Contributors to Volume 46 Bau Deen, Department of ( Pivic Heath, Reston. MA 02 wer Cal Bil. Harvard Sohne TESA (Beemples@t sph harvare uy ‘Peten Gu acer, Schoal af Pharmacy avd Phannaceutieal Sciences, Coupland TN Building, Coupland Stret, University of Manchester, ‘Mancheste, MIS9PT- UW Peter glberr@man aca) Tow D, Hassett. Depausent of Micot:ologs, Wing Hal, Comell Unversity, thaca, NY 14853-8101, USA (ait@eonell edd Java vax, Department of Micrabinlogy, University of lino, Urbans UL 61801, USA [jimlaydinins caw) Tomes Maes-Lirmas, Chantaing Lalwastry. Tttard Medival Sehook, Longvod Avene. Busou, BEA 02115, CSA, nous TMctauy, School of Phunaucy ad Phamceaizul Scietoes. Coupland IT Buildin, Couphoad Sweet, Vaiversity of Machetes, Manuhoster, MIS 99L, UK) Mane R. O'Hhax, Dapartmet of Hiachomisiy, 140 Varbor Hall, Sate aivensity of New York ac Bui, Buffalo, SY M4274, USA, (rnobrian@ busted Sauncain Buchai, Depacunent of Geaeties, Call Bilogy’and Develupmaenn, 250 Biological Slances Cenuee, M13 Germer Avenue, SSL Boul, MIN 35108, USA Panto 1. Pownosast0, Dapartrsnt uf Mictubinlogy, Mors Seieace Commer 1 Yim, University nf Masachsel, Amherst, MA OT, OSA; and Repartment of Cancer Cel olay, Hareare Shin! of Publi Health, Bs, MA £2115. USA Ratooxa Prasan, Membrane Bicigy Lahortory, Schell Cite Sclenses, owaha’fal Ne Univer, Neo Tei, 11OHS? Ina (p87@aeoail com) ALouiscin H. Receaso, Scvolof Phamaey and Phasnaceutval Scenees, Coupland 1 Banking, Coupland Steet, University of Manckesier, Manchester, MIS 920, LX {ane Senioenans, Heckman Cone, Room B241A, 30 Pasar Deve, Stanford, CA 94305-5424, L 5A [eehnaolnité emp stantord.edy} Sum, Membrane Biology Iaboracory, School of Life Scences. Sawaal Netw University, New Deli, (10067 Ladia Los Tio Moor, laste uf Microbiology, ETM Zicich, Schuselzberstruse 7, CH-8092 Zirich, Switecand ‘Peasts Wuv7h, School ef Phamaay and Phacraceuticl Selene, Covplane TIL Boiling, Conplans Sve, University of Maaclesen, Manchester MI3 FL. OK Microarray Analysis of Bacterial Pathogenicity Gary K. Schootnike Davonmeat of Matos Monat a eneige “Stes aren lS ‘Stagind coasts, UA anSTRACT Tue DNA sucroursy a sufice Ul cone an onde erangenent of ‘ath ialed open ceading rate of uvegnesced yx. he engine Tuneticmal enimics, sau the expression pn Dovid a genome wide sapeshacef the ranserptome, Refine by faeay-spvii sttica nsuumeats and dta-uiaed by hsteling Agorins sad meabolic putsay databases, te expression pole *Escloses, alte tnascrptonal evel, bow the okcrobe adap Ws ce ‘online of growth Te repultury raturks that eovern the ‘daptve response und the melubule nu bing heic pla thal fect the now phenotype Adapelinn tn hast icmnemsoRES arias Me eapasiy nf infects agent mo eit am ami hast eens. Whe mniaring th whale genome Hanseriptinal response of bu eel pts itu nected ses has a heen oieved Lis ely tht tbe coraplen ssue-speilie sponses but the can of individual renpomses ofthe bncteia wo spore FPryscochomica Kotte thet chacaztevze ths st alow ese ake smenabltasvperimertatio. ie vi and whole-genariesxpression ‘lies of hk ave fined the trarscrignnad resp io staatan, lw guy, acid pH, quotomrening pheromnns an ‘elie oefgpn ieee These have dislesed new information bout even wl-sued process nl prnvida a portrait ofthe ‘lgling hacer a¢ a "yen rath tran dhe prod of em fen even a few rela. Athongst the reguted gene ompove this acaptive cpt as ustecription furor, Expres pining experiments of tanscripon factor outa ligt the frees pending rulers caseade. Tas genic bai Sor pulhopensity an as he staied hy sing misre-ray hosed eampuratve Seno tn “hsxcorive and quai the exten of rene vaneiiey iin nt [opllnns te gene Tel af eon en Alea emi ar Allleeoces betwen pape sal earnest pout pase ‘nulence dlernanes oslo ceuhnunary stuns: Te hua igerouly engages dhe puoxen: expression suis using hse eenoms wisps an hatailly nett cl alas he hat the ital Tia ston ie dnt rhe asinine rexpee, Hester ‘wih he sompas spree poe st fh hotell ae shmigonente rained by patbugenspecite detcinaty Tie ete he conned vse of bite adel microarrays Uw ludy he sume Infbsted tisve wel vou! he dle Demon ebogen and oi ipne-by-geue and sand tinsapacfc mat "Irapsltng tis Sversction will othe eazy and wll pohly require» eorinaton St pruerilBiieteenate te aud aati experinenil Swppruacles al considerable elute Imcrosray mete 2A, Higdon obo Be Nepeal aaa of miro dia 32 Anevalso' mlctearty gate cLeethgakoome Falrogeayazreein ruin of yoni ceri 48. Ae tue maton aoe ene pay tha ° Renin of adr ge a G1 Comaraivegenamins fH tuberous A vis and MICROARRAY ANALYSIS OF BACTERIAL FATHOGERICITY 3 52. Comnmntion pennien at malta Mtborousiclise elle «3 53M pnortevan avery ey ronan ABBREVIATIONS FST. pease sequence ig INH Leenowrinioacis hyéarioe WIG Topiops1f Dbiogatacopycaneside LPS Lipopolysaccbanue MO) Muliplcny uf infection PE Perumsinuonin ROL Receive oxygen internet os STS Bln very ste 1. INTRODUCTION 1.1. Microarray Experimental Paradtigens Ue apidly exgaaing aur uf pevounesequene dtabasss of pahogenic tucteia und hee bests provides sm uireceteated appomssity fin As suds of pathogenesis from a sbule-penome pensictve heuvgh ie w3ect DNA, ‘ictearay® For the purposes af bie discussion, x uicroureay will be cu "klered obs any device tut provides a ona eonialang representations all (or osasey of the Sdoaiied apen reading frames (ORES) vf a equcoces “zx otal geaorms The suage sad andthe method by which OR F-pe fife DNA ie hound, onietad ard aranged on cts sevface cary sith the Spe employe, Hosever, all ane tomate sarein common the eapecity fo parallel reengition, hy soraplemencary base paling. of euch of fhe DNA, ‘or RNA spocies in 4 complex sample wich hive comesponding OB-pe- tiie sequonces an she auruy vues. ‘Iie snipe expevimertal system provide tho bss ore, ite stne applica: coweaaine gomomies 4 GARY K. SCHOOL and fictional perotics. by eapeeasion pening. The @suinetionhetwcen itnetisnal ead cuimparative. genomics has hed nthe we2 of the terms “yentine’ ad “lrpnsciipiore ta Aiffevoniste and slaseifyinfsmstion omting, eapestively,fusn the stad OF an organicn’s genome eoctenr eid lransempion reper, Below, ever expel paradgns ati fuseed n geeral aod: preter eta a subseyuenl secs, Net ced In this ehapeer are felted seas “protege” unl “phelucne. wach, ‘eter he ocin ceding ex>aciy of the genome and the pecnotspe diver bil These plein eonfe 1.2. Comparative Gonor “To genomes can hs rapidly campare atthe ORF: aontent level of rvolodan by hyvtizing labled DNA from anon sequenced straint a asr0a7y cOD- ing tepeesenar nos of each ORF tiem @ savonced, rtercnes Aria Despite il itations ‘dissed in section 5, his application cat be a8 (> deduce the evulionimy relalslies af tums ad spetes by providing cu tmutive measure of diversity al infareaton about how that diversigy wake Ke tenn aso inminate anvil oacaldierences between pathogenic ad 00 palhogcais srine hy idonifying patbogoa-spccte pons fa might code foe nvlene detroit 1.3. Functional Genomics The ulus of microarcuy-hased Fanctional peuoris< comes trom ths ronari- tion thatthe taneriptional state ach gene nf a genome ands wipe, Aiforentcontrions oF grat Ts far mers noreptive Than the gene eaqeialF even the det hage-by- bis comparisou of ne eeu govomes (Rea and Poowu, 1999}, Miewatsy-based functional eausic Sludics wwe RNA prepare fiwas ot Jatt ulezo-onganisin fn w particular row sue, Hybridizasim of tbe beled RNA Cor coresponding labeled {EDNA to ehe aay sunlae lloras ta an organisstanserptane by Providing 2 quansitive metourementaf mRNA. The hybriicuton inten ty slevly reflects ene-speuiic RNA abundiove and serves as a neeomahle roeesute a Ue gees unusripioal uciiy for te cumliun of pow tthe Tig ae preety sso henge viding ean eugene ‘rrplojed sha insaetaacously slop RNA WiusceipGeu aad prevent RNA, Seeradaton). Incontvae to gene-he-goneassa¥e uf anstrpl aad al be long heen used by rndoeuiar rrobisogist (Fieen sd Brow, 1999), ‘whole genome DNA rocroarays measur the transcript sbundaace of ach, ORE saultnccusly (Le. in paraleD,ehus providing 2 condition-meciic and lime-soecitic gennme-scule snapshot of transeriguional activity. The resulting information is comma teed the "expression profile’ an di? Iecences betwen exptession ptubles aluminate the repubory deaths and ‘metuoie and hinsynthouepolnvays und are ative sing the ye grant) sate. Hers if amt he emphas 70d That the peor of iis method does at esi in fe stuohue quantitaiveneasiremert af angst wamber pe cel, bt rather in the compacsun uf expressioa pries wbuuined Irom vageully scleted, sbRerent experimen! conditions. Commonly studied comparisons include expression profiles ttom contains conditions of srosth at erent fime-poines ina imosourse ov a diflernt stages ima mtistage dsvelop= tenia progr, ‘The relitvey sal nuatace of miorsaray axpressin studios of bcrria pliogens pushed Uns Tn aad evened Welw are swpesingly aves ith Fapecl ivespersorntal cweston ul Vespa, Nol eile fs he 89 nian) ‘oF indvidual tude are tse tends cn emerge fom w survey ofthe Gell a patcgar. "Fes, the expanding micrartay-based expression datas fr» puniclar sein or species bs geassled au arise that I Ihe foto Wi soree a8 2 ‘eholz-genome mansion referee of an urganisn’splsivlgioal cpr ‘oir. Comparison af new expression proties obained from baie Hsing omple® and ondefiaed micreervreareents sith these archived, candi speciio profiles fiom ia tne experiments shaeld help deine the hssivacenc etones f st istes and ese. Ths ex essin profile tos eres 1 ogrobe of a uganian's nderooavieen:nen fan the perspective of Ue ere at in rvunstaaces uct ena y dost msaeoromane with ‘hse sensor. ‘Secand. ths comperson uf expression pull obtaied fom mip cu dans of growth and Te analyst af he pris with varus clsteting bobs dau 210 the denilicatiou tf sts of genes wih coxnmrn express patterns Prive the muraer uf wompazed adios is gs tees ing che ge Ties m contri genes tha re e-reculed, Ling of ous, operons, bat alee nen-nparoals se of genes colle hy the se wansotia fata, SSoch gane sere ean he penpewed as regulon. By otra a “stinulow is he tunnolstena expression pre #8 9 whole ut wa evoked by a psu ‘unttion of gle anf prohublycneampasies several revulns CT ¢ LBPI:, Meseeship ina costar not only tmpieset-egulation, bul ako sue esis Uist be products af the clustoved genes ary ou sime cummat faclabute, beeper paths anscon (sen eta, 19: Lea und IBrown, 199%). Mveneer, becuse approimalelyne-hul of tae ORFs com posine Most seqonced genomes endo Kor protein of uakaowl Fallen esa annoraten eehypnhetical or emservcu hypothetical ORFS), wan be srussgated ac th same proportion of pene Si any eluses will aso be of luakiuvin function. Hewexer, reeanse gones in cluster are likely 10 be functionally slat, hese: mth nea Functions sgpeseéuneions fh he caller Fares an er, 19901 thie wR rhe ete analyse af essa roles can conta Wt the waa. aon pes 'A poset ational appncaiun ol expresiva prbue i cise ut gulazory networks, Whi such net ne apd 6 he star nay “Sserhod above, eqpression piling cun be used n experimentally ienty lene srhnss raguacon daponcs on paricola: transcription fzter. Ore Somach comes fing the razagiitnn tac taneritn fasion are oer en= elated wih the gene: th in ha segulat. Thesis, loser alysis an Jead tothe leaiation os gee onan 9 pulse temsrigtion Fete vitkina eluster uf ober yeues Ts hypolteiy cm be este by dete if upton of the maser fut aboot expen of the gene = ter nf which eis.arsraber. In this ear, bath the ype and hs ose com> fiom expression expesimtants.Arenatvsly,gename seqionse pices Ti wally sassy goues acendiqg fuss Final steguriee atl aot tse se surous lasses uf egulutors.Esprsvien experiments hat deat sorltions that chung tbe expression Joel of a puative tanseription fate can be fl Toned bythe ase Ye sarc rac ns os ans face vata, ‘The resating evession profiles, compara dh hos laid with Rs Wild spe paren, shat isc gone sty regard Wy he teatacripion ft, TE ‘nel stomatal. ths appvachcatinprinple ee zoes governed by, Ter arog, cach woudl allele ipa ctr or bce compment rege Ialur in the gewome. Becasse some vf tae reguluted gener will he ther teanactiption factors, i shale be passble ta recansuct hierarchies oF 30 lanry networks tin these dats. ‘While the sok ws aan sal aaicouusay ses will ints suse iptiw Letrs an Ee pene thes regulate in est caes the results dv dstagunst hetsen rel wd ndinet meshanisms of reglaion aise regulatun can simply be a eomsecoonce of» mikétieed regliory sxcade ale postion ofthe rnsrpcon flor hgh nthe irre. Hor, the ds action Beseen odie und net elec ean lye mae wh ela the use fuss ur dtermire i ths menserpénn fate psa bids te rombler ofthe ons itis presumed nde regulate, Alcnatvely iia Teulon enn ccm: fie #2 use af conditions ahd ssi thal cause pleomophicdwmstreum effect, Thi soute> of ambiguiscan be gael the se of sv ime coumses and conto ha noc aoe overall HESS corgi zute ula mutant compared with ths wilde parca. Accor to avail ideuulsing uilleentity expressed geosth phase gest e500 ‘be gets Ue expression pv nse workere new recomend these Of “spetisenal conic hac dy na enue dferness grat beret Oro ‘ested corsidums ‘bis ull poses sublle und aiieme experimental desig pioblom snl ea frequen sabe of eapeesson pros thal omen 2 Zora tation of psc ant nope eens Micreuray expression profiling gr se not neces emt cennpfion far woe elle cn eapres si de a pstanuaalaiod event [Lcamples uf thse scl psp reas rough os compurent system ta the activation of prcfort tanscription Facts hy anges io their ent mation. AdAiionaly, bevause mst mieuaeray formals el represeal repons of tne etsuneutspeify sl. tog-tunslated RNs, her nee ingly ecugnized recubtory lutions rufa to be identified by tis metho Wacarmun eral. U0, 2. MICROARRAY FORMATS, ‘hs studies dsseribed in he (ollowing sections tye ployed ene of three array formuts For te purpows ofthis siseutsien, ts three ronnats will be refers wo af ‘high Sous ligoanclenide rays, "isiarrey and sme braue macs ste exresson sis xlependcut ue aay Curnes eoal Ue prepa ruta of labeled cDNA probes Sr ttal RNA because, uulke eukaryttes, hactvial mRNA isnot palyadenvled uthos exnnet he purified seectivels frst the same manne ax eakeryee mRNA Cen eta, WH: Arf # a “oat, Since “AS ot ral RNA ie corpnsed af RNA and CRNA, the uss oF sos besauers he nes pctoonkdvieg <DNA ayes wl rain al denaRNA, Wait nl chor Ds devacases sie By iaceasing he Teicha esq aig Cus ENA spun means tas may Be 3 ftense tn ajar pols age ube Thun ee anally Ie preecenaly lune! mRNA im astral pte hove been exam and Us sles hare wed primer pect Fe the $end ech Fol! ORF Wy prime Fst SNA spats rom total RNA To ra, 1983: Ain ea, 210). On Sse sues crag Ue 0 pte arethods a Ftd hat dhe Hic ion sizes rus with pies prodcond with ORF-spevle peimes wa only ‘e-ind Ma pres pluced with rams ecu Ain 2004, Figh-tensty aiganaeteotde arrays. manotaorured hy Affymetrix (Sant Plan, CA, NA cone of rn 20 Afoemt 2-ge ohigomalenides fae fish ORF i 4 sequenced genome, Alo represented inthe Sirs ue Te ine gsme regions greater dan 200 pe Lackhart taf, 1: Lipsbut {99 Haengzon of, 2004, The election of gene-specific ulgemucleies is tase in parton egenze uniqueness in adr spredce cosy luzaion bias hetwooe pxatogs ether eones a the wename hu vontain related sequunes Fach aignanclenrites pale ith a wcll isms’ ent) ‘lganveleolive at ifr: fom ae ‘perfor mats parma only nna, eon telly kate baie, Coaapinon of the hyiiatin insite th petoce rath and mismulch oligouucletice provides tet fur detenaiaie # Sobrasing background Mueescence ‘ffymcai amuys cre maufactured by combinutoral soid-phuse. Ugh veered aljgomaclotide shen has performed oa «1.28 1 2h em gas srface. Syhuheis inkers containing a protecting rocp that can be phot hemvally remowsd are attached glass. Tight dlscted tvough preleeenined windows an aie opie mash cases Wealined dopo lection lewwing behinds renuive snoop lzough which 9 mwuclive ioxymuvlerside can bo coupled, The sepoise application of row mh, depretsenon of the growing ehyemucleotide and coupling wa an adel fenxymeleowde yilde a ricmanay containing thansands of difrert. bet Drelerming oiganuclevies each comglementary toa sequence o ice ‘The cupleterepeatvice of sm ary consisting of 25 base vigonuceviles al eprsseabng each ORM ruc nb¥ng nervemc repr ubueteil eam sequence. can be symsized bya mame uf UD Eyles 48, ero Vis ee ‘sligomucietie length) “To abain expcesion profs from hacia using high-donsiyeligan ‘coeds anay.¢ recency asa isinUelled cDNA Team eral RNA that ‘as ot heat onaraed fos eyes esefition i enced pring FRNA De Suizioy ea, 2000), Hybrietion of bisa Iabella DNA Wo tbe ary wis Uollowed by Aoresen, tring sith steptavidin-pycoery ibrin. Hinecessar, the signal can be further ampliied with w bindnylazed untscepaviin anh oll Wy the smeyraidin-pniceershsn oanogate DeSavien or of 20M. The gan aud iatosity uF de sesuing sig ix Alerecte by ler seaming ui Ue Allyneis pete cag same Ln vrarase tothe teu eeu hvbridletvn uderourayayotan desstibed in ke next nec- nk, INS Cus ea sae Is hvtvdicel oa sepa aay ering cf, 2000) au Uns mn eaperimertconpann, th expression profile om ‘oneal souls eequies wu Ligtedenstyoligiauslewule as 2.2, Glass-Spotted DNA Microarrays ‘The DNA forum fest described by Prawn and colleagues at Stanford Univesity (Seheus ea, 1996; Dek er af 1997; Eisen und Brown. 1999) Sonsists os wlrmeupe sie ove suluce conus a x by » outa of Dike pots, en spe confi pulycerie chao reatton (PCR dcrved ubiy-trand aooou thal caresponds (a all ur part of an ORE of the sequenced gennae. Alternatively, snglestand syiftiy 50 10 70 base ‘ligoauceotides comaining unique ORF:spocitis sequohces Lsve als Noe