 3388 Fax: 386 1 257 3390 E-mail: Darja.Zgur.Bertok@bf.uni-lj.si Stephen J W Busby School of Biosciences The University of Birmingham Edgbaston Birmingham B15 2TT UK Tel: +44 (0)121 41 45439 Fax: +44 (0)121 41 45925 E-mail: s.j.w.busby@bham.ac.uk Biographical Sketch MatejButala obtained his PhD from the Medical Faculty, University of Ljubljana, Slovenia. Upon graduation he studied regulation of expression of the SOS genes encoding colicins with Dr. D ZZgur-Bertok, and became interested in the LexA biochemical processes. For his PhD thesis he was awarded a 2009 PathoGenoMics PhD award. He did his postdoctoral work in Dr. SJW Busby's lab in Birmingham, UK. He was a teaching assistant for molecular biology at the Biotechnical Faculty in Ljubljana, where he is currently a postdoctoral researcher. He is studying the dynamics of the interaction between the LexA repressor and the RecA filament. Darja ZZgur-Bertok obtained her PhD from the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia. She is a professor at the Biotechnical Faculty, University of Ljubljana. She has worked on regulation of bacteriocin synthesis in Escherichia coH and their antimicrobial activity. She has also worked on plasmids and regulation of plasmid conjugative transfer. Darja ZZgur-Bertok is also involved in teaching undergraduate courses in microbial genetics and microbial pathogenesis. Steve Busby became interested in transcriptional regulation in bacteria when he was a postdoctoral scientist at the Institut Pasteur, Paris. He subsequently joined the academic staff at the University of Birmingham, UK, and is currently professor of biochemistry in the School of Biosciences. He has worked on many different bacterial transcription factors but, recently, his work has focused on how different signals are integrated at promoters, and the application of novel genomic methods to study the global regulation of transcription. Author Query Form Book: Encyclopedia of Biological Chemistry Article No.: 00278 Dear Author, During the preparation of your manuscript for typesetting some questions have arisen. These are listed below. Please check your typeset proof carefully and mark any corrections in the margin of the proof or compile them as a separate list. Your responses to these questions should be returned within seven days, by email, to MRW Production, email: BCH2proofs@elsevier.com Query Details Required Author's response AU1 Please check the long affiliations for accuracy. These are for Elsevier's records and will not appear in the printed work. AU2 Please provide in-text citations for Figures 1-4. AU3 Please provide the place of publication of the reference 'Foti et al. (2010)'. AU4 Do Figures 1-4 require permission? If yes, please provide the relevant correspondence granting permission. [If you have already provided this information, please ignore this query.] a0010 |Au2,4| The LexA Regulatory System M Butala and D ZZgur-Bertok, University of Ljubljana, Ljubljana, Slovenia S J W Busby, The University of Birmingham, Birmingham, UK © 2012 Elsevier Inc. All rights reserved. This article is a revision of the previous edition article by Veronica G. Godoy, Penny J. Beuning, and Graham C. Walker, volume 2, pp. 546-550, © 2004, Elsevier Inc. Glossary Autoregulation A gene product regulates expression of its own gene. Chromatin immunoprecipitation Technique used to precipitate a protein antigen using specific antibody to identify protein-DNA interactions at the genome level. DNA microarrays A surface carrying an array of probes, DNA oligonucleotides corresponding to genes of interest, which are hybridized with cDNA from RNA isolated from cells under a given condition. Operator Specific DNA site where transcription factor binds and modulates initiation of gene transcription. Promoter Sequence located upstream of a gene to which RNA polymerase binds to initiate transcription. Regulon Group of genes whose expression is regulated by a common regulator(s). Repressor Protein that inhibits gene expression by sterically interfering with binding of RNA polymerase or by binding to RNA. sooio Introduction p00i0 The Escherichia coli LexA regulon is a regulatory network, encompassing at least 57 genes whose products govern a coordinated bacterial response to DNA damage. The induced LexA regulatory system has also been designated the SOS response to emphasize its role in the cellular response to distress. The expressed SOS functions not only repair DNA damage but also enhance adaptation through mutagenesis and genetic exchange. The SOS response thus plays a broad role, modulating evolution and dissemination of drug resistance and virulence factor genes, as well as the synthesis and secretion of virulence factors. In addition, the SOS system controls persistence and multidrug tolerance in a subpopulation of bacterial cells. The SOS system is widespread among bacteria but exhibits considerable variation with regard to its components and regulation. This article outlines regulation by LexA in E. coli, which is the best-understood SOS system and has been studied most extensively. sooi5 The E. coli LexA Regulatory System two key proteins, a repressor and an inducer, is mounted upon DNA damage. The product of the lexA gene (locus for X-ray sensitivity A) is the repressor of the regulon while recom-binase A (RecA) is involved in sensing DNA damage and induces inactivation of the LexA repressor. During normal bacterial growth, LexA downregulates expression of its own gene and, in E. coli, the expression of more than 50 unlinked genes. In response to DNA damage, RecA (bound to adenosine triphosphate (ATP)) polymerizes onto single-stranded DNA (ssDNA) exposed upon repair or replication of damaged DNA, creating a helical nucleoprotein filament. The active ssDNA-ATP-RecA filament (RecA*) interacts with LexA and activates its latent self-cleaving activity. Cleavage inactivates LexA, instigating repressor dissociation from its DNA targets (SOS boxes) and induction of the LexA regulon. Subsequently, as DNA damage is repaired or bypassed, the level of ssDNA, the SOS-inducing signal, decreases and the co-protease activity of RecA filaments disappears (note, RecA* does not participate directly in the proteolysis reaction but instead stimulates LexA cleavage and is thus termed a 'co-protease'). Functional LexA rapidly re-accumulates, returning the system to its repressed state. p00i5 Control of gene expression in response to environmental assaults, and the maintenance of the structural and functional integrity of the genome are essential for cell survival. The bacterial SOS system is an inducible DNA repair and damage-tolerance response triggered either by extrinsic treatments that elicit DNA damage or by intrinsic events that disrupt DNA replication. p0020 A comprehensive response to DNA lesions was first described in detail in E. coli. Evelyn M. Witkin postulated that cellular filamentation and phage induction are regulated by a common repressor, which is inactivated in response to DNA damage. In the 1970s, Miroslav Radman proposed that a coordinated cellular response controlled by the interplay of Defining the LexA Regulon s0020 Genes of the SOS regulon are characterized by (1) basal-level p0025 expression during normal bacterial growth and induction following DNA damage; (2) absence of induction in the lexA (ind-) mutant strain with noncleavable LexA protein; (3) constitutive induction in strains carrying the lexA (def) allele, due to impaired repressor dimerization and unstable DNA association; and (4) promoter regions that carry DNA targets that resemble the conserved LexA operator sequence. The first investigations to show that the SOS response is p0030 a global genomic response to DNA damage were performed in dt0025 dt0010 dt0030 dt0015 dt0035 dt0020 dt0040 Graham C Walker's laboratory. Through random insertion of a lacZ reporter gene into the E. coli chromosome, they identified genes whose expression was induced following DNA damage. Characterization of genes upregulated in a recA/ lexA-dependent manner revealed a 20-base-pair consensus LexA-binding site in promoter regions of SOS genes. Whole genome technologies that use microarrays to analyze transcrip-tome or chromatin immunoprecipitation experiments have now identified the full catalog of genes regulated by LexA. While the roles of most of the newly identified LexA-regulated genes are still unknown, unraveling their particular functions will yield insight into the molecular mechanisms underlying the SOS response. Several gene transcripts are decreased following DNA damage and some, while exhibiting a similar expression profile as genes of the LexA regulon, are not directly regulated by LexA. It thus seems that the SOS response is part of a larger, coordinated response network. s0025 The LexA Regulatory System in the Repressed State p0035 LexA exerts repression by binding to target sites located near promoters of SOS genes, blocking access of RNA polymerase. The C-terminal domain (CTD) of LexA is involved in dimer-ization and the N-terminal domain (NTD) in DNA binding. Intact LexA dimerizes by the CTD, and binds to DNA via a helix-turn-helix in its NTD. p0040 LexA binding motifs are conserved in many Gram-negative bacteria. The consensus DNA target in E. coli is a palindromic dyad taCTGT-(at)4-ACAGta and is designated the LexA box or SOS box. Functional LexA repressor is a homodimer while intracellular monomer levels are very low. Each of the two symmetrically inverted DNA-binding elements accommodates one LexA subunit. For stabile and specific DNA binding, a conformational change in LexA must occur. Binding to consensus targets with dyad symmetry requires LexA subunit-subunit interactions that enable high specificity and stabilizes interactions with both halves of the DNA duplex. p0045 The LexA box exhibits considerable diversity; thus, no two sequences are alike and LexA binds with different affinities to the various variants enabling differential induction of the LexA regulon genes. The location of SOS boxes at promoters varies with respect to the transcription start site; some are positioned between the - 35 and - 10 elements, some overlap with the promoter elements, while others are adjacent to the target promoter. Although most E. coli LexA regulon genes possess a single LexA operator site, the number can range up to three SOS boxes. For example, the promoter region of the lexA gene carries separated tandem operators. LexA autoregulation sets a control of its own intracellular level via a feedback mechanism, enabling a rapid response to even small amounts of the inducing signal. s0030 Triggers of the SOS Response p0050 SOS genes can be induced by diverse exogenous treatments such as irradiation or chemicals, and can also be induced by DNA damage, caused by metabolic intermediates within the cell, by stalled replication forks, or by defects following recombination or chromosome segregation. Physical stress, such as high pressure that induces activity of the type IV restriction endonuclease, and even certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, are also known to induce the SOS response. Note that the SOS-inducing signal is persistent regions of ssDNA that are generated when growing cells attempt to replicate damaged DNA. Depending upon the nature of the inducing signal, either the RecBCD or the RecFOR complex expose ssDNA to RecA. The SOS response can also be triggered independently of p0055 RecA at low intracellular pH when LexA forms aggregates, which results in induction of LexA-repressed genes. Transient failure of pH homeostasis occurs in E. coli upon shifts of extracellular pH or in mutants with improper intracellular pH regulation. Presumably, this is a bacterial survival strategy when crossing the gastric acid barrier. Sensing the Signal and Inducing LexA Inactivation s0035 The major SOS-inducing signal is the accumulation of ssDNA. p0060 During normal growth a limited amount of ssDNA is tolerated; however, above this threshold, the SOS system is induced in a LexA-dependent manner. Long-lived ssDNA is protected and stabilized by the ssDNA-binding (SSB) protein. Tetrameric SSB migrates along ssDNA, transiently melting short DNA hairpins and stimulating RecA filament elongation on DNA. Association of ATP-liganded RecA protomers constitutes an activated nucleoprotein filament (RecA*). RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis (note that RecA protein besides working as a co-protease and activator of the DNA polymerase V plays a central role in recombination and is involved in a surprising range of other reactions in E. coli). LexA is recognized by proteases only following self- p0065 cleavage, when otherwise latent protease recognition signals are exposed in the cleaved fragments. The self-cleavage of LexA results generates LexA N- and C-terminal fragments of 83 and 118 amino acids, respectively. The fragments are rapidly degraded by the ClpXP protease and the degradation of the cleaved C-terminal fragment is facilitated by the Lon protease. Proteolysis ensures proper regulation of induction of the SOS response, since the LexA N-terminal fragment, that contains the DNA binding domain, still retains some repressor function. Insights into the Key Step in the SOS Response soo4o The LexA repressor is stable in normal growing cells, with a p0070 half-life of nearly 1 h. E. coli contains approximately 1300 LexA molecules. Repressor self-cleavage commences approximately 1 min after exposure to UV and, after 5 min, the level of LexA falls 10-fold. Self-cleavage takes place only after LexA has dissociated from its target, since dimers that are bound at specific operator targets cannot be inactivated. Upon LexA interaction with the deep helical groove of p0075 RecA*, intramolecular cleavage of the repressor occurs. LexA is specifically cleaved at its Ala84-Gly85 bond. John W Little and colleagues proposed a Ser-Lys dyad mechanism for LexA autodigestion. The uncharged form of Lys156 helps remove a proton from the Ser119 hydroxy! group, which then acts as a nucleophile to attack the Ala84-Gly85 bond. In vivo cleavage requires RecA but, in vitro, it can proceed independently of RecA at alkaline pH (a reaction termed autocleavage). p0080 Crystal structures of LexA mutants revealed that the cleavage site can adopt two conformations. In the cleavable state, the cleavage site is located adjacent to the catalytic center, the Ser119-Lys156 dyad, while in the noncleavable conformation it is ~20 A away from the active site. It has been suggested that interaction with RecA* induces a conformational change in LexA and deprotonation of Lys156. It was also suggested that RecA* may preferentially interact with and stabilize the LexA cleavable state. However, recent evidence suggests that RecA* can bind to LexA in both the cleavable and noncleavable states. Residue Lys156 is solvent exposed and likely protonated in the LexA noncleavable conformation. The energetic cost of burying the charged group of Lys156, which is required for cleavage, provides another layer of regulation of LexA cleavage and helps to prevent autodigestion. Thus, by acting as a co-protease, RecA inactivates LexA, thereby inducing its expression, together with more than 50 other SOS gene products. s0045 DNA Damage Repair p0085 The level, timing, and duration of expression of each individual LexA regulon genes differ significantly. Most genes of the LexA regulon, including recA, are, in the absence of induction, expressed at a basal level. Specifically bound LexA molecules cannot be inactivated, which accounts for the precise timing of expression of the SOS genes following induction. Genes with high-affinity SOS boxes are expressed late in the SOS response due to a persistent decrease in the intracellular LexA pool. On the contrary, selective derepression of SOS genes with weaker operators occurs in response to minor inducing signals. p0090 The SOS response is characterized by temporal control. Initially, SOS products (recA, ssb) sense DNA damage to protect and maintain the structural integrity of the replication fork. The LexA repressor is also induced immediately. Active RecA* initially signals the upregulation of SOS genes involved in high-fidelity DNA repair. Early induced genes include nucleotide excision repair genes uvrA, uvrB, uvrD that enable single-strand repair catalyzed by the UvrABCD proteins. To facilitate the resumption of processive replication, genes recA, recN, ruvAB of recombina-tional repair are induced. In order to circumvent lesions that inhibit DNA replication even after enhanced recombinational repair, low-fidelity DNA damage tolerance pathways are induced and DNA polymerases, Pollll (poIB), PollV (dinB), PolV (umuC, umuD) that operate in a poorly processive and error-prone manner are synthesized. Their ability to perform translesion DNA synthesis, allows a lethal event to be bypassed and replication to recover. These polymerases are the main contributors to SOS mutagenesis, which is an active process. p0095 Precise temporal modulation of SOS gene expression is coordinated with DNA repair processes and influences many other cellular processes. Damage inflicted on bacterial DNA leads to fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. These intracellular assemblies are the functional target for DNA repair and are responsible for protection of the cell's DNA heritage. Cell-Cycle Checkpoints soo5o The expression of SOS genes is turned on in a pattern of p0i00 discrete activation pulses; therefore, the system is not simply induced and turned off when DNA damage is repaired. To prevent the overlap of cell-cycle processes, the SOS system regulates DNA damage and cell division checkpoints. E. coli cell-cycle checkpoints are regulated by the umuDC and p0i05 sulA gene products. Uncleaved UmuD2 in complex with UmuC activates a DNA damage replication checkpoint. UmuD2C inhibits DNA synthesis directly by associating with the DNA replication complex. If high-fidelity repair is insufficient, the UmuD'2C complex, PolV polymerase, is formed. Following SOS induction, dimeric UmuD is converted to functionally active UmuD' by RecA*-induced self-cleavage that is similar to inactivation of LexA. However, RecA*-mediated self-cleavage of UmuD is much slower than self-cleavage of LexA, providing time for accurate repair prior to recovery of replication by translesion DNA synthesis. The UmuD'2C complex is activated by interacting with a single RecA-ATP transferred from the RecA* filament. Translesion DNA synthesis by the PolV polymerase enables replication over any remaining DNA lesions. During the DNA repair process, cell division is inhibited p0ii0 which leads to the formation of cellular filaments. Notably, upon damage to the genome, the LexA-regulated sulA gene product is highly expressed and interacts with the FtsZ protein, involved in septum formation prior to cell division. Most likely, this checkpoint serves to delay cell division until DNA damage has been repaired. In addition, by inhibiting cell division the two daughter chromosomes are not separated enabling recombinational repair. Turning Off the SOS Response s0055 Once DNA damage is repaired and replication resumed, the p0ii5 co-protease activity of RecA disappears resulting in reaccumulation of LexA and repression of the SOS genes. Intracellular proteolysis of SOS gene products is also triggered to control and restrict their activity during the repair and recovery phases of the SOS response respectively. Members of the LexA Super-Family s0060 Jeffrey W Roberts and colleagues demonstrated that exposure p0i20 of lysogens containing bacteriophage 1 to DNA-damaging treatments results in RecA-mediated cleavage of the ICI repressor. SOS regulation enables temperate l-like bacteriophages to sense the physiological condition of the host cell and switch the phage from lysogenic to lytic growth. LexA, UmuD, and several ICI-like repressors, exhibit CTD homology and undergo completely parallel cleavage reactions in helical groove of the RecA* filament. Self-cleavage of LexA is intramolecular while UmuD is cleaved in an intermolecular reaction. Note that upon self-cleavage, dimeric UmuD is converted to the functionally active UmuD', in contrast to repressors that are inactivated by cleavage. Remarkably, compared to LexA, RecA* catalyzes slow self-cleavage of the CI repressor and UmuD; hence, prophage induction and mutagenesis are induced only when DNA is severely damaged. f0010 SOS system repressed DNA damage p'-SOS gen^ "" "" RecA I Specific DNA binding ssDNA-ATP-RecA filament LexA repressor SfiS Q tD SOS system induced LexA dissociates from operator(s) + -36-1^ SOS gene I Protease degradation Decrease in LexA pool DNA-damage repair Arrest of cell-division RecA and LexA synthesis Translesion DNA synthesis Phage induction I LexA monomer accumulates DNA repaired, drop in signal level Figure 1 An overview of the SOS response in E. coli. In the uninduced state, LexA repressor binds to the promoter regions of SOS genes and sterically precludes their transcription. The polymerase III holoenzyme (Pol) carries out DNA replication. In the induced state, at the site of DNA damage PolIII arrests, ssDNA accumulates and active RecA filament is formed. Due to induced LexA self-cleavage, specifically bound LexA repressor dissociates from operators, leading to de-repression of SOS genes. Subsequently, as DNA damage is repaired, SOS induction is reversed. Adapted from Butala M, Žgur-Bertok D, and Busby SJW (2009) The bacterial LexA transcriptional repressor. Cellular and Molecular Life Sciences 66: 82-93. s0065 Plasmid-Encoded Genes of the LexA Regulon p0i25 Some plasmid-encoded genes, with broader functions than defense against DNA damage and adaptation through mutagenesis, are also part of the LexA regulon. For example, colicins are plasmid-encoded bacteriocins, synthesized by and active against E. coli strains and its close relatives. Colicins are released into the environment only after lysis of the host cell. Expression of operons encoding colicin functions are always strongly repressed by LexA, and slow dissociation from the operators may account for the late induction of colicin genes during the SOS response. RecA-mediated production of bacteriocins thus resembles pro-phage induction, leading to cell lysis upon persistent, high level DNA damage. Many colicins can promote genetic diversity in E. coli populations pointing to a role in evolution. p0i30 The qnr genes, which encode fluoroquinolone-resistance determinants, provide another example of plasmid-borne LexA-repressed genes. These are widespread in Enterobacteria-ceae and are all directly regulated by LexA. Since fluoroquino-lones induce self-cleavage of LexA, this is the first example of SOS-dependent regulation of an antibiotic-resistance mechanism in response to the antibiotic itself. C-terminal domain Hinge region N-terminal domain Figure 2 Model of the E. coli LexA repressor bound to the operator DNA f0015 site. LexA dimerises by the carboxy-terminal domain, and interacts with DNA by the amino-terminal domain. The two domains are linked by a flexible hinge region. Adapted from Butala M, Žgur-Bertok D, and Busby SJW (2009). The bacterial LexA transcriptional repressor. Cellular and Molecular Life Sciences 66: 82-93. soo7o Bacterial LexA Regulon Diversity p0i35 Although the SOS system is highly conserved among bacteria, the genes controlled by LexA, their regulation and consensus LexA-binding sites differ significantly. In Bacillus subtilis LexA regulates 26 operons encompassing 63 genes (note that the B. subtilis LexA protein is also designated DinR). In comparison, the E. coli LexA regulon comprises 57 genes and has only eight orthologs in B. subtilis. To further illustrate the diversity p0140 found in SOS networks, in both Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp., the LexA paralogue can function both to repress and to activate transcription. The Virulent Side of the SOS Response Besides high-fidelity repair pathways, SOS genes encode low-fidelity translesion DNA polymerases (in E. coli, Polll [polB], PollV [dinB], and PolV [umuC, umuD]) that enable bacteria to increase their mutation rate in times of stress. Studies employing therapeutic drugs showed that low or subinhibitory concentrations of certain antibiotics, that interfere with DNA replication as well as cell wall synthesis, can trigger the SOS response. Hence, antibiotics can accelerate evolution by, for example, the acquisition of point mutations that result in inactivation or efflux of the drug. SOS-inducing antibiotics also affect virulence in several pathogenic bacteria. Antibiotics that activate RecA*-mediated inactivation of LexA also trigger self-cleavage of phage repres-sors of resident prophages in E. coli, Vibrio cholerae, and Staphy-lococcus aureus. Consequently, certain antibiotics promote the horizontal spread of temperate phage and associated pathoge-nicity islands. In addition, the lateral transfer of integrating p0145 RecA5 (a) (b) Figure 3 Crystal structure of the active E. coli RecA filament (pdb ID: 3CMU), the front (a) and the side view (b). The six RecA protomer monomers (numbered) form a filament on the 18 nt ssDNA (nucleotides are in yellow). ADP-aluminum fluoride-Mg (ADP-AF4-Mg) is a nonhydrolyzable ATP analog. ADP-AlF4-Mg is sandwiched between two adjacent RecA protomers (ADP in yellow, Mg in red). Dotted arrow indicates deep helical groove. Figure prepared with visual molecular dynamics (VMD). From Humphrey W, Dalke A, and Schulten K (1996) VMD: Visual molecular dynamics. Journal of Molecular Graphics 14: 33-38. Lys156 (a) (b) Figure 4 Two distinct conformations of the LexA cleavage site region and a detailed view of the active site. (a) Cleavage site region in the noncleavable state (pdb ID: 1jhh, chain A) is presented in blue and the CTD (pdb ID: 1jhe, chain A) in the cleavable state in red. The catalytic dyad, Ser119 and Lys156, is presented as a sick model and cleavage site Ala84-Aly85 as a ribbon presentation in yellow. (b) Model of the LexA self-cleavage mechanism. Neutral base Lys156 activates the nucleophile LexA119. Hydroxyl group of the activated nucleophile attacks the carbonyl carbon of the scissile peptide bond (arrow), followed by the transfer of the proton to the newly generated amino group (dotted line). The figure was generated by VMD and adapted from Butala M, Žgur-Bertok D, and Busby SJW (2009) The bacterial LexA transcriptional repressor. Cellular and Molecular Life Sciences 66: 82-93. s0075 10020 10025 conjugative elements, for example, the V. colerae SXT element encoding antibiotic resistance, can be induced. Thus, SOS-induced mobilization and high-frequency horizontal transfer of DNA elements accelerate the spread of virulence factors and drug resistance genes. In E. coli, induction of the LexA regulon has been shown to be required for the acquisition of resistance to ciprofloxacin and rifampicin. In addition, recombination of integrons, genetic elements capable of incorporating and expressing promoterless genes, was shown to be controlled by the SOS response. p0i50 Cells in a bacterial population can survive antibiotic stress by forming dormant cells, designated as persisters that are highly tolerant to antibiotics. Persisters are not mutants but rather phenotypic variants of sensitive cells. Recently, a small membrane-acting peptide encoded by the LexA-regulated gene, tisB, was suggested to control persister formation. p0i55 Distinct from drug-induced mobilization of DNA elements, the SOS system also induces chromosomal virulence gene expression. For example, prophage encode the E. coli Shiga toxin. In enteropathogenic E. coli, SOS regulates a type III secretion system responsible for secretion of virulence-associated factors into host cells. Interestingly, in some S. aureus strains, a LexA-regulated gene encodes the fibronectin binding protein (FnbB) that mediates tissue attachment and the establishment of infection. See also: 00419; 00233; 00238; 00253; 00486. Further Reading Butala M, Zgur-Bertok D, and Busby SJW (2009) The bacterial LexA transcriptional repressor. Cellular and Molecular Life Sciences 66: 82-93. Courcelle J, Khodursky A, Peter B, Brown PO, and Hanawalt PC (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158: 41-64. Erill I, Campoy S, and Barbe J (2007) Aeons of distress: An evolutionary perspective on the bacterial SOS response. FEMS Microbiology Reviews 31: 637-656. Foti JJ, Simmons LA, Beuning PJ, and Walker GC (2010) Signal transduction in the Escherichia coli SOS response. In: Bradshaw RA and Dennis EA (eds.) Handbook of Cell Signaling, vol. 3, pp. 2127-2136. Elsevier. Kelley WL (2006) Lex marks the spot: The virulent side of SOS and a closer look at the LexA regulon. Molecular Microbiology 62:1228-1238. Little JW (1991) Mechanism of specific LexA cleavage: Autodigestion and the role of RecA coprotease. Biochimie 73: 411-421. Luo Y, Pfuetzner RA, Mosimann S, et al. (2001) Crystal structure of LexA: A conformational switch for regulation of self-cleavage. Cell 106: 585-594. Sassanfar M and Roberts JW (1990) Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. Journal of Molecular Biology 212:79-96. [Ana Allosteric Regulation 2 Aminopeptidases 3 Aspartic Proteases 4 B12-Containing Enzymes Biotin Chemiluminescence and Bioluminescence 7 Coenzyme A 8 Collagenases 9 Cysteine Proteases 10 Disulfide Bond Formation 11 Enzyme Inhibitors 12 Enzyme Kinetics Enzyme Reaction Mechanisms: 13 Stereochemistry 14 Flavins 15 Heme Proteins 16 Kinetic Isotope Effects 17 Low Barrier Hydrogen Bonds 18 Metalloproteases 19 Peptide Amidation 20 Proteases in Blood Clotting Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister 1 5 6 21 Protein N-Myristoylation 22 Protein Palmitoylation 23 Pteridines 24 Pyridoxal Phosphate 25 Selenoprotein Synthesis 26 Substrate Binding Catalysis and Product Release 27 Zinc Fingers Pentose Phosphate (Hexose Mono 28 Phosphate) Pathway Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Metabolism Vitamins and Hormones NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister Michael Toney Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION M. Daniel Lane 29 Amino Acid Metabolism Metabolism Vitamins and Hormones Luc Cynober M. Daniel Lane 31 Bile Salts and their Metabolism Metabolism Vitamins and Hormones Ulrich Beuers M. Daniel Lane 32 The Chemistry of Alzheimer Disease Carbohydrate responsive element 33 binding protein Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones George H. Sack Jr. M. Daniel Lane Kosaku Uyeda M. Daniel Lane 34 Coenzyme A Metabolism Vitamins and Hormones M. Daniel Lane M. Daniel Lane 35 Diabetes Metabolism Vitamins and Hormones David W. Cooke M. Daniel Lane Fat Mobilization: Perilipin and Hormone-36 Sensitive Lipase Metabolism Vitamins and Hormones Alan Kimmel M. Daniel Lane 37 Fatty Acid Metabolism and Cancer Metabolism Vitamins and Hormones F Kuhajda M. Daniel Lane 38 Fatty Acid Synthesis and its Regulation Metabolism Vitamins and Hormones Steven D. Clarke M. Daniel Lane 39 Folate & Vit B12 Metabolism Vitamins and Hormones B. Shane M. Daniel Lane 40 Gluconeogenesis Metabolism Vitamins and Hormones Richard W. Hanson M. Daniel Lane 41 Glucose/Sugar Transport in Mammals Metabolism Vitamins and Hormones Jeffrey Pessin M. Daniel Lane 42 Glycogen Metabolism 43 Glycogen Storage Diseases 44 Glycolysis Overview 45 Gut orex-anorex NPs Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Peter J. Roach M. Daniel Lane George H. Sack Jr. M. Daniel Lane Robert A. Harris M. Daniel Lane T. Moran M. Daniel Lane Insulin- and Glucagon-Secreting Cells of 46 the Pancreas Metab/orexigenic & anorex 47 neuropeptides 48 Metabolomic profiling 49 Photosynthesis 50 Photosynthetic Carbon Dioxide Fixation Phosphofructokinase-2/Fructose 51 Bisphosphatase-2 52 Porphyrin Metabolism 53 Pyruvate Kinase Regulation of Gene Transcription by 54 Hypoxia-Inducible Factor 1 55 Role of Aquaporins 56 Vitamin A (Retinoids) 57 AAA-ATPases 58 Calpain 59 HIV Protease Lipid Modification of Proteins: Targeting 60 to Membranes 61 Phage Display for Protein Binding Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Franz M. Matschinsky M. Daniel Lane G Morton M. Daniel Lane C. Newgard M. Daniel Lane Richard C. Leegood M. Daniel Lane Matthew J. Paul M. Daniel Lane Daniel M. Raben M. Daniel Lane Harry A. Dailey M. Daniel Lane Kosaku Uyeda M. Daniel Lane Greg Semenza M. Daniel Lane Peter Agre M. Daniel Lane Joseph L. Napoli M. Daniel Lane Andrei Lupas Wolfgang Baumeister Wolfgang Baumeister Hiroyuki Sorimachi Ben M. Dunn Wolfgang Baumeister Marilyn D. Resh Wolfgang Baumeister Wolfgang Baumeister Henry B. Lowman 62 Protein Carboxyl Esterification 63 Protein Degradation 64 Protein Folding and Assembly Regulated Intramembrane Proteolysis 65 (Rip) 66 Two-Hybrid Protein-Protein Interactions 67 Tyrosine Sulfation 68 Ubiquitin-Like Proteins 69 Protein Data Resources Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Jeffry B. Stock Wolfgang Baumeister Wolfgang Baumeister Alfred L. Goldberg David P. Goldenberg Wolfgang Baumeister Jin Ye Wolfgang Baumeister Ilya Serebriiskii Wolfgang Baumeister Denis Corbeil Wolfgang Baumeister Edward T. H. Yeh Wolfgang Baumeister Janet Thornton Wolfgang Baumeister 70 Cholesterol Synthesis Metabolism Vitamins and Hormones P Espenshade M. Daniel Lane 71 Fatty Acid Oxidation Metabolism Vitamins and Hormones NO REVISION M. Daniel Lane 72 Branched-Chain amino acids Metabolism Vitamins and Hormones David T. Chuang M. Daniel Lane 73 Hexokinases/Glucokinases Metabolic Control during Ischemia of the 75 Heart Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Emile Van Schaftingen Garry Lopaschuk M. Daniel Lane M. Daniel Lane Carbohydrate Metabolism in the Central 76 Nervous System Metabolism Vitamins and Hormones I Simpson M. Daniel Lane 77 Regulation by Fatty Acids/Malonyl-CoA in the brain Metabolism Vitamins and Hormones M Wolfgang M. Daniel Lane 78 Role of the micro RNAs in Metabolism Structure and Regulation of Pyruvate 79 Dehydrogenase Complex 80 Chaperonins 81 Mass Spec of Native Complexes 82 Mass spec and proteomics Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation G. Wong J. Milne M. Daniel Lane M. Daniel Lane Ulrich Hartl Wolfgang Baumeister Albert Heck Wolfgang Baumeister Matthias Mann Wolfgang Baumeister 83 Sphingolipid Metabolism and Disease Metabolism Vitamins and Hormones Roscoe O. Brady M. Daniel Lane 84 Biochem of liver regeneration Metabolism Vitamins and Hormones A-M Diehl M. Daniel Lane 85 T Cell Receptor Signaling to NF-kappaB Metabolism Vitamins and Hormones Joel Pomerantz M. Daniel Lane 86 Biliary Cirrhosis Primary Metabolism Vitamins and Hormones Marshall M. Kaplan M. Daniel Lane 87 Starvation Metabolism Vitamins and Hormones Richard W. Hanson M. Daniel Lane 88 Biochem of hematopoiesis Metabolism Vitamins and Hormones Alan Friedman M. Daniel Lane 89 Adipogenesis Metabolism Vitamins and Hormones M. Daniel Lane M. Daniel Lane 90 Biochemistry of muscle contraction Metabolism Vitamins and Hormones DD Thomas M. Daniel Lane 91 Biochemistry of development: Muscle Metabolism Vitamins and Hormones Rhonda Bassel- Duby M. Daniel Lane 92 Vitamin C Metabolism Vitamins and Hormones Francene Steinberg M. Daniel Lane 93 Insect metabolism/hormones Metabolism Vitamins and Hormones RL Miesfeld M. Daniel Lane 94 Biochem of neurogenesis Metabolism Vitamins and Hormones H Song M. Daniel Lane Vitamin K: Biochemistry Metabolism and 95 Nutritional Aspects Metabolism Vitamins and Hormones J.W Suttie M. Daniel Lane 96 Adiponectin: metabolic role Metabolism Vitamins and Hormones PE Scherer M. Daniel Lane 97 Vitamin D Metabolism Vitamins and Hormones H DeLuca M. Daniel Lane 98 Color Vision / Biochem of vision Metabolism Vitamins and Hormones Gerald Jacobs M. Daniel Lane 99 Ketogenesis Metabolism Vitamins and Hormones Charles Hoppel M. Daniel Lane 100 The Fatty Acyl-CoA Synthetases Metabolism Vitamins and Hormones P. Watkins M. Daniel Lane 101 Urea cycle: Disease Aspects Metabolism Vitamins and Hormones Marc Yudkoff M. Daniel Lane 102 Biochemistry: thiamine/thiamine-PP Metabolism Vitamins and Hormones L.Bettendorff M. Daniel Lane 103 Biochemistry: Niacin/NAD(P) 104 Peroximsomes: Metabolic Role 105 Riboflavin: flavoproteins-FAD/FMN Gastrointestinal digestion And 106 Absorbtion 107 Mucins in Embryo Implantation 108 Glycosylation Congenital Disorders of Glycoprotein-Mediated Cell Interactions 109 O-Linked Glycoprotein Folding and Processing 110 Reactions GlcNAc Biosynthesis and Function O- 111 Linked 112 Prions Overview 113 Proteoglycans 114 Lipid Bilayer Structure 115 Glycoproteins N-Linked 116 Insulin: Mech/Metab actions Glycolipid-Dependent Adhesion 117 Processes 118 Lipases 119 Sugar Nucleotide Transporters 120 Glycation 121 Endocytosis Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Metabolism Vitamins and Hormones Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Protein/Enzyme Structure Function and Degradation Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Metabolism Vitamins and Hormones Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins C Brenner M. Daniel Lane Steve Gould M. Daniel Lane Andrea Mattevi M. Daniel Lane J Keller M. Daniel Lane Dan Carson William Lennarz Hudson Freeze William Lennarz Robert Haltiwanger William Lennarz Armando Parodi William Lennarz Kaoru Sakabe William Lennarz Detlev Riesner Wolfgang Baumeister Jeffrey D. Esko William Lennarz Erwin London William Lennarz Mark Lehrman William Lennarz Derek LeRoith M. Daniel Lane NO REVISION William Lennarz NO REVISION William Lennarz Carlos Hirschberg William Lennarz John Baynes William Lennarz Julie Donaldson William Lennarz 122 Luft's Disease Bioenergetics NO REVISION Ernesto Carafoli 123 Calcium Biological Fitness of????? Bioenergetics NO REVISION Ernesto Carafoli 124 Spectrophotometric Assays Bioenergetics NO REVISION Ernesto Carafoli 125 Membrane Transport General Concepts Bioenergetics NO REVISION Ernesto Carafoli 126 Mitochondrial DNA Bioenergetics NO REVISION Ernesto Carafoli 127 Oxygenases Bioenergetics NO REVISION Ernesto Carafoli 128 V-ATPases Bioenergetics Michael Forgac Ernesto Carafoli 129 Superoxide Dismutase 130 Cell-Matrix Interactions Bioenergetics Lipids Carbohydrates Membranes and Membrane Proteins Irwin Fridovich Ernesto Carafoli Janet Askari William Lennarz 131 Cytochrome Oxidases Bacterial Membrane Transporters:Na+/Ca2+ 132 Exchangers 133 Ion Channel Protein Superfamily Bioenergetics Bioenergetics Lipids Carbohydrates Membranes and Membrane Proteins Peter Brzezinski Ernesto Carafoli Jonathan Lytton Ernesto Carafoli William A. Catterall William Lennarz 134 Chlorophylls and Carotenoids Bioenergetics ATP Synthesis in Plant Mitochondria: 135 Substrates Inhibitors Uncouplers Bioenergetics Nicotinamide Nucleotide 136 Transhydrogenase Bioenergetics Hugo Scheer Ernesto Carafoli Kathleen Soole Ernesto Carafoli Jan Rydstrom Ernesto Carafoli 137 Plastocyanin Bioenergetics NO REVISION Ernesto Carafoli 138 Neuronal Calcium Signal Bioenergetics Hilmar Bading Ernesto Carafoli 139 Calcium-Modulated Proteins (EF-Hand) Bioenergetics 140 Calcium Sensing Receptor Bioenergetics Robert H. Kretsinger Edward M. Brown Ernesto Carafoli Ernesto Carafoli 141 Chloroplasts Bioenergetics Nicoletta Rascio Ernesto Carafoli 142 Respiratory Chain Complex II and Succinate: Quinone Oxidoreductases Bioenergetics Roy Lancaster Ernesto Carafoli Mitochondrial Membranes Structural Carmen A. 143 Organization Bioenergetics Mannella Ernesto Carafoli 144 Voltage-Dependent K+ Channels 145 Heme Synthesis 146 ER/SR Calcium Pump: Structure Bioenergetics Bioenergetics Bioenergetics Ramon Latorre Ernesto Carafoli Gloria C. Ferreira Ernesto Carafoli Chikashi Toyoshima Ernesto Carafoli Calcium Buffering Proteins: ER Luminal 147 Proteins Bioenergetics Purple Bacteria: Photosynthetic Reaction 148 Centers Bioenergetics Mitochondrial Metabolite Transporter 149 Family Bioenergetics 150 IP3 Receptors Bioenergetics The mitochondrial permeability transition Marek Michalak Ernesto Carafoli Roy Lancaster Ernesto Carafoli Ferdinando Palmieri Katsuhiko Mikoshiba Ernesto Carafoli Ernesto Carafoli 151 pore Bioenergetics Paolo Bernardi Ernesto Carafoli 152 Chloroplast Redox Poise and Signaling Bioenergetics Jean-David Rochaix Ernesto Carafoli 153 Calcium Oscillations 154 Trp channels 155 Respiratory Chain Complex I Mitochondrial calcium transport : 156 historical aspects Bioenergetics Bioenergetics Bioenergetics Bioenergetics Ole Petersen Ernesto Carafoli Indu S. Ambudkar Ernesto Carafoli Ulrich Brandt Ernesto Carafoli Ernesto Carafoli Ernesto Carafoli 157 Structure of P-type ATPases Bioenergetics Poul Nissen Ernesto Carafoli 158 Cytochrome b6f Complex Bioenergetics Green Sulfur Bacteria: Reaction Center 159 and Electron Transport Bioenergetics William Cramer Ernesto Carafoli Donald A. Bryant Ernesto Carafoli 160 P-Type Pumps: H+/K+ Pump Bioenergetics Mitochondrial Outer Membrane and the 161 VDAC Channel Bioenergetics Jai M Shin Marco Colombini Ernesto Carafoli Ernesto Carafoli 162 Uncoupling Proteins Bioenergetics Daniel Ricquier Ernesto Carafoli Nuclear Genes in Mitochondrial Function 163 and Biogenesis Cytochrome bc1 Complex (Respiratory 164 Chain Complex III) Photosystem II Light Harvesting System: 165 Dynamic Behavior 166 Ubiquitin System 167 Amyloid 168 Biotinylation of Proteins 169 Collagens 170 Elastin 171 Proteasome Overview 172 Secretases 173 Affinity Tags for Protein Purification Calcium Signaling: Calmodulin- 174 Dependent Phosphatase Calcium in the regulation of the gene 175 expression Bioenergetics Bioenergetics Bioenergetics Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Protein/Enzyme Structure Function and Degradation Bioenergetics Bioenergetics Alexander Tzagoloff Ernesto Carafoli NO REVISION Ernesto Carafoli Peter Horton Ernesto Carafoli Aaron Ciechanover Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister NO REVISION Wolfgang Baumeister Claude Klee Ernesto Carafoli Jose Ramon Naranjo Ernesto Carafoli 176 Ferredoxin Bioenergetics Giuliana Zanetti Ernesto Carafoli 177 Ferredoxin-NADP+ Reductase Bioenergetics Giuliana Zanetti Ernesto Carafoli 178 Pyrimidine Biosynthesis Bioenergetics Monika Loffler Ernesto Carafoli 179 Peroxidase catalysis and redox signaling Bioenergetics Alberto Bindoli Ernesto Carafoli 180 Chemiosmotic Theory Bioenergetics Green Bacteria: Secondary Electron 181 Donor (Cytochromes) Bioenergetics Keith Garlid Ernesto Carafoli Hirozo Oh-oka Ernesto Carafoli 182 Amine Oxidases Bioenergetics Giovanni Floris Ernesto Carafoli 183 Voltage-Sensitive Ca2+ Channels 184 187 Photosystem I: FX FA and FB Iron-Sulfur Clusters 185 Lipid Rafts 186 Neoglycoproteins Store operated calcium channels. 2 : ORAI 1 188 P-Type Pumps: Copper Pump Bioenergetics Bioenergetics Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Bioenergetics Bioenergetics Intracellular Calcium Channels: cADPR-189 Modulated (Ryanodine Receptors) Bioenergetics Harald Reuter Ernesto Carafoli John H. Golbeck Ernesto Carafoli NO REVISION William Lennarz NO REVISION William Lennarz Anjana Rao Ernesto Carafoli Svetlana Lutsenko Gerhard Meissner Ernesto Carafoli Ernesto Carafoli 190 192 193 194 Giant Mitochondria (Megamitochondria) Bioenergetics Bioenergetics Mitochondrial Genes and their Expression: Yeast Calcium-Binding Proteins: Cytosolic (Annexins Gelsolins C2-Domain Proteins) P-Type Pumps: Plasma-Membrane H+ Pump Bioenergetics Bioenergetics Bernard Tandler Ernesto Carafoli Giovanna Carignani Ernesto Carafoli Joachim Krebs Ernesto Carafoli Carolyn W. Slayman Ernesto Carafoli 195 Troponin 196 197 Bioenergetics The Arachidonic Acid Regulated Calcium Channel Bioenergetics Plasma-Membrane Calcium Pump: Structure and Function Bioenergetics Iwao Ohtsuki Ernesto Carafoli Trevor Shuttleworth Ernesto Carafoli Marisa Brini Ernesto Carafoli 198 P-Type Pumps: Na+/K+ Pump Bioenergetics Steve Karlish Ernesto Carafoli 199 ES/SR Calcium Pump: Function Bioenergetics Giuseppe Inesi Ernesto Carafoli 200 Mitochondrial Auto-Antibodies Bioenergetics Harold Baum Ernesto Carafoli 201 Cytochrome P-450 Bioenergetics Respiratory Processes in Anoxygenic 202 and Oxygenic Phototrophs Bioenergetics Rita Bernhardt Ernesto Carafoli Roberto Borghese Ernesto Carafoli 203 Protein Import into Mitochondria Bioenergetics Walter Nfeupert Ernesto Carafoli 204 Quinones Bioenergetics Giorgio Lenaz Ernesto Carafoli 205 Hydrogenase structure and function Bioenergetics Wolfgang Lubitz Ernesto Carafoli 206 Calcium Signaling: NO Synthase 207 Membrane-Associated Energy Transduction in Bacteria and Archaea Bioenergetics Bioenergetics Dennis Stuehr Ernesto Carafoli Guenter Schaefer Ernesto Carafoli 208 Sodium Channels Bioenergetics William A. Catterall Ernesto Carafoli 209 Lipid signaling and ion channels Bioenergetics Bertil Hille Ernesto Carafoli 210 Calcium Transport in Mitochondria Glycosylphosphatidylinositol (GPI) 211 Anchors Carbohydrate Chains: Enzymatic and 212 Chemical Synthesis Bioenergetics Rosario Rizzuto Ernesto Carafoli Lipids Carbohydrates Membranes and Membrane Proteins Anant Menon William Lennarz Lipids Carbohydrates Membranes and Membrane Proteins Chi-Huey Wong William Lennarz Bioenergetics: General Definition of 213 Principles Bioenergetics NO REVISION Ernesto Carafoli 214 Respiratory Chain and ATP Synthase Bioenergetics Anthony Moore Ernesto Carafoli Lipids Carbohydrates Membranes and Nathan C. 215 MDR Membrane Proteins Membrane Proteins Rockwell William Lennarz 216 Mitochondrial dynamics 217 Lectins Bioenergetics Lipids Carbohydrates Membranes and Membrane Proteins Luca Scorrano Ernesto Carafoli Nathan Sharon William Lennarz Periplasmic Electron Transport Systems 218 in Bacteria Bioenergetics David Richardson Ernesto Carafoli 219 Chemolithotrophy?? Cyclic ADP ribose and NAADP in 220 calcium signaling Bioenergetics Bioenergetics Alan Hooper Ernesto Carafoli Luigia Santella Ernesto Carafoli 221 Excitation-contraction coupling Bioenergetics Donald Bers Ernesto Carafoli 222 Iron-Sulfur Proteins Bioenergetics Richard Cammack Ernesto Carafoli 223 Vitamin E Metabolism Vitamins and Hormones Jeffrey Atkinson M. Daniel Lane 224 ABC Transporters Bioenergetics André Goffeau Ernesto Carafoli 225 Phosphatidylinositol-3-Phosphate 226 227 228 229 230 231 232 Free Radicals Sources and Targets of: Mitochondria Photoinhibition and photoprotection in plants, algae, and cyanobacteria The sodium/calcium exchanger : structural aspects Chromatin: Methyl-CpG-DNA binding proteins Bioenergetics Bioenergetics Photosystem II: Assembly and Turnover of the D1 Protein Bioenergetics Calcium Buffering Proteins: Calbindin Bioenergetics Bioenergetics Bioenergetics Molecular Biology Chromatin: Nucleosome positioning - the GAL Promoter Molecular Biology Michael Czech Ernesto Carafoli Alberto Aboveris Ernesto Carafoli Eva-Mari Aro Ernesto Carafoli Sylvia Christakos Giorgio Giacometti Kenneth Philipson Ernesto Carafoli Ernesto Carafoli Ernesto Carafoli David G. Skalnik Nancy L. Craig Dennis Lohr Nancy L. Craig 233 DNA Damage: Alkylation DNA Methyltransferases Structural 235 Themes DNA Methyltransferases: Eubacterial 236 GATC DNA Mismatch Repair and Homologous 237 Recombination DNA Mismatch Repair and the DNA 238 Damage Response Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology John Tainer Nancy L. Craig Nancy L. Craig Xiaodong Cheng Martin G. Marinus Ivan Matic Guo-Min Li Nancy L. Craig Nancy L. Craig Nancy L. Craig 239 DNA Mismatch Repair in Bacteria Molecular Biology A-Lien Lu Nancy L. Craig 240 DNA Oxidation 241 DNA Polymerase p Eukaryotic Molecular Biology Molecular Biology Dmitry Zharkov Nancy L. Craig Nancy L. Craig Samuel H. Wilson 242 DNA Replication Fork Eukaryotic Molecular Biology DNA Restriction and Modification: Type 243 III Enzymes Molecular Biology Zvi Kelman Nancy L. Craig Desirazu N. Rao Nancy L. Craig 244 DNA Supercoiling Molecular Biology Tao-shih Hsieh Nancy L. Craig 245 DNA Topoisomerases: Type I Molecular Biology James J. Champoux Nancy L. Craig 246 DNA Topoisomerases: Type I Molecular Biology Neil Osheroff Nancy L. Craig 247 HIV-1 Reverse Transcriptase Structure Molecular Biology 248 Homologous Recombination in Meiosis Molecular Biology 249 lac Operon Nonhomologous recombination: DNA 250 transposons Nuclear Organization Chromatin 251 Structure and Gene Silencing 252 Nucleolus Overview Nucleotide Excision Repair Bacterial: 253 The UvrABCD System 254 Nucleotide Excision Repair: Biology 256 Prions and Epigenetic Inheritance Recombination-Dependent DNA 257 Replication Reverse Transcriptase, Integrase and 258 Retroviral Replication 259 Ribosome Assembly 260 Riboswitches Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Steven Hughes Nancy L. Craig Nancy M. Hollingsworth Nancy L. Craig Kathleen Matthews Michael Chandler Nancy L. Craig Nancy L. Craig Lori L. Wallrath Nancy L. Craig Thoru Pederson Nancy L. Craig Nancy L. Craig Bennett Van Houten Errol C. Friedberg Reed B. Wickner Kenneth N. Kreuzer Nancy L. Craig Nancy L. Craig Nancy L. Craig Simon Litvak Nancy L. Craig Nancy L. Craig John L. Woolford Adrian R. Ferré-D'Amaré Nancy L. Craig 261 Ribozymes and Evolution 262 RNA Editing 263 264 Molecular Biology Molecular Biology RNA Polymerase I and RNA Polymerase III in Eukaryotes Molecular Biology RNA Polymerase II Structure in Eukaryotes Molecular Biology Niles Lehman Nancy L. Craig Nancy L. Craig Charles E. Samuel Robert J. White Nancy L. Craig Patrick Cramer Nancy L. Craig 265 RNA Polymerase Structure Bacterial Molecular Biology Sergei Borukhov Nancy L. Craig 266 Sigma Factors Molecular Biology John D. Helmann Nancy L. Craig 267 T7 RNA Polymerase Molecular Biology Rui Sousa Nancy L. Craig 268 Telomeres: Maintenance and Replication Molecular Biology Molecular Biology Translation Initiation in Bacteria: Factors 269 and Mechanisms David Shore Nancy L. Craig Claudio Gualerzi Nancy L. Craig 270 trp Operon and Attenuation XPV DNA Polymerase and Ultraviolet 271 Damage Bypass Non-Homologous End Joining in 272 Eukaryotes Ligand-Operated Membrane Channels: 273 GABA Molecular Biology Molecular Biology Molecular Biology Bioenergetics Paul Gollnick Nancy L. Craig Nancy L. Craig Alan R. Lehmann David J. Chen Nancy L. Craig Erwin Sigel Ernesto Carafoli 274 DNA Sequence Recognition by Proteins Molecular Biology Lipids Carbohydrates 275 Glycoproteins Plant Membranes and Membrane Proteins Greg van Duyne Nancy L. Craig NO REVISION William Lennarz 276 RecQ Helicase Systems Pre-tRNA and Pre-rRNA Processing in 277 Bacteria 278 LexA Regulatory System 279 DNA Glycosylases: Mechanisms Transcription-Coupled DNA Repair 280 Overview Molecular Biology Molecular Biology Molecular Biology Molecular Biology Molecular Biology Ian Hickson Nancy L. Craig Zhongwei Li Nancy L. Craig Matej Butala Nancy L. Craig Alex Drohat Nancy L. Craig Silvia Tornaletti Nancy L. Craig 281 282 283 284 285 Messenger RNA Degradation in Bacteria Molecular Biology Bioenergetics Energy Transduction in Anaerobic Prokaryotes Metabolite Channeling: Creatine Kinase Microcompartments Bioenergetics Calcium/Calmodulin-Dependent Protein Kinase II Bioenergetics ATP Synthesis: Mitochondrial Cyanide- Resistant Terminal Oxidases Bioenergetics David Bechhofer Nancy L. Craig Gottfried Unden Ernesto Carafoli Uwe Schlattner Ernesto Carafoli Howard Schulman Ernesto Carafoli Jim Siedow Ernesto Carafoli 286 Photosystem II: Water Oxidation Overview Bioenergetics Fabrice Rappaport Ernesto Carafoli 287 Photosystem I Structure and Function Bioenergetics Ligand-Operated Membrane Channels: 288 Calcium (Glutamate) Bioenergetics 289 Mitochondrial Channels 290 291 Bioenergetics Light-Harvesting Complex (LHC) I and II: Pigments and Proteins Bioenergetics Mitochondrial Genome Evolution, Inheritance Bioenergetics Petra Fromme Ernesto Carafoli Elias K. Michaelis M. Catia Sorgato Ernesto Carafoli Ernesto Carafoli Stefan Jansson Ernesto Carafoli Douglas C. Wallace Ernesto Carafoli 292 Intracellular Calcium Waves 293 Extracellular Calcium Waves Bioenergetics Bioenergetics Luigia Santella Ernesto Carafoli Michael Sanderson Ernesto Carafoli 294 F1-F0 ATP Synthase Bioenergetics John Walker Ernesto Carafoli 295 Respiratory Chain Complex IV Bioenergetics Hartmut Michel Ernesto Carafoli 296 Mitochondria in myocardial ischemia Complex I of the mitochondrial 297 respiratpory chain 298 DNA Replication Fork Bacterial Bioenergetics Bioenergetics Molecular Biology Fabio Di Lisa Ernesto Carafoli Leonid A. Sazanov Stephen J. Benkovic Ernesto Carafoli Nancy L. Craig 299 Spastic Paraplegia Bioenergetics Green Bacteria: The Light-Harvesting 300 Chlorosome Bioenergetics Store operated calcium channels . 1 : 301 STIM1 Bioenergetics Renewable Hydrogen Energy from 302 Biomass Bioenergetics Elena Rugarli Ernesto Carafoli Mette Miller Ernesto Carafoli Michael Cahalan Ernesto Carafoli Mike Seibert Ernesto Carafoli 303 DNA Ligases: Mechanism and Functions Molecular Biology DNA mismatch repair in disease and 304 ageing Molecular Biology Alan Tomkinson Nancy L. Craig Peggy Hsieh Nancy L. Craig 305 DNA Polymerase 5 Eukaryotic Molecular Biology Peter Burgers Nancy L. Craig 306 DNA Polymerase I Bacterial DNA Mismatch Repair: E. coli Vsr and 307 Eukaryotic G-T Systems Ribozyme Structural Elements: Group I 308 Introns DNA Restriction and Modification: Type I 309 Enzymes Molecular Biology Molecular Biology Molecular Biology Molecular Biology Catherine Joyce Nancy L. Craig Peggy Lieb Nancy L. Craig Barbara Golden Nancy L. Craig David T. F. Dryden Nancy L. Craig 310 DNA Polymerase III Bacterial Molecular Biology Hisaji Maki Nancy L. Craig 311 Organization of the Bacterial Necleoid Molecular Biology Molecular Biology DNA Polymerases: Kinetics and 312 Mechanism Charles Dorman Nancy L. Craig Nancy L. Craig Kenneth A. Johnson 313 DNA Replication: Initiation in Bacteria Molecular Biology Bioenergetics Inositol-tris-phosphate in calcium 314 signaling Jon M. Kaguni Nancy L. Craig Michael Berridge Ernesto Carafoli 315 DNA Mismatch Repair in Mammals Alternative Splicing: Regulation of Sex Determination in Drosophila 316 melanogaster Peroxisome Proliferator-Activated 317 Receptors 318 G12/G13 Family Molecular Biology Molecular Biology Signaling Signaling Eric Alani Nancy L. Craig Processivity Clamps in DNA Replication: 319 Clamp Loading Molecular Biology DNA Restriction and Modification: Type 320 II Enzymes Molecular Biology Paul Schedl Nancy L. Craig Mary C Sugden Joel Moss Joel Moss Stefan Offermanns Michael O'Donnell Stephen E. Halford Nancy L. Craig Nancy L. Craig 321 322 323 324 Alternative Splicing Ras Family Molecular Biology Signaling Nitric Oxide Signaling Signaling Thyroid-Stimulating Hormone/Luteinizing Hormone/Follicle-Stimulating Hormone Receptors Signaling 325 B-Cell Antigen Receptor Signaling Kristen Lynch Lawrence A. Quilliam Michael A. Marletta Deborah L. Segaloff Thomas M. Yankee Nancy L. Craig Joel Moss Joel Moss Joel Moss Joel Moss 326 Dopamine Receptors Signaling Kim A. Neve Joel Moss 327 Src Family of Protein Tyrosine Kinases Signaling NO REVISION Joel Moss 328 Calcitonin Receptor Signaling G Protein-Coupled Receptor Kinases 329 and Arrestins Signaling Samia I. Girgis Joel Moss Jeffrey L. Benovic Joel Moss 330 Photoreceptors Signaling King-Wai Yau Joel Moss 331 Platelet-Activating Factor Receptor 332 FAK Family 333 Von Hippel-Lindau (VHL) Protein Signaling Signaling Signaling Katherine M. Howard Steven K. Hanks Ronald C. Conaway Joel Moss Joel Moss Joel Moss 334 Adrenergic Receptors Signaling David B. Bylund Joel Moss 335 Nuclear Factor kappaB Signaling 336 Muscarinic Acetylcholine Receptors Signaling Thomas D. Gilmore Neil M. Nathanson Joel Moss Joel Moss 337 Glutamate Receptors Metabotropic 338 Protein Kinase C Family Signaling Signaling P. Jeffrey Conn Joel Moss Alexandra C. Newton Joel Moss 339 GABAA Receptor Signaling Richard W. Olsen Joel Moss 340 Serotonin Receptor Signaling Signaling Parathyroid Hormone/Parathyroid 341 Hormone-Related Protein Receptor Signaling Chemotactic Peptide/Complement 342 Receptors Signaling Paul J. Gresch Joel Moss Thomas J. Gardella Joel Moss Eric R. Prossnitz Joel Moss 343 Cyclic GMP Phosphodiesterases Signaling Cyclic Nucleotide-Dependent Protein 344 Kinases Signaling Sharron H. Francis Sharron H. Francis Joel Moss Joel Moss 345 Neurotransmitter Transporters Signaling Kevin Erreger Joel Moss 346 Phospholipase C Signaling Fujio Sekiya Joel Moss 347 Opioid Receptors 348 c-fes Proto-Oncogene 349 Endocannabinoids 350 P2Y Purinergic Receptors 351 Emerging Concepts of Leptin Inositol Phosphate Kinases and 352 Phosphatases 354 ARF Family 355 Brassinosteroids 357 DNA Replication Mitochondrial 358 Tumor Necrosis Factor Receptors Vascular Endothelial Growth Factor 359 Receptors 360 Phospholipase D Signaling Signaling Signaling Signaling Signaling Signaling 353 Cyclic Nucleotide Phosphodiesterases Signaling Signaling Signaling 356 BMP signaling and Vascular Disease Signaling Molecular Biology Signaling Signaling Signaling P. Y. Law Thomas E. Smithgall Joel Moss Joel Moss Daniele Piomelli Joel Moss George R. Dubyak Heike Muenzberg-Gruening Stephen B. Shears Vincent C. Manganiello Gustavo Pacheco- Rodriguez Steven D. Clouse Mark de Caestecker David A. Clayton Kenneth A. Thomas Michael A. Frohman Joel Moss Joel Moss Joel Moss Joel Moss Joel Moss Joel Moss Joel Moss Nancy L. Craig Carl F. Ware Joel Moss Joel Moss Joel Moss 361 Ran GTPase Signaling UNDER INVITE Joel Moss 362 363 Mitogen-Activated Protein Kinase Family Signaling Signaling Calcitonin Gene-Related Peptide and Adrenomedullin Receptors 364 Tachykinin/Substance P Receptors Signaling Silvio Gutkind Joel Moss Debbie L. Hay Joel Moss Madan M Kwatra Joel Moss 365 Small GTPases Signaling Channing Der Joel Moss 366 Protein Tyrosine Phosphatases 367 Hematopoietin Receptors Signaling Signaling Jack Dixon Barbara A. Miller Joel Moss Joel Moss 369 Interferon Receptors Signaling 370 p53 Protein Signaling Purple Bacteria: Electron Acceptors and 371 Donors Bioenergetics NO REVISION Joel Moss Jennifer Pietenpol Roberto De Philippis Joel Moss Ernesto Carafoli 372 ABC transporters : structure Bioenergetics 373 Cell Death by Apoptosis and Necrosis Bioenergetics André Goffeau Ernesto Carafoli Pierluigi Nicotera Ernesto Carafoli 374 Cytochrome c Bioenergetics NO REVISION Ernesto Carafoli 375 Monoamine oxidase Plasma membrane sodium/calcium 376 exchanger . 2 : structural aspects Intracellular Calcium Channels: 379 NAADP+-Modulated Bioenergetics Bioenergetics Bioenergetics Andrea Mattevi Ernesto Carafoli Ken Philipson Ernesto Carafoli Luigia Santella Ernesto Carafoli 380 Mitochondria and the NO radical Bioenergetics Dr Brown Ernesto Carafoli 381 Photosystem II: Protein Components Bioenergetics 382 Proton Pumping in the Respiratory Chain Bioenergetics James Barber Ernesto Carafoli Marten Wikstrom Ernesto Carafoli 383 Glutathione Peroxidases Bioenergetics Fulvio Ursini Ernesto Carafoli 384 Conservative site-specific recombination Molecular Biology Molecular Biology Recombination: Helicases and 385 Nucleases 386 Gi Family of Heterotrimeric G Proteins Signaling 387 mTOR and its downstream targets 388 Chromatin: Physical Organization Signaling Molecular Biology Maggie Smith Nancy L. Craig Grzegorz Ira Nancy L. Craig Maurine E. Linder Christopher G. Proud Christopher L. Woodcock Joel Moss Joel Moss Nancy L. Craig 389 Chemokine Receptors Control of RNA Polymerase II Elongation 390 in Eukaryotes 391 Somatostatin Receptors Signaling Molecular Biology Signaling Ann Richmond Joel Moss David Price Nancy L. Craig Agnes Schonbrunn Joel Moss 392 Steroid/Thyroid Hormone Receptors Signaling 393 DNA Helicases: Dimeric Enzyme Action Molecular Biology Molecular Biology DNA Helicases: HexamericEnzyme 394 Action 395 Anaplerosis 396 Vitamin D Receptor Bioenergetics Signaling Nancy L. Weigel Joel Moss Nancy L. Craig Timothy M. Lohman Smita Patel Nancy L. Craig Raymond R. Russell III Ernesto Carafoli Diane R. Dowd Joel Moss 397 Taste Receptors (possibly better title) Signaling 398 Proteinase-Activated Receptors Signaling John Boughter Joel Moss Morley D. Hollenberg Joel Moss 399 T-Cell Antigen Receptor Signaling Dario Vignali Joel Moss 400 Ribosome Structure 401 Adenylyl Cyclases Molecular Biology Signaling Brian Wimberly Nancy L. Craig Ron Taussig Joel Moss 402 Natriuretic Peptides and their Receptors Signaling Signaling Fibroblast Growth Factor Receptors and 403 Cancer-Associated Perturbations Lincoln Potter Joel Moss Marko Kornmann Joel Moss 404 Rab Family 405 Neurotrophin Receptor Signaling Phosphatidylinositol Bisphosphate and 406 Trisphosphate Non-Homologous End Joining in 407 Bacteria Signaling Signaling Signaling Molecular Biology Mary McCaffrey Joel Moss Bruce Carter Joel Moss NO REVISION Joel Moss Aidan Doherty Nancy L. Craig 408 DNA Polymerase a Eukaryotic Molecular Biology Bik Tye Nancy L. Craig Diacylglycerol Kinases and Phosphatidic 409 Acid Phosphatases Ribozyme Structural Elements: Groups II 410 Introns and the Spliceosome 411 Olfactory Receptors Signaling Molecular Biology Signaling Matthew K. Topham Christina Waldsich Sigrun Korsching Joel Moss Nancy L. Craig Joel Moss 411 DNA Ligases: Structures Molecular Biology John Pascal Nancy L. Craig 412 Eicosanoid Receptors Signaling Richard M. Breyer Joel Moss 413 Cyclic AMP Receptors of Dictyostelium Signaling 414 Reactive Oxygen and Nitrogen Species and Their Interactions With Mitochondria 415 Actin-Capping and -Severing Proteins Bioenergetics Cell Architecture and Function Dale Hereld Joel Moss Victor Darley- Usmar Ernesto Carafoli James Bamburg P. Coulombe + C. Parent 416 Autophagy in Fungi and Mammals Cell Architecture and Function Dan Klionsky P. Coulombe + C. Parent 417 Cell Cycle Controls in G1 and G0 Cell Cycle: Control of Entry and 418 Progression Through S Phase 419 Cell Cycle: DNA Damage Checkpoints 420 Cell Migration Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Steve Dowdy P. Coulombe + C. Parent Susan L Forsburg P. Coulombe + C. Parent Jean Wang P. Coulombe + C. Parent John Victor Small P. Coulombe + C. Parent 421 Chemotaxis Cell Architecture and Function Carole Parent P. Coulombe + C. Parent 422 Chromosome Organization and Structure Overview 423 Dynactin 424 Dynein Endoplasmic Reticulum-Associated 425 Protein Degradation Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Sarah Elgin P. Coulombe + C. Parent Trina A. Schroer P. Coulombe + C. Parent Kenneth K. Pfister Maurizio Molinari P. Coulombe + C. Parent P. Coulombe + C. Parent 426 Exosomes 427 Heat/Stress Responses Cell Architecture and Function Cell Architecture and Function Stephen Gould P. Coulombe + C. Parent Davis Ng P. Coulombe + C. Parent Intermediate Filament Linker Proteins: 428 Plectin and BPAG1 Cell Architecture and Function Gerhard Wiche P. Coulombe + C. Parent 429 Intermediate Filaments Cell Architecture and Function Pierre Coulombe P. Coulombe + C. Parent 430 Keratins and the Skin Cell Architecture and Function Pierre Coulombe P. Coulombe + C. Parent 431 Kinesin Superfamily Proteins 432 Live Imaging of Nuclear Dynamics Major Sperm Protein and Sperm 433 Locomotion Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Nobutaka Hirokawa P. Coulombe + C. Parent Karen Reddy P. Coulombe + C. Parent Tom Roberts P. Coulombe + C. Parent 434 Microtubule-Associated Proteins Cell Architecture and Function Nobutaka Hirokawa P. Coulombe + C. Parent 435 Myosin Motors 436 Neuronal Intermediate Filaments Cell Architecture and Function Cell Architecture and Function Roy Edward Larson P. Coulombe + C. Parent Ron Liem P. Coulombe + C. Parent Nuclear Pores and Nuclear 437 Import/Export 438 Phagocytosis and Pinocytosis Rho GTPases and Actin Cytoskeleton 439 Dynamics 440 Tight Junctions 441 Vacuoles Sliding Clamps in DNA Replication: E. 442 coli p-Clamp and PCNA Structure 443 Friedreich's Ataxia Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Molecular Biology Bioenergetics Anita Corbett P. Coulombe + C. Parent Chris Janetopoulos P. Coulombe + C. Parent Anne Ridley P. Coulombe + C. Parent Sachiko Tsukita P. Coulombe + C. Parent Scott D. Emr P. Coulombe + C. Parent Linda Bloom Nancy L. Craig Anthony Schapira Ernesto Carafoli 444 Indicators of intracellular calcium Bioenergetics Tullio Pozzan Ernesto Carafoli 445 Ribosome regulation by EF-G and EF-Tu Molecular Biology 446 Hydrogen production Bioenergetics Steven Gregory Nancy L. Craig Maria L. Ghirardi Ernesto Carafoli 447 Integrin Signaling Signaling Larry Goldfinger Joel Moss 448 Chromatin Remodeling Molecular Biology Erica Hong Nancy L. Craig 449 GABAB Receptor Signaling S. J. Enna Joel Moss 451 Retinoic Acid Receptors Signaling Martin Petkovich Joel Moss 452 Serine/Threonine Phosphatases Signaling Tom Ingebritsen Joel Moss 453 Pheromone Receptors (Yeast) Signaling James Konopka Joel Moss 454 Gq Family Cyclic Nucleotide-Regulated Cation 455 Channels 456 Cytokinin Signaling Signaling Signaling Wanling Yang Joel Moss Martin Biel Thomas Schmulling Joel Moss Joel Moss 457 G Protein Signaling Regulators Signaling No Revision Joel Moss 458 Vasopressin/Oxytocin Receptor Family Signaling Mike Brownstein Joel Moss 459 Glycogen Synthase Kinase-3 Signaling Jim Woodgett Joel Moss 460 Retinoblastoma Protein (pRB) Signaling Nick Dyson Joel Moss 461 Cadherin Signaling 462 Sphingolipid Catabolism Signaling Lipids Carbohydrates Membranes and Membrane Proteins David B. Sacks Joel Moss Jim Shayman William Lennarz 463 Glycine Receptors Signaling Bodo Laube Joel Moss 464 Immunoglobulin (Fc) Receptors Signaling P. Mark Hogarth Joel Moss 465 Lysophospholipid Receptors 466 Signaling Cell Architecture and 26S Proteasome Structure and Function Function Gabor Tigyi Joel Moss Friedrich Forster P. Coulombe + C. Parent 467 Actin Organization 468 Actin-Related Proteins Cell Architecture and Function Cell Architecture and Function Tatyana Svitkina P. Coulombe + C. Parent Dyche Mullins P. Coulombe + C. Parent Bax and Bcl2 Cell Death Enhancers and 469 Inhibitors Cell Architecture and Function David Vaux P. Coulombe + C. Parent 470 Cell Cycle: Mitotic Checkpoint 471 Centromeres Cell Architecture and Function Cell Architecture and Function Tim Yen P. Coulombe + C. Parent Beth Sullivan P. Coulombe + C. Parent 472 Desmosomes and Hemidesmosomes Cell Architecture and Function Kathleen Green P. Coulombe + C. Parent 473 Focal Adhesions 474 Meiosis Cell Architecture and Function Cell Architecture and Function Benny Geiger P. Coulombe + C. Parent Neil Hunter P. Coulombe + C. Parent 475 Metalloproteinases Matrix 476 Nuclear Compartmentalization 477 Nuclear Envelope and Lamins 478 Septins and Cytokinesis 479 Tubulin and its Isoforms 480 Unfolded Protein Responses 481 Translation Elongation in Bacteria 482 Translation Initiation in Eukaryotes: Factors and Mechanisms 483 Siglecs 484 Prostaglandins and Leukotrienes 485 Flippases UmuC D Lesion Bypass DNA 486 Polymerase V RNA Polymerase II and Basal 487 Transcription Factors in Eukaryotes Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Cell Architecture and Function Molecular Biology Molecular Biology Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Molecular Biology Molecular Biology Gillian Murphy P. Coulombe + C. Parent P. Coulombe + C. Parent Jeanne Lawrence Bryce M. Paschal David Ron Scott C. Blanchard Christopher Hellen Ajit Varki Charles Waechter P. Coulombe + C. Parent Christine Field P. Coulombe + C. Parent Eva Nogales P. Coulombe + C. Parent P. Coulombe + C. Parent Nancy L. Craig Nancy L. Craig William Lennarz William Smith William Lennarz William Lennarz Penny Beuning Nancy L. Craig Jeff Corden Nancy L. Craig 488 Melanocortin System Signaling Roger D. Cone Joel Moss 489 Angiotensin Receptors Signaling NO REVISION Joel Moss 490 Bradykinin Receptors Signaling Ronald Burch Joel Moss Nucleotide Excision Repair in 491 Eukaryotes Molecular Biology Phosphoinositide 4- and 5-Kinases and 492 Phosphatases Signaling Dr. Goosen Nancy L. Craig Shawn F. Bairstow Joel Moss 493 Calcium/Calmodulin-Dependent Protein Kinases Signaling Alfred Robison Joel Moss Platelet-Derived Growth Factor Receptor 494 Family Signaling No Revision Joel Moss 495 3D Migration Cell Architecture and Function Patricia Keely P. Coulombe + C. Parent 496 Actin Assembly/Disassembly Cell Architecture and Function Henry N. Higgs P. Coulombe + C. Parent 497 Cadherin-Mediated Cell-Cell Adhesion Cell Architecture and Function W. James Nelson P. Coulombe + C. Parent 498 Caspases and Cell Death Cell Architecture and Function Gerry Melino P. Coulombe + C. Parent 499 Cytokinesis Cell Architecture and Function Douglas Robinson P. Coulombe + C. Parent 500 Cytoskeletal motors: general principles Cell Architecture and Function Ronald S. Rock, Jr. P. Coulombe + C. Parent 501 GAP Junctions Cell Architecture and Function Bruce J Nicholson P. Coulombe + C. Parent 502 Mitosis Cell Architecture and Function Pat Wadsworth P. Coulombe + C. Parent 503 Peroxisomes Cell Architecture and Function Suresh Subramani P. Coulombe + C. Parent 504 Toll-Like Receptors Signaling Himanshu Kumar Joel Moss Kinesins as Microtubule Disassembly 505 Enzymes Cell Architecture and Function Ryoma Ohi P. Coulombe + C. Parent 506 Phosphoinositide-Dependent Protein Kinases 507 Secretory Pathway Signaling Lipids Carbohydrates Membranes and Membrane Proteins No Revision Joel Moss Karen Colley William Lennarz Nonhomologous Recombination: 508 Retrotransposons Molecular Biology Suzanne Sandmeyer Nancy L. Craig 509 Recombination: Strand Transferases 510 Phospholipid Metabolism in Mammals N-Linked Glycan Processing 511 Glucosidases and Mannosidases Oligosaccharide Chains: Free N-Linked 512 O-Linked 513 Phospholipid Synthesis in Yeast 514 Protein Glycosylation Inhibitors 515 Sphingolipid Biosynthesis 517 tRNA Synthetases DNA Replication: Eukaryotic Origins and 518 the Origin Recognition Complex Molecular Biology Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Molecular Biology Molecular Biology Wolf-Dietrich Heyer Dennis R. Voelker Nancy L. Craig William Lennarz Tadashi Suzuki William Lennarz Tadashi Suzuki William Lennarz George Carman William Lennarz UNDER INVITE William Lennarz Alfred Merrill William Lennarz Rebecca Alexander Nancy L. Craig Igor Chesnokov Nancy L. Craig 519 A-Kinase Anchoring Proteins Signaling UNDER INVITE Joel Moss 520 Abscisic Acid (ABA) Signaling UNDER INVITE Joel Moss 521 Adenosine Receptors Signaling UNDER INVITE Joel Moss 522 Cytokines Epidermal Growth Factor Receptor 523 Family Signaling Signaling UNDER INVITE Joel Moss UNDER INVITE Joel Moss 524 Fatty Acid Receptors Signaling Glucagon Family of Peptides and their 525 Receptors Signaling UNDER INVITE Joel Moss UNDER INVITE Joel Moss 526 Glutamate Receptors Ionotropic Signaling UNDER INVITE Joel Moss 527 Gs Family of Heterotrimeric G Proteins Signaling Hepatocyte Growth Factor/Scatter Factor 528 Receptor Signaling UNDER INVITE Joel Moss UNDER INVITE Joel Moss 529 Histamine Receptors Signaling UNDER INVITE Joel Moss 530 Insulin Receptor Family Signaling UNDER INVITE Joel Moss 531 JAK-STAT Signaling Paradigm Signaling UNDER INVITE Joel Moss 532 Neuropeptide Y Receptors Signaling UNDER INVITE Joel Moss 533 Neurotensin Receptors Signaling UNDER INVITE Joel Moss 534 Nicotinic Acetylcholine Receptors Signaling UNDER INVITE Joel Moss 535 P2X Purinergic Receptors Signaling UNDER INVITE Joel Moss 536 Phosphoinositide 3-Kinase Signaling UNDER INVITE Joel Moss 537 Phospholipase A2 Signaling UNDER INVITE Joel Moss 538 Plant Signaling Peptides Signaling UNDER INVITE Joel Moss 539 Protein Kinase B Signaling UNDER INVITE Joel Moss 540 Syk Family of Protein Tyrosine Kinases Signaling UNDER INVITE Joel Moss 541 Tec/Btk Family Tyrosine Kinases Signaling UNDER INVITE Joel Moss 548 Biochemistry of bone formation/turnover Metabolism Vitamins and Hormones UNDER INVITE M. Daniel Lane 549 Biochemistry of development: Bone Metabolism Vitamins and Hormones UNDER INVITE M. Daniel Lane 571 Graves disease Metabolism Vitamins and Hormones UNDER INVITE M. Daniel Lane 580 Glucose/Sugar Transport in Bacteria Metabolism Vitamins and Hormones Ronald Kaback M. Daniel Lane 608 Tricarboxylic Acid Cycle Metabolism Vitamins and Hormones NO REVISION M. Daniel Lane 610 Urea Cycle Inborn Defects of Metabolism Vitamins and Hormones UNDER INVITE M. Daniel Lane Vitamin K: Blood Coagulation and Use in 612 Therapy Metabolism Vitamins and Hormones UNDER INVITE M. Daniel Lane 618 DNA Base Excision Repair Molecular Biology Bruce Demple Nancy L. Craig 621 DNA Secondary Structure Molecular Biology Albino Bacolla Nancy L. Craig 623 MicroRNA's in Eukaryotes Molecular Biology UNDER INVITE Nancy L. Craig 624 mRNA Polyadenylation in Eukaryotes Molecular Biology UNDER INVITE Nancy L. Craig 627 RNA Processing in Eukaryotes Molecular Biology Jo Ann Wise Nancy L. Craig 628 Micro RNA's Molecular Biology UNDER INVITE Nancy L. Craig 629 RNA Polymerase Reaction in Bacteria Molecular Biology UNDER INVITE Nancy L. Craig 630 RNA splicing 631 Small RNAs in Bacteria Molecular Biology Molecular Biology UNDER INVITE Nancy L. Craig Nancy L. Craig John van der Oost 632 Transcription Termination Eukaryotic Protein Biosynthesis: The 633 Elongation Cycle Genome-Wide Analysis of Gene 634 Expression 638 Lipoproteins HDL/LDL 639 Membrane Fusion 670 Mucin Family of Glycoproteins 671 Polysialic Acid 672 Detergent Properties 673 Golgi Complex Molecular Biology Molecular Biology Molecular Biology Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Lipids Carbohydrates Membranes and Membrane Proteins Cell Architecture and Function Tom Santangelo Nancy L. Craig Anton A. Komar Nancy L. Craig UNDER INVITE Nancy L. Craig UNDER INVITE William Lennarz UNDER INVITE William Lennarz Tony Hollingsworth William Lennarz UNDER INVITE William Lennarz Darrell McCaslin William Lennarz Mark Stamnes P. Coulombe + C. Parent 19-Dec-2011 Dear Dr. Butala, Re: Manuscript MMI-2011-11926 Thank you again for submitting your manuscript "Double-locking of the Escherichia coli colicin K gene promoter by two repressors prevents premature cell lysis after DNA damage" for publication in Molecular Microbiology. The reviewers appreciate the topic, and they generally feel convinced that IscR is a regulator. It is less clear to them (and to me) that IscR is solely responsible for the colK transcription delay. I am also left wondering how IscR levels are being controlled, since your model points to those levels as being the ultimate determinant of expression. Please see the comments from the reviewers and myself, which are appended below. If you can respond to all of the referees' points - by making the requested changes or by providing a compelling argument why a change cannot or should not be made - then I encourage you to submit a revised manuscript. Please note that multiple revisions are rarely permitted and acceptance of your revised manuscript is not guaranteed. In general, revised manuscripts should be returned within three months. If you anticipate that significantly more time will be needed, please let me know. To resubmit, log into Molecular Microbiology's Electronic Editorial Office, enter the Author Centre, enter Manuscripts with Decisions, click on the manuscript link, and upload the following: 1. A Supplemental File in which you have copy-pasted and responded to the editor's and referees' comments point-by-point. This file must be in Word format. 2. A single file containing the revised Text, Figure legends, and Tables. This file should not contain any Figures. This file must be in Word format. 3. Single, high-resolution files for each Figure. These files must be in TIFF or EPS format. 4. (optional) Supplementary material for online presentation in a single PDF file. Movies and other material that cannot be converted to PDF should be in separate files. Please indicate in the text where the supplementary material is cited (Fig. S1, Table S1, etc.). There are no color charges for supplementary material. 5. (optional) Authors are encouraged to submit a proposed journal front cover illustration. Submissions should be high resolution (600 dpi) image files (e.g. TIFF) and should be accompanied by a short description of up to 30 words. Authors who provide an image chosen for the cover will be eligible for free colour art work in a subsequent research paper. IT IS IMPORTANT TO SUBMIT THESE MATERIALS IN THE REQUIRED FORMATS. FAILURE TO COMPLY WILL DELAY THE PROCESSING OF YOUR MANUSCRIPT. Citation of a personal communication must be pre-authorized by the concerned party. Use or adaptation of a previously published figure must be pre-authorized by the copyright holder. If substantial linguistic changes were recommended, you may wish to consult a language service (http://www.blackwellpublishing.com/bauthor/english language.asp ). If you use a language service, please send them the final version of the manuscript and avoid making further changes thereafter. Please let me know if you have any questions or if I can assist you in any way. We look forward to hearing from you again as soon as possible. Best wishes, Jim Imlay [Editor's comments] 1. Even in the iscR mutant one sees a delay in colK expression (e.g., Fig. 2B). If lexA were the sole remaining regulator, why isn't the gene induced in the manner of sulA (Fig. 1)? 2. Is the deactivation of IscR control driven specifically by something that nalidixic acid does to IscR level or activity, or is its deactivation simply driven by a decline of cellular nutritional status or growth rate? In your experiments the induction of colK occurred roughly commensurate with entry into stationary phase. It is not clear whether this reflects a cause-effect relationship or whether the timing was adventitious. To check: Add Nal, but maintain the cells in a nutritionally rich environment by periodic subculturing (e.g., not allowing cell density to exceed 0.4 OD). Is colK induction affected? Does IscR continue to repress? This is an important point, because the overarching notion that is articulated in the Abstract is that IscR will stop repressing if the DNA damage is overwhelming. Yet the body of the paper seems to imply that IscR status reflects how well-fed the cell is, not whether DNA damage is irreparable. Conversely, one might ask whether nutritional starvation by itself depletes IscR titer enough that colK expression becomes somewhat activated even in the absence of DNA damage. Indeed, one might make that case from the no-NAL control in Fig. 1. Use of a lexA3 mutation might enable one to verify that the low-but-significant induction is not due to DNA damage. 3. I would flatly assert that the holo- and apo-IscR overproduction experiments do not demonstrate that both forms of the protein can repress transcription. When you overexpress any Fe/S protein, a substantial fraction exists in the apo-protein form, both because there is necessarily a delay between translation and Fe/S insertion, and because overproduction can overtax the Isc system. On top of that, overproduction of IscR has the additional effect of shutting down the transcription of the genes that encode the Isc assembly system--so that accumulation of apo-protein is inevitable. Therefore, while the genetic experiment does demonstrate that apo-IscR can repress the gene, it does not demonstrate that holo-IscR can do the same thing. One could approach this question by measuring binding constants in vitro, as reviewer 3 suggests. To do so one must build Fe/S clusters in IscR (which is not hard, using purified IscS--we could provide reagent enzyme if you want to attempt this). I think this uncertainty shines a light on an important point: Why was IscR chosen to control colK expression? The most obvious possibility is to link expression to Fe/S status in some way. It seems less likely that the system is built to detect a modest (3-fold) decline in IscR as a way of sensing a slow-down in protein synthesis. Do you have any thoughts about this? Comments to Author from Referees: Referee: 1 REPORT FOR TRANSMISSION TO AUTHORS In contrast to many LexA-controlled SOS genes such as sulA which is induced immediately after treatment with DNA-damaging antibiotics (eg, nalidixic acid), another LexA-controlled cka gene that encodes colicin K is delayed in induction. In this work, the authors found that an additional regulator IscR represses the cka gene, and proposes that the decrease in the amount of IscR is the reason for delayed induction. To prove repression by IscR through direct binding, they showed that mutation of putative IscR binding site caused similar effect as iscR deletion in elevating cka-lacZ expression. Through SPR analysis in vitro, the authors showed that the IscR-binding affinity decreased more than 10-fold by the binding site mutations. The IscR binding was proposed to be independent of the presence of Fe-S, on the basis of similar repression effect between the wild type and the constitutive apo-mutant. They observed decrease in the amount of IscR protein when cells entered stationary phase, and proposed that this is the mechanism behind the delayed induction of cka gene after nalidixic acid treatment. This is an interesting finding that adds a new function to IscR, which induces its target genes at later phases of growth, possibly through reduction in its amount. It is convincing that IscR functions as a repressor in controlling the cka gene. However, there are several observations that are not well explained and hence needs to be better resolved. Major points. 1. The mechanism behind delayed induction. The behavior of DiscR in derepressing cka-lacZ expression upon SOS induction (Fig. 2B) is puzzling, since it still shows some delay in induction as in the wild type. If the amount of IscR is all that matters to enhance cka gene expression when LexA is inactivated rapidly (by nalidixic acid), why is the full induction of cka has to wait until the stationary phase in the absence of IscR? It appears that there still exists another controlling factor that depends on the growth phase. The delayed induction is again observed when the cis-acting binding site mutants were examined (Fig. 3C, p-44G, p-28C mutants). This phenomenon has to be explained and investigated. It has been previously reported that the stationary phase induction of cka depends on ppGpp and IHF (Kuhar and Zfur-Bertok, 1999). What would the relationship between ppGpp and IscR regulation? What about IHF? 2. Dependence of IscR system on SOS induction. Even though IscR was fished out by using LexA-bound DNA, it seems to function independently of SOS response. What would be the induction pattern of cka in DiscR mutant in normal growth without nalidixic acid treatment? How would the expression profile look like in comparison with the SOS-induction data in Fig. 2B? 2. Effect of p-12C ("-10" promoter element mutant) in Fig. 5. First of all, it is not clear why the authors used p-12C mutant as a genetic background in all constructs examined. This needs be explained. When -10 promoter box is mutated, would the transcription initiation site be changed? What would the effect of nalidixic acid in p-12C background? Explanation for UP3 mutation is lacking. Has +1 site ever been determined for cka gene even in the wild type? If not, it is better to be determined experimentally, to verify that the promoter elements and their mutations mean as they are called. 3. Considering many factors that affect cka gene expression, the two repressor model for SOS induction appears too simple. Since IscR repression seems independent of LexA repression, incorporation of IscR in the model for cka gene regulation needs not necessarily be confined in the context of SOS response. The model pathway in Fig. 6B needs be elaborated by including other factors that affect cka gene regulation. Minor points Fig. 6. How many experiments were done to get the average numbers? Fig. 7. (B) The method for quantifying the increased amount of colicin in DiscR mutant needs be explained. Page 4, line 4, and page 16, Fig. 1; trigerring -- triggering? Referee: 2 REPORT FOR TRANSMISSION TO AUTHORS In the manuscript entitled "Double-locking of the E. coli colicin K gene promoter by two repressors prevents premature cell lysis after DNA damage" enlightens the colicin K expression control, describing its regulation by the IscR regulator. Moreover the authors also describe the presence of the IscR binding sites in the promoter region of other colicins, showing that it may be a widespread control mechanism to delay the colicin expression after SOS induction. The results described in the manuscript are interesting and enhance the knowledge about the SOS response and its relationship with other genetic networks and regulators that permit to adjust precisely the gene expression. Nevertheless I have some concerns about the results showed in this manuscript, some controls are missing and sometimes there are discordances between the results presented by the authors. So I think that all these problems must be solved. Major concerns: 1. The authors detect the proteins that are involved in colicin C regulation using the cka promoter region attached to streptavidin Dynabeads. After crude extract addition and washing, the authors compare the bands observed using beads without DNA with those containing the Pcka - LexA promorter-protein complex. Why do the authors use the Pcka associated with LexA protein? Will the same bands appear if LexA was not already associated to the promoter? May the presence of LexA interfere with the attachment of other proteins by competence? In fact, the authors added SOS induced crude extract, so RecA* was present and would activate the auto-hydrolysis of LexA, also those that were bound to the Pcka promoter. Why do they use the Pcka associated with LexA protein? On the other hand, in the text, the authors say that they ignored the proteins with less than 20% identity but also "the ones that were previously shown not to regulate pcka", but the references that support this idea are not stated either in the text or in supplementary material. In the list there are some hypothetical proteins that may be regulators and they are not studied. Why do the authors choose some and some other not? If previous works discard those proteins they must be cited. 2. In Fig. 4 it is shown that the presence of an "empty" plasmid it generates great differences with respect the same strain without the plasmid. Have the authors any explanation of this fact? On the other hand, the Fig. 4 results showed that the strain with the "empty" plasmid has not only a decrease in its expression level but also a delay on it. So, is it really comparable the expression of the sulA fusion and the cka fusion in Fig 1? Actually, the sulA::lacZ fusion is not in a plasmid as cka, but in the chromosome of the E. coli strain. I'm not questioning the delay of the colicin induction (that is fully described), but perhaps the experiment performed here is not the more appropriated to show the delay since the strains used are not isogenic and do not contain the same copies of the lacZ fusion. For instance, quantitative RT-PCR experiments measuring sulA and cka mRNA levels may be suitable in this case to determine the induction moment of each promoter after inducer addition. 3. A major concern is the discordance between results showed in the manuscript. Apparently there are some lacZ fusions that are used in different experiments. For instance, the wtpRW50cka is used in the experiments that are shown in Fig 1, 2 and 3. The beta-galactosidase assays are performed in these three experiments following the same strategy: the SOS inducer was added when the cultures grew up to OD 0.2-0.3, and the betagalactosidase activity was measured several times after the induction. In all case the same amount of inducer was used (37uM NAL). And also in all cases the results are shown with ±SEM. But when one looks carefully to each Figure realizes there are great differences between the results obtained in each experiment. See below: Betagalactosidase Enzimatic units for wt pRW50cka. Fig1: 2h post-induction : 200 U 3h PI: about 1400U 4h PI: about 1800U Fig 2: 2h post-induction : less than 100 U 3h PI: about 750U 4h PI: about 750U Fig 3B: 2h post-induction : less than 100 U 3h PI: about 400U 4h PI: about 400U Fig 3C: 2h post-induction : about 200 U 3h PI: about 1500U 4h PI: about 1700U The less betagalactosidase activity registered in Fig. 3B may be caused by the addition of arabinose. But apparently Fig 1, 2 and 3C are exactly the same experiment using different mutants. Differences between Fig 1 and Fig2 wt pRW50cka results could be attributed to the different strains used (DCB387 pRW50cka and BW25113 pRW50cka, respectively), but in Fig 3C results are similar to Fig 1 and the strain used in this case was BW25113 the same that is used in Fig 2 so the problem must not be the strain. How the authors can explain that? Why this difference is not seen in the SEM that represents 3 different experiments? Why the authors change the strain between the experiments? Are the other fusion results also so variable? The differences are not negligible since in most cases they would reduce the differences observed in the analyzed mutants. 4. It would be interesting the relationship between IscR and LexA protein. Are both proteins bind together to the Pcka? Is there a competence for the Pcka Promoter region? Could an excess of IscR avoid the LexA binding? 5. The authors describe that either apo-IscR or holo-IscR are able to block the Pcka since no induction of Pcka expression is observed when iscR or iscR-CTM complement the DiscR mutation. Nevertheless the iscR expression levels in the complemented strain have to be high, since they are controlled by PBAD promoter, so great amount of each protein are present, more than in a wild type strain producing IscR. Do the apo-IscR and holo-IscR proteins present the same affinity for the promoter region of cka? EMSAs or SPR analysis will be suitable to determine this. 6. Finally, the authors describe a model for the delayed expression of Pcka: Basically, when SOS system is induced, the IscR retains the cka expression. If the DNA damage is released, then LexA blocks again the cka expression even when the cell is on stationary phase. If the DNA damage persists, when the nutrients decrease, the levels of IscR will go down and so, the cka expression will be no longer blocked and the cell will die. What has it happen if a sulA strain was used in these experiments? It is described that the OD increase in a cell with activated SOS response is due to filamentation that is responsible of sulA gene, which product interacts with FtsZ protein avoiding the cell division. Are cells with an activated SOS response in stationary metabolic state? Minor concerns: 1. At the end of the results, there is an incomplete sentence: " In contrast, only a small difference in colicinA production was detected, which could be due to additional posttranscriptional", the reviewer assumes that the authors do mean, posttranscriptional control. 2. I think it will be easier for the reader that the graphics where performed using post induction time. 3. In M&M, the secondary antibody of the western blot, once anti-RecA is added as primary antibody is missing. 4. Legend of Fig.6. It is not MG1655 the strain that is used in this experiment, it is PK10016, isn't it? 5. Table S1 must be cited just after "„.delay in induction of the cka gene promoter (pcka)" not at the end of the sentence since Table S1 has not expression results, only contains the description of the promoters. 6. MG1655 is not cited in the Table S1. Referee: 3 REPORT FOR TRANSMISSION TO AUTHORS The current dogma surrounding the release of colicins involves induction of the SOS response in response to DNA damage that causes the RecA-mediated cleavage of LexA that de-represses colicin transcription leading to the synthesis of colicin. Colicins are released into the environment through induction of a lysis gene leading to the production of a phospholipase that permeabilizes the outer membrane, culminating in cell death. Induction of the lysis gene is often coupled to that of the colicin gene and synthesis of both occurs concurrently but previous studies have shown that this induction may be delayed following SOS induction. The work of Butala et al in this paper has reinvestigated the SOS induction of colicin K, a pore forming colicin, and reported a second repressor called IscR binding to a region upstream of the SOS promoter that is involved in delayed expression of Colicin K following DNA damage. They highlight a region of the promoter that has palindromic symmetry that is involved in binding of IscR leading to a 'double locking' of the colK promoter that is responsible for the delayed expression of the colicin following DNA damage. The experimental approach is logical and largely convincing. The inferences are novel and despite some sloppy spelling mistakes the paper is well written, and should be considered for publication in Molecular Microbiology. However, I have some issues that the authors should consider for revision: 1) Colicin release in response to DNA damage is dependent on the lysis gene. The authors recognize the role of cell lysis in colicin release but do not associate this with the induction of the lysis gene. There is a range of data published in the 80's that report on the organization of the colicin operons, and provide evidence on the role of the lysis gene in colicin release in different systems. Depending on the organization of the ColK operon (ie. relationships between cka and ckl) and despite the data in Fig. 1, would the authors consider that repression of cell lysis by IscR might be repression of ckl, and that the newly identified binding region be a promoter for induction of ckl? There is evidence that cells expressing colicin Ia produce large amounts of colicin that is only released on cell lysis. 2) Both holo and apo IscR appear to regulate cka. This is somewhat surprising as it might seem that loss of the Fe-S cluster might affect folding of IscR and be important for binding the DNA. The authors should repeat their SPR experiments using apo-enzyme to show that loss of the Fe-S cluster does not affect binding to the ligand, or check the relative protein structures using CD spectroscopy. 3) The SPR experiments appear convincing but the response units are arbitrary and do not provide any indication as to the strength of the interaction. I would have liked to have seen an affinity binding constant (Kd) to allow a proper comparison of the binding of wt fragment with mutated DNA fragments. Also the data for wt DNA in Fig. 4C is identical to the 1 mM IscR sample in Fig. 4B. Was the data in Figs. 4B and C obtained from the same experiment? 4) The predicted target for IscR binding has homology to the consensus sequences and the authors showed the importance of two residues within this region by b-gal assays and SPR. I was surprised that mutating just a single residue had such a dramatic effect on IscR binding, but they obtained similar results for both p-44G and p-28C. I would have mutated one or two more residues over the remainder of the consensus, and indeed one outside to confirm the effect. Alternatively they could consider adding the IscR repressor binding site to the promoter of the sulA-lacZ fusion reporter in ENZ1257 to confirm that there is sufficient delay of expression of b-galactosidase by this construct. 5) Colicin is expressed spontaneously in a small percentage <5% of naturally occurring colicin producing cells. Does IscR have any role in the production of colicin by these colicinogenic cells when grown in the absence of an SOS inducing agent? 6) I would be a little reluctant to state similar inferences between ColN and colicins K and E1 when discussing the induction of these colicins by NA. Colicin production in wt and deletion iscR in response to NA is not that dissimilar in ColN unlike the same data with ColK and E1 induction! 7) There is no effect of IscR on ColA induction despite there being a strong candidate IscR binding site with palindromic symmetry similar to ColK. Is there any difference in the ColA operon that differs from ColK and allows any speculation on these differences? Minor: Pg 3 Remove 'Recall that^' Numerous spelling mistakes: Pg 5 upstream Pg 6 line 7, fragment; line 13 repression; line 22 below Pg7 line 10 sentence not completed, 'additional posttranscriptional^.'P Factors? Pg 8 line21, maintain Pg11, diluted Pg11, line21, Is 'injected' the correct word here, would aliquoted or added be more suitable? Pg13 line 1 throughout; line2, harvested not harvested Pg 16 fig 1 legend 'triggering' Supplementary information Fig. S2., dilution Pg 10 line 5. Built by models..? Sentence in complete? Pg10 line 17 collection, line24, sub-inhibitory molecular microbiology Double-locking of the Escherichia coli colicin K gene promoter by two repressors prevents premature cell lysis after DNA damage Journal: Molecular Microbiology Manuscript ID: MMI-2011-11926 Manuscript Type: Research Article Date Submitted by the Author: lO-Nov-2011 Complete List of Authors: Butala, Matej; University of Ljubljana, Department of Biology Browning, Douglas; UB, Biosciences Sonjak, Silva; University of Ljubljana, Department of Biology Hodošček, Milan; National Institute ofChemistry, Laboratoryfor Molecular Modeling Žgur-Bertok, Darja; University of Ljubljana, Department of Biology Busby, Steve; University Of Birmingham, School Of Biosciences Key Words: Colicins, DNA damage. Induction of gene expression, LexA regulon. Transcription factor IscR SCHOLARONE" Manuscripts for Molecular Microbiology 14th November, 2011 Double-locking of the Escherichia coli colicin K gene promoter by two repressors prevents premature cell lysis after DNA damage Matej Butala^*, Douglas F. Browning^, Silva Sonjak^, Milan Hodošček^, Darja Žgur-Bertok^, Stephen J. W. Busby^ ^Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia, ^School of Biosciences, University of Birmingham, Birmingham B15 2TT, U.K., ^National Institute of Chemistry, 1000 Ljubljana, Slovenia *Corresponding author: Matej Butala: Phone: +386 1 320 3397; Fax: +386 1 257 33 90; e-mail: matej .butala@bf.uni-lj .si RUNNING TITLE IscR mediates delayed induction of colicin genes SUMMARY The synthesis of Eschericha coli colicins is lethal to the producing cell and is repressed during normal growth by the LexA transcription factor, which is the master repressor of the SOS system for repair of DNA damage. Following DNA damage, LexA is inactivated and SOS repair genes are induced immediately, but colicin production is delayed and induced only in terminally damaged cells. The cause of this delay is unknown. Here we identify the global transcription repressor, IscR, as being directly responsible for the delay in colicin K expression during the SOS response and identify the DNA target for IscR at the colicin K operon promoter. Hence, this promoter is 'double locked' to ensure that suicidal colicin K production is switched on only as a last resort. KEYWORDS Colicins; DNA damage / Induction of gene expression / LexA regulon / Transcription factor IscR INTRODUCTION The bacterial SOS response enables cells to deal with DNA damage and associated stresses. The response is controlled by the LexA global transcription factor that represses transcription of dozens of SOS genes that are involved in coping with and repairing DNA damage (Courcelle et al., 2001, Wade et al., 2005). In response to DNA damage, RecA polymerizes onto exposed single-stranded DNA, creating the active helical nucleoprotein filament (RecA*), which mediates cleavage of LexA (Little, 1991), and instigates repressor dissociation from its DNA targets and induction of the LexA regulon (Butala et al., 2011). In Escherichia coli and related bacteria, where the SOS response has been most studied, it has been found that the LexA regulon includes many genes encoding colicins (Ebina et al., 1982, Lloubes et al., 1986). Recall that colicins are toxic suicide proteins that kill other bacteria by a single-hit mode of action, targeting either cell walls, DNA or RNA (Kleanthous, 2010, Cascales et al, 2007). In Escherichia coli, most colicins are encoded by plasmids and transcribed from strong promoters whose activity is firmly repressed by LexA, and hence colicin expression is triggered by agents that induce the SOS response (Cascales et al., 2007, Ebina et al., 1982). Most LexA-repressed promoters are induced immediately upon DNA damage (Courcelle et al., 2001) but induction of the majority of colicin genes is delayed and triggered only upon severe and persistent DNA damage (Salles et al, 1987, Herschman and Helinski, 1967). This makes sense as colicins play no role in DNA repair, but rather, the producer cell dies as they are released, and their role appears to be to assist surviving cells by killing potential competitors (Majeed eř a/., 2011). It has been postulated that the lag period in colicin production after SOS induction provides cells with time for damage repair before induction of the lethal colicin (Salles et al., 1987), but the cause of the delay is unknown. In previous work, we established how LexA represses the promoter of the E. coli cka gene that encodes colicin K, a pore-forming toxin that kills susceptible cells by collapsing the membrane potential (Jerman et al., 2005, Kuhar and Zgur-Bertok, 1999, Mulec et al., 2003). Here, we have studied the timing of cka transcription after SOS induction and we report that the IscR global transcription repressor is directly responsible for delaying cka expression. We show that the cka promoter is 'double locked' to ensure tight and timed regulation of colicin K expression and that induction is triggered by the decrease in IscR levels that occurs as cell growth slows. RESULTS Delayed induction of the colicin K gene during the SOS response When growing E. coli cells are treated with DNA damaging agents, initially, LexA regulon genes are induced that relieve DNA damage, arrest cell-division and enhance adaptation through mutagenesis (Courcelle et al., 2001). Consistent with several published studies of colicin induction (Salles et al., 1987, Herschman and Helinski, 1967), after trigerring the SOS response with nalidixic acid, we observed a pronounced delay in induction of the the cka gene promoter {çcka), compared with expression of the sulA LexA-regulon gene (Fig. 1, Table SI). We previously showed that LexA represses ^cka by binding to tandem DNA sites for LexA located downstream from the -10 promoter element (Mrak et al., 2007). Results in Fig. SI show that LexA can both block RNA polymerase binding at ^cka and displace pre-bound polymerase, but this cannot explain the observed kinetics of çcka induction. Thus, we searched for another regulator by using affinity chromatography methods, using a DNA fragment containing ^cka in complex with LexA as bait and cleared SOS-induced cell extracts (see Experimental Procedures). After elution of bound proteins and analysis by SDS-polyacrylamide gel electophoresis (Fig. 2A) and mass spectroscopy, we identified the nucleoid associated factor H-NS and the transcription regulators NsrR, Lrp, GlcC, UlaR, DeoR, IscR, and LexA as factors that had associated with the bait (Table S2). Since LexA was expected, and H-NS, NsrR, UlaR and Lrp were previously shown not to be involved in the cka regulation (Kuhar and Zgur-Bertok, 1999; unpublished observations), we focussed on GlcC, DeoR and IscR and assayed cka promoter activity following SOS induction from a ^cka-lacZ fusion in the corresponding deletion mutant strains from the Keio collection (Baba et al., 2006). The results show little effects of the glcC and deoR deletions, but disruption of iscR resulted in induced çcka activity immediately after addition of sub-inhibitory concentration of nalidixic acid (Fig. 2B), indicating that IscR represses expression from ^cka. IscR regulates cka expression The IscR (iron-sulfur cluster regulator) protein, was originally identified as a transcription repressor that regulates genes involved in the formation and the repair of iron-sulfur clusters in proteins (Schwartz et al., 2001). It has homologues in eukaryotes which sustain fundamental life processes (Lill and Muhlenhoff, 2005), IscR exists in two forms, holo IscR that contains an Fe-S cluster, and apo IscR, which is formed upon destruction of the Fe-S cluster, for example, in response to oxidative stress. It is now known that certain targets require holo IscR for repression, whilst the majority of targets are repressed by both forms (Nesbiteřa/.,2009). To determine directly whether IscR can bind to the cka regulatory region (Fig. 3A) and restore repression of ^cka in the MscR strain, we complemented the latter strain with a plasmid encoding an arabinose-inducible IscR or an IscR mutant locked in the apo- form due to alanine substitutions of the cysteine Fe-S cluster ligands (IscR-CTM) (Wu and Outten, 2009). With the highest concentration of L-arabinose that had a minimal effect on cell growth, both wild-type IscR and IscR-CTM complemented the iscR deletion and strongly repressed ^cka in spite of DNA damage (Fig. 3B). Thus we conclude that both apo- and holo-IscR can repress çcka, and inspection of the base sequence identified a perfect palindrome, overlapping the -35 promoter element (Fig. 3A), that corresponds well to the established consensus sequence (Nesbit et al., 2009). To dissect the nucleotides required for the IscR-dependent repression, we modified the two most critical nucleotides in the predicted site (Fig. 3A): the base at position 44 upsteram of the çcka transcript start (p-44C to G) and the symmetric modification at position 28 (p-28G to C). Results illustrated in Fig. 3C show that the 5 mutations have similar effects on the expression of çcka as the iscR deletion, strongly suggesting that the palindrome is the target for IscR binding. Next we purified IscR protein and performed surface plasmon resonance (SPR) analysis directly to measure IscR binding at çcka using the DNA fragments illustrated in Fig. 4A. Our results show that IscR interacts with the chip-immobilized DNA fragment in a concentration dependent manner (Fig. 4B). Association of IscR with the DNA fragment harbouring mutation p-44G was decreased by ~10-fold in comparison to the wild-type cka fregment, and the affinity of IscR for the DNA fragment harbouring both the p-44G and the p-28C mutations was neglible (Fig. 4C). To measure the effects of oxidative stress on IscR-dependent repression of çcka in vivo, we used a cka promoter variant with mutated LexA operators (pRW50UP3) unable to bind LexA specifically (Mrak et al., 2007) and the p-12C substitution in the promoter -10 element (Fig. 3A). Results illustrated in Fig. 5 show that IscR represses çcka and that this represion is unaffected by oxidative stress from hydrogen peroxide. Since our data indicate that ^cka is repressed by both holo- and apo-IscR, we considered that relief of IscR-dependent repression could be due to changes in IscR levels. Thus, we used western blotting to determine intracellular concentrations of IscR during normal growth or during the SOS response in E. coli MG1655 strain expressing the FLAG-tagged IscR from the native iscR promoter. A 3-fold decrease of the IscR level was observed when cells entered into the late exponential phase and early stationary phase after early exponential growth (Fig. 6A). This suggest that cka transcription in SOS induced cells is induced when concentrations oflscR fall bellow a threshold level (Fig. 6B). IscR controls the expression of different colicins To investigate the effects of IscR on the expression of other colicins, we introduced the AiscR allele into strains that produce the pore forming colicins K, El, A and N. Following SOS induction of the colicinogenic cultures, cell growth and colicin production was compared in the starting strains and the AiscR mutants. We observed that IscR confers viability to the most of the tested strains (Fig. 7A). Crude cell extracts were prepared from cultures before and after SOS induction and colicin levels were compared by bioassays (Fig. 7B) or by SDS-PAGE (Fig. 7C). The results show that nalidixic acid induces an immediate increase in colicin K, El and N levels in the AiscR strains in comparison to the delayed colicin production in the wild-type strains. In contrast, only a small difference in colicin A production was detected, which coud be due to additional posttranscriptional (Yang et al., 2010). Colicin promoter regions were sequenced and alignment of these sequences (Fig. 7D) revealed SOS boxes and IscR binding sites present in the same organisation and location. DISCUSSION Many E. coli strains carry plasmids which encode colicins that are expressed in response to extreme stress conditions (Cascales et al., 2007). Colicin production by a bacterial cell is suicidal and it is thought that this is an example of bacterial altruism (Majeed et al., 2011). Thus, in response to extreme stress, a small proportion of the population of a strain sacrifice themselves and produce colicin toxins that kill susceptible competitor strains. Clearly then, colicin synthesis needs to be tightly regulated and it is well known that transcription of most E. coli colicins is repressed by the LexA global repressor that coordinates the SOS response to DNA damage. This is understandable since colicins have evolved as a last resort emergency response, but this creates the problem of how to uncouple the induction of colicin expression from temporal induction of the SOS response to deal with repairable DNA damage. Our work 7 with çcka shows that the solution to this is a second repressor, IscR, that binds to a target that overlaps the -35 element. Hence ^cka is double locked. Interestingly, such double locking of promoters is rare in E. coH and appears to be reserved for gene products whose ectopic expression would be harmful, the best characterised examples being the silencing of certain plasmid-encoded genes (Bingle and Thomas, 2001). Previous studies identified IscR as a regulator of the expression of gene products involved in the synthesis or repair of Fe-S proteins (Tokumoto and Takahashi, 2001, Schwartz et al., 2001). IscR exists in two states apo-IscR and holo-IscR which contains an Fe-S cluster (Schwartz et al., 2001). For some targets, the ability of IscR to repress is dependent on the Fe-S cluster. This is the case for the iscR promoter itself and hence IscR levels vary greatly depending on the oxidation status of the cell (Nesbit et al., 2009). For most targets, both apo-and holo-IscR bind and repress transcription, and regulation appears to be due to changes in the cellular concentration of IscR. Our data suggest that this is the case for IscR binding at the cka promoter. It was previously shown that cka and colcin El gene are induced due to lack of nutrients and not by an inducer released from the surrounding cells (Eraso et al., 1996, Kuhar and Zgur-Bertok, 1999). Thus, IscR levels remain high until nutrients become depleted upon entry into stationary phase, and hence, in metabolically active cells in the absence of DNA damage, colicin K synthesis is carefully locked by the IscR and LexA. However, following a prolonged SOS response, when nutrients are depleted and metabolism slows, colicin synthesis is turned on and defective cells are eradicated. This may be in order to donate nutrients to related neighbors or to maintenan a low mutation rate in a microbial community. To conclude, here we have shown that IscR has a role in programmed bacterial cell death, which is part of the developmental process in a number of bacterial species (Lewis, 2000). Our data show that IscR affects the expression of many colicin opérons by carefully orchestrating colicin gene induction folloing the SOS response. EXPERIMENTAL PROCEDURES The following materials and methods are described in the Supplementary Experimental Procedures: plasmids and promoter constructs, computer modeling, p-galactosidase assay and electromobility shift assays. Proteins E. coH RNA polymerase holoenzyme containing was purchased from Epicentre Technologies (Madison). The LexA protein was overexpressed and purified as described (Butala et al., 2011). The MHl strain and the pQ-ORF2-95 plasmid to overexpress the IscR protein were donated by Yonesaki T. The IscR protein was expressed as described (Otsuka et al., 2010) and isolated to >95% purity by the Ni-NTA affinity cromatography and stored at -20°C in 20 mM Tris-HCl (pH 8.0), 0.1 mM NaCl, 0.5 mM EDTA, 40% glycerol, 0.2% Triton-X. Concentrations of the LexA and IscR repressor were determined using NanoDroplOOO (Thermo SCIENTIFIC) and the extinction coefficients of 6990 M"^ cm"^ and of9970 M"^ cm"^ at 280 nm, respectively. DNA affinity purification E. coli JCB387 harboring the pRW50c^a plasmid (0.5 1) were induced with 8.5 ^g/ml nalidixic acid when the ODeoo reached 0.5, and after 45 min, cells were harvested and cell extracts prepared as described (Butala et al., 2009). Biotinylated -180 bp cka promoter fragments were generated by PCR using primers Pull F, Pull R and pRW50c^a as a template and purified by GeneJET PCR purification kit (Fermentas), was attached to 2.5 mg of M-280 9 streptavidin Dynabeads (Invitrogen) according to the manufacter's instructions. In binding buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA) 60 ^g LexA repressor was bound to 50 ^g of DNA immobilized to the magnetic beads and excess LexA was washed off in wash buffer (20 mM Hepes-Na (pH 7.4), 100 mM NaCl, 0.1% (v/v) Tween 20). Binding reactions were performed in binding buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA) containing: beads either with or without the immobilized cka promoter-LexA complex. Dynabeads were collected with a magnet and washed four times with wash buffer. Proteins were eluted from the DNA with buffer containing 800 mM NaCl, and concentrated by TCA precipitation. Proteins were resolved on a 12% SDS-PAGE gel (Invitrogen), 1 mm gel slices were excised and analysed by the Birmingham Functional Genomics and Proteomics Unit (http://www.genomics.bham.ac.uk/) using a Thermo-Finnigan LTQ Orbitrap mass spectrometer. Three protein bands specific for the cka promoter-LexA complex were recovered from the high stringency 0.8 M NaCl eluate. These bands, that corresponded to molecular weights of approximately 15 kDa, 19 kDa and 35 kDa (Fig. 2A), were recovered and analysed. We ignored candidate proteins with less than 20% identity and selected those that exhibited DNA binding properties but ignored the ones that were previously shown not to regulate çcka (Table S2). Surface plasmon resonance assays SPR measurements were performed on a Biacore X (GE Healthcare) at 25°C. The streptavidin (SA) sensor chip (GE Healthcare) was equilibrated with buffer containing SPR l buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.005% surfactant P20 (GE Healthcare). Approximately 100 response units (RU) of 3'-biotinylated SI primer was immobilized on the flow cells of the SA chip. To prepare double stranded DNA with the predicted IscR operator or its two mutant derivates, complementary primers IscR F and IscR R or IscRm F and IscRm R or IscR2m_F and IscR2m_R (Table SI) in 20 mM Tris-HCl (pH 7.5), 0.1 mM NaCl were mixed in 1:1.5 (mol:mol) ratio, respectively. Primers were annealed in temperature gradient from 94°C to room temperature (-1.5 h) in PCR machine (Eppendorf). So prepared 31-bp duplex DNA with a 15 nucleotide overhang complementary to the streptavidin chip-immobilized SI primer was passed for 2 min at 2 ^1/min across the flow cell 1 to immobilize -90 RU of either IscR operator DNA fragment or its derivates. The interaction between the IscR repressor and the chip-immobilized DNAs was studied by injecting solutions of the desired concentration of the IscR in 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 4 mM MgClz, 0.5 mM EDTA, 5 mM DTT, 0.005% surfactant P20 at 100 ^1/min for 1 min. Dissociation was followed for 2 min. The DNA-sensor chip surface was regenerated by injecting buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl and the hibridised DNA fragments separated by 50 mM NaOH. SPR experiments were performed at the Infrastructural Centre for Surface Plasmon Resonance, University of Ljubljana. Colicin production assays Colicin synthesis was monitored in the wild-type or the AiscR strain harboring naturally occuring colicinogenic plasmids by a colicin production assay as described previously (Jerman et al., 2005). Cultures of colicin-producing strains were grown in LB broth supplemented with ampicillin (100 ^g/ml) with aeration at 37°C. Samples were collectedjust before nalidixic acid treatment at an ODeoo of 0.3 and 1, 2 and 3 hours after induction. Cells were dilluted in LB broth to the ODeoo of 0.3 to make a milliliter of the sample and crude colicin extracts were prepared by sonication (Sonics VCX750) at 40% power for 30 second on ice. Subsequently, 100 of the crude extracts were injected into wells in an LBTc plate overlayed with 4 ml of soft agar with 40 of the indicator strain DH5a harbouring pBR322 (laboratory stock). For an estimation of colicin production ratio among the strains, a tenfold dilution series of crude colicin extracts were prepared and 5 samples were spotted on the LBAp plate overlayed with the indicator strain. To determine the the ratio of colicin production in wt or AiscR strain, the sizes of the colicin produced lysis zones were compared and dillution of the cell lysates were taken into account. The remaining crude colicin K extracts were TCA precipitated and protein bands resolved on the 12% SDS-PAGE gel (Invitrogen) and visualized as described above. Experiments were performed in duplicate. Colicin promoter regions were sequenced with primers used previously (Kamensek et al., 2010). Western blot analysis The PK10016 strain (iscR-¥LAG) harbouring the pRW50c^a was grown in LB broth supplemented with Tc (12.5 ^g/ml) with aeration at 37°C. Samples were collected at an ODgoo of 0.3 and after 0.5, 1.0, 1.5, 2.5 and 4.0 hours of growth in normal or SOS induced conditions. DNA damage was elicted with 8.5 ^g/ml nalidixic acid at an ODeoo of 0.3, where relevant. Samples were equilibrated to an ODeoo of 0.6 to detect protein levels in equal number of cells during bacterial growth. Cell pellets were resuspended in 10 NuPAGE LDS sample buffer, 10 of DTT and 20 of dHjO and heated (95°C, 5min) before loading equal ammount of the samples on a 12% SDS-PAGE gel (Invitrogen). For blotting, proteins were transferred to polyvinylidene difluoride membranes (Millipore), blocked in 4% bovine serum albumin at room temperature. Primarly the proteins were stained with monoclonal mouse anti-flag M2 antibody (Sigma-Aldrich) and secondary antibodies conjugated by horseradish peroxidase. The same membrane was re-stained by primary anti-RecA antibody (Anti-RAD51 polyclonal antibody, Thermo Scientific). Antibodies were used at a concentration of 0.5 ^g/ml. Bands were stained using 4-chloro-l-naphtol/H202. The resolved bands were quantified using a G:Box (Syngene). 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(2010) Posttranscriptional repression of the eel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli. Nucleic Acids Res 38: 3936-3951. FIGURE LEGENDS Fig. 1. Delayed induction of the cka gene promoter after trigerring the SOS response. Measured P-galactosidase activities (full lines) of JCB387 carrying pRW50c^a, with a cka-lacZ transcriptional fusion, and of strain ENZ1257 harboring a sulA-lacZ fusion, as indicated. Each value represents the mean ± SEM of at least three independent measurements, the arrow indicates the time of addition of nalidixic acid (NAL) where relevant and the dashed lines represent optical density measured at 600 nm. Fig. 2. LexA and IscR regulate induction of the cka gene promoter. (A) Coomassie stained protein profile of flow through (FT), protein standards (M), denatured beads and LexA (lane 1) and eluates from the control (lane 2) or çcka affinity chromatography (lane 3). Proteins in three gel slices (denoted by boxes) were trypsin digested and analyzed by mass spectrometry. Proteins in the bands marked A, B and C were identified as DeoR, GlcC, UlaR; Lrp and IscR; H-NS and NsrR, respectively. (B) Expression of the cka-lacZ fusion either in wild type BW25113 (wt), or in the NdeoR, ^glcC or ^iscR mutants. Each value represents the mean ± SEM of at least three independent measurements, the arrow indicates the time of addition of nalidixic acid (NAL) and the dashed lines represent optical density measured at 600 nm. Fig. 3. Role of IscR in regulating cka expression. (A) Regulatory elements of the cka promoter region. The boxes indicate the predicted palindromic target for IscR binding which corresponds to the consensus (Nesbit et al., 2009). The promoter -10 and -35 elements are in bold type, and the SOS box targets for LexA, the Shine Dalgamo sequence (S.D.) and the translation start site {cka) are also indicated. Positions of the site-directed mutations described in the paper are indicated above the sequence. (B) Expression of the cka promoter in strain BW25113 (wt) or the MscR mutant derivative complemented with holo-IscR (pz5c^) or apo-IscR (pz5c^-CTM). Empty parent vector pFW02 was used as a control plasmid. L-arabinose was added at the time of inoculation and the arrow indicates the time of addition of nalidixic acid (NAL). For panels B and C each value is the average ± SEM of at least triplicate experiments and the optical density measured at 600 nm is shown as dashed lines. (C) Measured P-galactosidase activities in BW25113 (wt) or the MscR mutant carrying either the pc^a, ^cka p-44G or ^cka p-28C fragments subcloned into pRW50. The arrow indicates the time of addition of NAL as indicated. Fig. 4. IscR interacts with the cka regulatory region. (A) Biotinylated DNA fragments used in the SPR analysis. The DNA linker by which fragments were attached to the chip surface is indicated in blue font, the palindromic sequence in red and the point mutations in green. (B) SPR sensorgrams of interactions of IscR (62 nM to 1 ^M) with chip-immobilized wt DNA fragment. (C) Sensorgram of 1 ^M IscR interacting with either wt DNA fragments or derivatives with mutations in the predicted IscR site. Fig. 5. Regulation of IscR activity. Measured P-galactosidase activities in BW25113 (wt) or ^iscR cells carrying pRW50c^a with the p-12C mutation in çcka, with or without the UP3 substitutions that stop LexA binding (see Fig. 3A). The arrow indicates the time of addition of 0.2 mM H2O2, where relevant. Each value is the average ± SEM of at least triplicate experiments and the optical density measured at 600 nm is shown as dashed lines. Fig.6. IscR levels decrease as ceil growth slows. (A) Western blot analysis of the growth phase-dependent variation in the levels of RecA and FLAG-tagged IscR at various growth phases in MG1655. Samples were taken at time intervals before or after induction of the SOS reponse with nalidixic acid (NAL) at ODeoo 0.3 (Oh), as indicated, or during normal growth. Cells entered the stationary phase of growth after 2.5 h. Purified RecA(His)6 (0.18 ^g) loaded in the last lane was used as a control. Quantitation of proteins is presented below the gels as the ratio (%) of the protein density value of the initial sample (Oh) relative to the density value obtained from the samples harvested throught the bacterial growth, shown with the standard deviation. (B) Model for the delayed expression of çcka. During normal growth, LexA and IscR bind and repress transcription from ^cka. Upon DNA damage, e.g. caused by antibiotics, SOS DNA repair commences due to the decrease in intracellular LexA concentrations, but IscR levels are not affected, ^cka becomes de-repressed after long-lasting DNA damage due to decreased IscR levels as cell growth ceases. Fig. 7. IscR protein manages temporal induction of different colicins. (A) Growth curve of BW25113 (wt) or MscR cells harboring naturally occurring plasmids encoding pore forming colicins either K (pColK), El (pColEl), A (pColA) or N (pColN). The arrow indicates the time of addition of nalidixic acid (NAL), each value is the average ± SEM of duplicate experiments. (B) Assays of colicin production in cells harboring colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points from the time of addition of nalidixic acid (0 h) and cell extracts were placed into wells in an LBTc plate overlaid with soft agar harboring an indicator strain. Numbers below the lysis zones indicate the fold increase of colicin production in the ^iscR strain compared to the wild type strain at the same time point, as determined from the dilution of crude colicin extracts (Fig. S2). Experiments were performed in duplicate. (C) SDS-PAGE analysis of total cell extracts of BW25113 or ^iscR cells carrying pColK. The arrow indicates the position of colicin K as determined in comparison to the size of the purified (His)6-tagged colicin K. (D) The sequence alignments highlight regulatory elements in colicin gene promoter regions, annotated as in Fig. 3A, and the predicted IscR sites are marked with dashed boxes. + 37uM NAL JCB387 2000^AENZ1257 pRW50c/ca 3 4 5 Time (h) 0,01 79x59mm (300 x 300 DPI) B + 37 [JM NAL 4500 -r B wt pRW50c/(S -4000 »AdeoR pRW50c/fa ♦a/scR pRW50c/ n.19r - H202: □ wt pRW50c/(a P-12C O AiscR pRW50 c/(a p-12C A wt PRW50UP3 P-12C AifinR nRWfini IP.-^ 79x68mm (300 x 300 DPI) A + 37 pM NAL normal bacterial growth M(kDa) 0 0.5 1.0 1.5 2.5 4.0h RecA M (kPa) 0 0.5 1.0 1.5 2.5 4.0h RecA 43 RecA 43f RecA o\o J^ í^í > / / / / / / / / / / / + 37 mM NAL M (kPa) 0 0.5 1.0 1.5 2.5 4.Oh RecA M (kPa) 0 0.5 "1.0 1.5 2.5 4.0h RecA normal bacterial growth 25 \o Q) V 'b ^ V / .ž?' # ^^ ISCR- 25 FLAG / / / / / / IscR-FLAC3 B LexA IscR J DNA damage SOS repair genes-qna repair colicin K gene ^persistent DNA dam^ _ 129x67mm (300 x 300 DPI) 43- + 37 pM NAL: —■—wtpColK —D- &iscR pColK -♦- wt pColEI &iscR pColEI wt pColA MscR pColA -•-wtpColN -O— &iscR pColN 3 4 5 Time (h) B Col K C0IEI ColA Coin wild-type AiscR Oh 1h 2h 3h Oh 1h 2h 3h 0 . 0 • # -100 -10 -5 • . -100 -10 C wild-type àiscR M (kPa) Oh 1h 2h 3h Oh 1h 2h 3h ColK 70 55 43 34 ColK IscR_ GAňAfl|TCCATGCTCTTGftCATGGa|:AATGCTGAGTAGTAGGTTTTTACTm:ACaTAAAa££ASTGGTTATATGTACAGTA(N),„AAAGAGGM^ -35 element -10 element distal SOS box proximal SOS box S.D. cka C0IEI GAAAAÍTCČAČAGGGTTCaCAiGG^ AAAGAGGATTTTATAATO -35 element -10 element distal SOS box proximal SOS box S.D. cea ColA AACTAAAAiACCATGTTGAC^ (N)gg AAAGAGGAAAGATTATG -35 element -10 element distal SOS box proximal SOS box S.D. caa Coin GGAAdTCCACAGTCTTGACAœ^^ -35 element -10 element distal SOS box proximal SOS box S.D. cna 168xll5mm (300 x 300 DPI)