STM2209-STM2208 (opvAB): A Phase Variation Locus of Salmonella enterica Involved in Control of O-Antigen Chain Length

STM2209-STM2208 (op AB) : A Phase Va ia ion Locus o Salmonella en e ica In ol ed in Con ol o O-An igen Chain Leng h Ignacio Co a 1 , Anne Be ´a ice Blanc-Po a d 2 , Josep Casadesu ´s 1 * 1Depa amen o de Gene ´ ica, Facul ad de Biologı ´a, Uni e sidad de Se illa, Se illa, Spain, 2Uni e ´Mix e de Reche ches 5235, Cen e Na ional de la Reche che Scien i ique e Uni e si e ´Mon pellie II, Mon pellie , F ance Abs ac STM2209 and STM2208 a e con iguous loci anno a ed as pu a i e p o ein-coding genes in he ch omosome o Salmonella en e ica. Lack o homologs in ela ed En e obac e ia and low G+C con en sugges ha S. en e ica may ha e acqui ed STM2209-STM2208 by ho izon al ans e . STM2209 and STM2208 a e co- ansc ibed om a p omo e ups eam STM2209, and hei p oduc s a e inne (cy oplasmic) memb ane p o eins. Analysis wi h he bac e ial adenyla e cyclase wo-hyb id sys em sugges s ha STM2209 and STM2208 may in e ac . Exp ession o STM2209-STM2208 is subjec ed o phase a ia ion in wild ype Salmonella en e ica se o a Typhimu ium. Swi ching equencies in LB medium a e 6.1610 25 (OFFRON) and 3.7610 22 (ONROFF) pe cell and gene a ion. Lack o DNA adenine me hyla ion locks STM2209-STM2208 in he ON s a e, and lack o he LysR- ype ac o OxyR locks STM2209-STM2208 in he OFF s a e. OxyR-dependen ac i a ion o STM2209- STM2208 exp ession is independen o he oxida ion s a e o OxyR. Salmonella cul u es locked in he ON s a e show al e a ion o O-an igen leng h in he lipopolysaccha ide, educed abso p ion o bac e iophage P22, impai ed esis ance o se um, and educed p oli e a ion in mac ophages. Pheno ypic he e ogenei y gene a ed by STM2209-STM2208 phase a ia ion may hus p o ide de ense agains phages. In u n, o ma ion o a subpopula ion unable o p oli e a e in mac ophages may es ain Salmonella sp ead in animal o gans, po en ially con ibu ing o success ul in ec ion. Ci a ion: Co a I, Blanc-Po a d AB, Casadesu ´s J (2012) STM2209-STM2208 (op AB): A Phase Va ia ion Locus o Salmonella en e ica In ol ed in Con ol o O-An igen Chain Leng h. PLoS ONE 7(5): e36863. doi:10.1371/jou nal.pone.0036863 Edi o : Dipshikha Chak a o y, Indian Ins i u e o Science, India Recei ed Feb ua y 29, 2012; Accep ed Ap il 15, 2012; Published May 11, 2012 Copy igh : ß2012 Co a e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This s udy was suppo ed by g an s BIO2007-67457-CO2-02 and CSD2008-00013 om he Spanish Minis y o Science and Inno a ion (MICINN) and he Eu opean Regional Fund, and g an CVI-5879 om he Conseje ı ´a de Inno acio ´n, Ciencia y Emp esa, Jun a de Andalucı ´a ( o J.C.). I.C. holds a FPU (Fo macio ´nde P o eso ado Uni e si a io) ellowship om he MICINN. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: [email p o ec ed] In oduc ion Phase a ia ion, he e e sible swi ch o gene exp ession a high equency (e. g., .10 25 pe cell and gene a ion), is a common phenomenon in bac e ia ( e iewed in [1,2]). Swi ching u ns gene exp ession om OFF o ON, o om low exp ession o high exp ession, and ice e sa. A consequence o phase a ia ion is pheno ypic he e ogenei y in clonal bac e ial popula ions, a phenomenon o pa amoun ele ance o bac e ial su i al in ha sh en i onmen s. In bac e ial pa hogens, o ins ance, pheno- ypic he e ogenei y in cell en elope componen s may acili a e immune e asion and modula ion [2,3]. Classical examples o phase a ia ion in pa hogenic bac e ia in ol e loci encoding su ace-exposed p o eins, cell appendixes such as imb iae, pili, and lagella, and lipopolysaccha ide modi ica ion unc ions [1,2,4,5]. Phase a ia ion, howe e , is no es ic ed o bac e ial pa hogens no o loci ha encode componen s o he cell su ace [6,7]. Bac e ia use a a ie y o mechanisms o p oduce phase a ia ion [1], and a ela i ely common ype o con ol in ol es swi ching be ween al e na i e epigene ic s a es. Each epigene ic s a e is p opaga ed by a eedback loop, and e e sed a e a ce ain numbe o gene a ions. Epigene ic egula ion o phase a ia ion sys ems is o en con olled by DNA adenine me hyla ion ( e iewed in [8,9]). Pa adigms o his kind o egula ion a e he pap ope on o u opa hogenic E. coli, which encodes imb iae o a achmen o he u ina y epi helium [10], and he agn43 gene o E. coli, which encodes a non- imb ial adhesin [11]. O he phase a ia ion loci unde Dam me hyla ion con ol a e he glycosyl ans e ase ope on (g ) o phage P22 [4], he clp ope on o en e o oxigenic E. coli [12], he pe ope on o he Salmonella i ulence plasmid [13], and pe haps he S. en e ica s d imb ial ope on [14]. This s udy desc ibes a new phase a ia ion locus in Salmonella en e ica se o a Typhimu ium. The locus, anno a ed as STM2209- STM2208 in he Salmonella genome da abase [15], is p esen in Salmonella en e ica bu no in Salmonella bongo i no in o he en e ic bac e ia. Aside om he anno a ion o STM2209-STM2208 as pu a i e p o ein-coding genes, he li e a u e con ains li le in o ma ion on STM2209-STM2208. An excep ion is a ansc ip- ome analysis in Dam + and Dam 2 s ains o S. en e ica which e ealed ha STM2209-STM2208 ansc ip s a e mo e abundan in a Dam 2 backg ound [16]. This obse a ion en a i ely classi ied STM2209-STM2208 as a locus ep essed by Dam me hyla ion [16]. Howe e , we show ha STM2209-STM2208 is ac ually a phase a ia ion locus whose exp ession is locked in he ON s a e in Dam 2 mu an s. We also show ha lack o he LysR-like ac o PLoS ONE | www.plosone.o g 1 May 2012 | Volume 7 | Issue 5 | e36863 OxyR locks STM2209-STM2208 exp ession in he OFF s a e. STM2209 and STM2208 a e pa o a single ansc ip ional uni , and encode inne memb ane p o eins. Cons i u i e exp ession o STM2209-STM2208 al e s lipopolysaccha ide O chain leng h, educes phage P22 adso p ion, dec eases esis ance o se um, and impai s p oli e a ion in mac ophages. Al oge he , ou obse a ions sugges ha phase a ia ion o STM2209-STM2208 may con ib- u e o pheno ypic he e ogenei y in Salmonella popula ions, p o id- ing de ense agains phages and es aining Salmonella sp ead in animal o gans. Me hods Bac e ial s ains, plasmids, bac e iophages, and s ain cons uc ion All he s ains o Salmonella en e ica used in his s udy (Table 1) belong o se o a Typhimu ium, and de i e om ATCC 14028. Fo simplici y, S. en e ica se o a Typhimu ium is o en abb e ia ed as S. en e ica.Esche ichia coli BTH101 (F 2 cya-99 a aD139 galE15 galK16 psL1(S )hsdR2 mc A1 mc B1) was used o bac e ial wo- hyb id assays. E.coli CC118 lambda pi [phoA20 hi-1 spE poB a gE(Am) ecA1 (lambda pi )] and E. coli S17-1 lambda pi [ ecA p o hsdR RP4-2-Tc::Mu-Km::Tn7(lambda pi )] we e used o di ec ed cons uc ion o poin mu a ions. Plasmids cons uc ed o his s udy a e lis ed in Table 2. Lu ia–Be ani (LB) b o h was used as liquid medium. Solid LB b o h con ained aga a 1.5% inal concen a ion. G een pla es [17] con ained me hyl blue (Sigma-Ald ich) ins ead o aniline blue. The indica o o moni o ing ß-galac osidase ac i i y in pla e es s was 5-b omo-4-chlo o-3-indolyl-ß-D-galac opy anoside (‘‘X-gal’’; Sigma-Ald ich, 40 mg/ml). An ibio ics we e used a he concen- a ions desc ibed p e iously [18]. To g ow OxyR 2 s ains on LB aga , 75 ml o a 10 mg/ml ca alase solu ion (Sigma-Ald ich) was sp ead on he su ace o he pla es. The oligonucleo ides used in his s udy a e lis ed in Table S1. Ta ge ed gene dis up ion was achie ed using plasmids pKD3, pKD4 and pKD13 [19] and oligonucleo ides PS1, PS2 o PS4. Oligonucleo ides E1 and E2 we e used o allele e i ica ion. An ibio ic esis ance casse es in oduced du ing s ain cons uc- ion we e excised by ecombina ion wi h plasmid pCP20 [19]. Fo he cons uc ion o ansc ip ional and ansla ional lac usions in he Salmonella ch omosome, FRT si es gene a ed by excision o Km casse es we e used o in eg a e ei he plasmid pCE37 o pCE40 [20]. Addi ion o 3xFLAG ag o p o ein-coding DNA sequences was ca ied ou using plasmid pSUB11 as a empla e [21] and oligonucleo ides F2209-5 and F2209-3 ( o STM2209), and F2208-5 and F2208-3 ( o STM2208). T ansduc ional c osses using phage P22 HT 105/1 in 201 ([22] and G. Robe s, unpublished da a) we e used o s ain cons uc ion ope a ions in ol ing ch omosomal ma ke s. The ansduc ion p o ocol has been desc ibed elsewhe e [23]. To ob ain phage- ee isola es, ansduc an s we e pu i ied by s eaking on g een pla es. Phage sensi i i y was es ed by c oss-s eaking wi h he clea -plaque mu an P22 H5. RNA ex ac ion RNA was ex ac ed om S. en e ica s a iona y phase cul u es (OD 600 ,3), using he SV o al RNA isola ion sys em (P omega) as desc ibed a h p://www.i .ac.uk/sa e y/mic oa ays/p o ocols. h ml. The quan i y and quali y o he ex ac ed RNA we e de e mined using an ND-1000 spec opho ome e (NanoD op Technologies). To diminish genomic DNA con amina ion, he p epa a ion was ea ed wi h DNase I (Tu bo DNA ee; Applied Biosys ems). Quan i a i e e e se ansc ip ase PCR and calcula ion o ela i e exp ession le els An aliquo o 0.6 mg o DNase I- ea ed RNA was used o cDNA syn hesis using he High-Capaci y cDNA A chi e ki (Applied Biosys ems). Quan i a i e e e se ansc ip ase (RT)-PCR eac ions we e pe o med in an Applied Biosys ems 7500 Fas Table 1. S ains o Salmonella en e ica se o a Typhimu ium. S ain Geno ype ATCC 14028 wild ype SV4536 Ddam-230 SV5573 STM2208::3xFLAG SV5574 Ddam-230 STM2208::3xFLAG SV5676 DSTM2209::lac ( ansc ip ional) SV5677 DSTM2208::lac ( ansc ip ional) SV5679 DSTM2208::lac ( ansla ional) SV5680 Ddam-230 DSTM2209::lac ( ansc ip ional) SV5681 Ddam-230 DSTM2208::lac ( ansc ip ional) SV5683 Ddam-230 DSTM2208::lac ( ansla ional) SV5734 DSTM2209::lac ( ansla ional) SV5735 Ddam-230 DSTM2209::lac ( ansla ional) SV5812 STM2209::3xFLAG SV5813 Ddam-230 STM2209::3xFLAG SV5925 DoxyR::Cm SV5989 DoxyR::Cm DSTM2208::lac ( ansla ional) SV5990 Ddam-230 DoxyR::Cm DSTM2208::lac ( ansla ional) SV6001 DoxyR::Cm STM2208::3xFLAG SV6002 Ddam-230 DoxyR::Cm STM2208::3xFLAG SV6004 DoxyR::Cm STM2209::3xFLAG SV6005 Ddam-230 DoxyR::Cm STM2209::3xFLAG SV6013 DSTM2209-STM2208 SV6397 oxyR C199S SV6401 mu . GATC SV6976 mu . GATC DSTM2209-STM2208 SV7031 mu . GATC DSTM2208::lac ( ansla ional) SV7032 Ddam-230 mu . GATC DSTM2208::lac ( ansla ional) SV7232 DoxyR::Cm mu . GATC DSTM2208::lac ( ansla ional) SV7233 Ddam-230 DoxyR::Cm mu . GATC DSTM2208::lac ( ansla ional) doi:10.1371/jou nal.pone.0036863. 001 Table 2. Plasmids cons uc ed o his s udy. Plasmid numbe Desc ip ion pIZ1758 pGEMT::[PE5-PE2209] pIZ1759 pGEMT::[PE5-PE2208] pIZ1812 pKT25::STM2209 pIZ1905 pUT18C::STM2208 pIZ1906 pUT18C::STM2209 pIZ1907 pKT25::STM2208 doi:10.1371/jou nal.pone.0036863. 002 Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 2 May 2012 | Volume 7 | Issue 5 | e36863 Real-Time PCR Sys em. Each eac ion was ca ied ou in a o al olume o 25 ml on a 96-well op ical eac ion pla e (Applied Biosys ems) con aining 12.5 ml Powe SYBR G een PCR Mas e Mix (Applied Biosys ems), 11.5 ml cDNA (1/10 dilu ion), and wo gene-speci ic p ime s (RT2209-5 and RT2209-3 o STM2209, RT2208-5 and RT2208-3 o STM2208) a a inal concen a ion o 0.2 mM each. Real- ime cycling condi ions we e as ollows: (i) 95uC o 10 min and (ii) 40 cycles a 95uC o 15 sec, and 60uC o 1 min. A no- empla e con ol was included o each p ime se . Mel ing cu e analysis e i ied ha each eac ion con ained a single PCR p oduc . Gene exp ession le els we e no malized o ansc ip s o ompA, a housekeeping gene ha se ed as an in e nal con ol. The S uden ’s es was used o de e mine i he di e ences in e o ansc ibed mRNA con en obse ed in di e en backg ounds we e s a is ically signi ican . ß-galac osidase assays Le els o ß-galac osidase ac i i y we e assayed as desc ibed p e iously [24], using he CHCl 3 -sodium dodecyl sul a e pe mea- biliza ion p ocedu e. The S uden ’s es was used o de e mine i he di e ences in ß-galac osidase ac i i ies obse ed in di e en backg ounds we e s a is ically signi ican . P o ein ex ac s and Wes e n blo ing analysis To al p o ein ex ac s we e p epa ed om bac e ial cul u es g own a 37uC in LB medium un il s a iona y phase (OD 600 ,3). Bac e ial cells con ained in 0.25 ml o cul u e we e collec ed by cen i uga ion and suspended in 50 ml o Laemmli sample bu e [1.3% SDS, 10% ( / ) glyce ol, 50 mM T is-HCl, 1.8% b- me cap oe hanol, 0.02% b omophenol blue, pH 6.8]. P o eins we e esol ed by T is-Glycine-PAGE using 12% gels ( o STM2208) o T is-T icine-PAGE 15% gels ( o STM2209). Condi ions o p o ein ans e ha e been desc ibed elsewhe e [14]. P ima y an ibodies we e an i-Flag M2 monoclonal an ibody (1:5,000, Sigma-Ald ich) and an i-G oEL polyclonal an ibody (1:20,000; Sigma-Ald ich). Goa an i-mouse ho se adish pe oxi- dase-conjuga ed an ibody (1:5,000; Bio-Rad) o goa an i- abbi ho se adish pe oxidase-conjuga ed an ibody (1:20,000; San a C uz Bio echnology) was used as seconda y an ibody. P o eins ecognized by he an ibodies we e isualized by chemolumines- cence using he luci e in–luminol eagen s (The mo Scien i ic). Subcellula ac iona ion Subcellula ac iona ion was pe o med as p e iously desc ibed [25], wi h some modi ica ions. B ie ly, bac e ia we e g own in LB medium a 37uC and spun down by cen i uga ion a 15,0006g o 5 min a 4uC, hen esuspended wice in cold phospha e- bu e ed saline (PBS, pH 7.4). The bac e ial suspension was ei he mixed wi h Laemmli bu e ( o al p o ein ex ac ) o dis up ed by sonica ion. Unb oken cells we e u he emo ed by low-speed cen i uga ion (5,0006g, 5 min, 4uC). The supe na an was cen i uged a high speed (100,0006g, 30 min, 4uC) and he new supe na an was eco e ed as he cy osol ac ion. The pelle con aining en elope ma e ial was suspended in PBS wi h 0.4% T i on X-100 and incuba ed o 2 h a 4uC. The sample was cen i uged again (100,0006g, 30 min, 4uC) and di ided in o he supe na an con aining mos ly inne memb ane p o eins and he insoluble ac ion co esponding o he ou e memb ane ac ion. An app opia e olume o Laemmli bu e was added o each ac ion. A e hea ing (100uC, 5 min) and clea ing by cen i u- ga ion (15,0006g, 5 min, oom empe a u e), he samples we e analyzed o p o ein con en by SDS-PAGE. P ime ex ension The oligonucleo ides PE2209 and PE2208, complemen a y o in e nal egions o he genes STM2209 and STM2208 espec i ely, we e end-labeled wi h [ 32 P]ATP and annealed o 10 mg o o al RNA p epa ed om S. en e ica s ains bea ing plasmids pIZ1758 (cons uc ed using oligonucleo ides PE5 and PE2209) and pIZ1759 (cons uc ed wi h PE5 and PE2208). The end-labeled p ime was ex ended wi h a ian myeloblas osis i us e e se ansc ip ase (Boeh inge Mannheim) unde condi ions desc ibed p e iously [26]. The p oduc s o e e se ansc ip ion we e analyzed in u ea-polyac ylamide gels and isualized using a FLA-5100 Imaging sys em (Fuji ilm). Di ec ed cons uc ion o poin mu a ions Mu a ion o he 4 GATC si es con ained in he p omo e egion o STM2209-STM2208 was achie ed using he QuikChangeH Si e-Di ec ed Mu agenesis Ki (S a agene). B ie ly, a ,1.3 Kb agmen o he STM2209-STM2208 egion con aining he 4 GATC si es was cloned in o he pGEMT plasmid using he oligonucleo ides Clo2208-5 and Clo-2208-3. Mu a ions in e e y GATC we e hen in oduced using oligonucleo ides ha bo ing CATC changes (labeled as DIRnue o and INVnue o). The esul ing plasmid con aining he agmen wi h 4 CATC si es was hen diges ed wi h XbaI and SacI, cloned on o he suicide plasmid pDMS197 [27] and p opaga ed in E. coli CC118 lambda pi . Plasmids de i ed om pMDS197 we e ans o med in o E. coli S17-1 lambda pi . The esul ing s ains we e used as dono s in ma ings wi h S. en e ica 14028 ha bo ing a Cm casse e in place o he 4 GATC si es (cons uc ed using oligonucleo ides delGATC- PS1 and delGATC-PS2) as ecipien s. Tc ansconjugan s we e selec ed on E pla es supplemen ed wi h e acycline. Se e al Tc ansconjugan s we e g own in nu ien b o h (wi hou NaCl) con aining 5% suc ose. Indi idual e acycline-sensi i e seg egan s we e hen sc eened o clo amphenicol sensi i i y and examined o he inco po a ion o he mu an allelle by Sau3AI diges ion and DNA sequencing using ex e nal oligonucleo ides. Cons uc ion o he oxyR C199S mu a ion was achie ed in he same way, using he oligonucleo ides ClooxyR-5 and ClooxyR-3 o cloning on o pGEMT, and he oligonucleo ides oxyRC199SDIR and oxy- RC199SINV o si e-di ec ed mu agenesis. A s ain wi h a Cm casse e in place o he oxyR gene (cons uc ed using oligonucle- o ides deloxyR199-PS1 and deloxyR199-PS2) was used as a ecipien in his case. Measu emen o he e iciency o phage adso p ion The e iciency o phage adso p ion was calcula ed as desc ibed by Gabig e al. [28]. B ie ly, P22 bac e iophages we e added o S. en e ica cells om an LB liquid o e nigh cul u e a a mul iplici y o in ec ion o 0.1, and he mix u e was incuba ed a 37uC. Samples we e aken e e y 2 min, cen i uged o 1 min a 13,000 pm in a mic ocen i uge, and he supe na an was i a ed on he S. en e ica wild- ype s ain ATCC 14028. The sample ob ained a ime ze o (a sample aken immedia ely a e addi ion o bac e iophages o he cell suspension) was conside ed o co espond o 100% unadso bed phages, and he emaining numbe s we e calcula ed ela i e o his numbe . The S uden ’s es was used o de e mine i he di e ences in adso p ion we e s a is ically signi ican . Elec opho e ic isualiza ion o lipopolysaccha ide p o iles To in es iga e lipopolysaccha ide (LPS) p o iles, bac e ial cul u es we e g own o e nigh in LB. Bac e ial cells we e ha es ed and washed h ee imes wi h 0.9% NaCl. The Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 3 May 2012 | Volume 7 | Issue 5 | e36863 O.D. 600 o he washed bac e ial suspension was measu ed o calcula e cell concen a ion. A bac e ial mass con aining abou 3.14610 8 cells was pelle ed by cen i uga ion. T ea men s applied o he bac e ial pelle , elec opho esis o c ude bac e ial ex ac s, and sil e s aining p ocedu es we e pe o med as desc ibed by Buendia-Cla e ia e al. [29]. Calcula ion o phase ansi ion equencies Phase ansi ion a es we e es ima ed as desc ibed by Eisens ein [30]. B ie ly, a s ain ha bo ing an STM2208::lac usion was pla ed on LB +X-gal and colonies displaying an ON o OFF pheno ype a e 16 h g ow h a 37uC we e selec ed, esuspended in PBS and esp ead on new pla es. Phase ansi ion equencies we e calcula ed using he o mula (M/N)/g whe e Mis he numbe o cells ha unde wen a phase ansi ion, N he o al numbe o cells, and g he o al numbe o gene a ions ha ga e ise o he colony. Mac ophage in ec ion expe imen s The a e o in amac ophage eplica ion a e 18 h in ec ion was pe o med in J774 mouse mac ophages as desc ibed in [31]. B ie ly, mac ophages we e seeded a a densi y o 5610 5 in 24-well pla es and g own in DMEM medium supplemen ed wi h 10% ( / ) e al bo ine se um a 37uC, 5% CO2. Bac e ia we e added o he wells a mac ophage- o-bac e ia a io o 1:10. Phagocy osis was allowed o p oceed o 30 min be o e washing h ee imes wi h s e ile PBS and adding esh DMEM media supplemen ed wi h 20 mg/ml gen amicin. Mac ophages we e lysed by using 1% T i on X-100, and he numbe o iable bac e ia ha su i ed he gen amicin ea men was de e mined by subsequen pla ing on o LB aga pla es. The eplica ion a e was de e mined as he a io be ween he numbe o bac e ia a ime 18 h and he numbe o in e nalized bac e ia a e 30 min phagocy osis. The S uden ’s es was used o de e mine i he di e ences in eplica ion a es obse ed in di e en backg ounds we e s a is ically signi ican . Measu emen o su i al in se um Su i al in guinea pig se um (Sigma-Ald ich) was analyzed as desc ibed in [32] wi h some modi ica ions. B ie ly, exponen ial cul u es o S. en e ica we e se ially dilu ed in PBS +2 mM MgCl 2 o 2610 4 c u/ml. Guinea pig se um was added o 30% inal concen a ion and he mix u es we e incuba ed a 37uC wi hou shaking. Samples we e aken a 30 min in e als by pla ing on nu ien aga , and iable coun s we e exp essed as a pe cen age o he ini ial concen a ion (% su i al). The S uden ’s es was used o de e mine i he di e ences in su i al o se um obse ed in di e en backg ounds we e s a is ically signi ican . Bac e ial wo-hyb id analysis The Bac e ial Adenyla e Cyclase Two-Hyb id (BACTH) sys em [33] was used o es in e ac ion be ween wo memb ane p o eins. The STM2209 and STM2208 genes we e PCR ampli ied using oligonucleo ides pKT25-STM2209-Ps I-5 and pKT25-STM2209- BamHI-3 (STM2209 cloned in o pKT25), pUT18C-STM2209- Ps I-5 and pKT25-STM2209-BamHI-3 (STM2209 cloned in o pUT18C), pUT18C-STM2208-Ps I-5 and pUT18C-STM2208- BamHI-3 (STM2208 cloned in o pUT18C), pKT25-STM2208- Ps I-5 and pUT18C-STM2208-BamHI-3 (STM2208 cloned in o pKT25), and cloned on o plasmids pUT18C and pKT25 using he Ps I and BamHI si es. Recombinan plasmids ca ying STM2209 and STM2208 we e sequenced using oligonucleo ides pKT25-seq5 and pKT25-seq3 and co- ans o med in o an E. coli CyaA 2 s ain (BTH101). T ans o man s we e pla ed on LB +ampicillin + kanamycin +X-gal medium a 30uC o 30 h. To quan i y he in e ac ion be ween hyb id p o eins, bac e ia we e g own o e - nigh a 30uCinLB+Ap +Km liquid medium supplemen ed wi h 0.5 mM isop opyl-ß-D-1- hiogalac opy anoside (IPTG). ß-galac- osidase assays we e ca ied ou as desc ibed abo e. A le el o ß- galac osidase ac i i y a leas i e old highe han ha measu ed o ec o s alone indica es a posi i e in e ac ion. Resul s STM2209-STM2208 is a Salmonella-speci ic locus STM2209 and STM2208 a e con iguous loci anno a ed as pu a i e p o ein-coding genes in he ch omosome o Salmonella en e ica. The STM2209 and STM2208 ORFs a e conse ed in Salmonella en e ica se o a Typhimu ium s ains ATCC 14028, SL1344, and LT2 (GenBank accession numbe s CP001363.1, FQ312003.1 and AE006468.1, espec i ely), in he icini y o he suga anspo gene se B [34]. The STM2209 and STM2208 ORFs a e also conse ed in o he Salmonella en e ica se o a s bu no in Salmonella bongo i no in he gene a Esche ichia and Shigella. Alignmen o he p edic ed amino acid sequences o STM2209 and STM2208 using BLASTP [35] de ec ed no ob ious homologs o STM2209-STM2208 ou side Salmonella en e ica. A diag am o he ch omosome egion in Salmonella en e ica and ela ed En e obac e- iaceae is shown in Fig. 1. Bo h STM2209 and STM2208 ha e low G+C con en (37% o STM2209 and 38% o STM2208) compa ed o bo h he a e age o he egion (53%) and ha o he Salmonella en e ica genome (52%) [36]. Because ho izon ally acqui ed genes o en ha e dis inc i e base composi ion, speci ically low G+C con en [37,38], hese obse a ions sugges ha STM2209-STM2208 may ha e been acqui ed by ho izon al gene ans e . The o ganiza ion o he STM2209 and STM2208 ORFs sugges s ha hey may be pa o a single ansc ip ional uni : bo h coding sequences a e on he same DNA s and, and a e sepa a ed by only one nucleo ide. Genome sequence analysis in silico p edic s ha STM2209 may encode a small pep ide o 40 amino acids, while STM2208 may be a la ge p o ein p oduc o 221 amino acids. In silico analysis o p o ein s uc u e using he TMHMM ansmemb ane p edic ion so wa e [39] p edic s he exis ence o one ansmemb ane domain in STM2209, and wo ansmemb ane domains in STM2208 (da a no shown). In silico analysis also indica es ha STM2208 sha es a domain wi h p o eins belonging o he Wzz supe amily o O- an igen chain leng h egula o s. This amily includes p o eins in ol ed in lipopolysaccha ide biosyn hesis ha con e a modal dis ibu ion o chain leng h on he O-an igen componen o lipopolysaccha ide [40]. This domain is also ound in bac e ial y osine kinases [41]. Exp ession o he STM2209-STM2208 locus is egula ed by Dam me hyla ion A p e ious s udy showed ha STM2209 and STM2208 a e exp essed a highe le els (13 old o STM2209 and 8 old o STM2208)inaS. en e ica Dam 2 mu an [16]. These obse a ions sugges ed ha exp ession o he pu a i e STM2209-STM2208 ansc ip ional uni migh be ep essed by Dam me hyla ion. To con i m Dam-dependen egula ion, ansc ip ional and ansla- ional lac usions we e cons uc ed in bo h loci. P o ein a ian s agged wi h he 3xFLAG epi ope we e also cons uc ed. The e ec o Dam me hyla ion on STM2209-STM2208 exp ession was moni o ed by ß-galac osidase assays, qRT-PCR, and Wes e n blo ing in isogenic Dam + and Dam 2 s ains. Highe le el o ß- galac osidase ac i i y, highe amoun o e o ansc ibed STM2209-STM2208 mRNA, and inc eased le el o he STM2208-3xFLAG p oduc we e de ec ed in he Dam 2 back- Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 4 May 2012 | Volume 7 | Issue 5 | e36863 g ound (Fig. 2). The STM2209-3xFLAG p oduc was easily de ec ed in a Dam 2 backg ound bu was ha dly isible in he Dam + backg ound, p esumably due he combined e ec s o i s low le el o exp ession and i s small size. STM2209-3xFLAG isualiza ion by Wes e n blo ing in a Dam + backg ound was howe e possible upon longe gel exposu e (da a no shown). Al hough he ex en o de ep ession di e ed sligh ly depending on he me hod, exp ession o STM2209-STM2208 was signi ican ly highe in a Dam 2 backg ound in all expe imen s. These esul s con i m ha Dam me hyla ion ep esses STM2209-STM2208. Fu he mo e, ou abili y o de ec Dam-dependen egula ion wi h bo h ansc ip ional lac usions and qRT-PCR sugges s ha Dam- dependen egula ion o STM2209-STM2208 may be ansc ip- ional. Cha ac e iza ion o he STM2209-STM2208 ansc ip ional uni To cha ac e ize he STM2209-STM2208 ansc ip ional uni , we mapped he 59 e minus o he pu a i e STM2209-STM2208 ansc ip using p ime ex ension (Fig. 3). Because STM2208 and STM2209 a e exp essed a low le els in Dam + S. en e ica [16], a DNA agmen con aining he egion ups eam STM2209- STM2208 and pa o he coding sequence o STM2209- STM2208 was cloned on he pGEMT mul icopy ec o o ob ain highe amoun s o ansc ip (s). The esul ing plasmids (pIZ1758 and pIZ1759) we e in oduced in he wild ype s ain, and wo p ime ex ension eac ions we e pe o med. One eac ion was p imed by an oligonucleo ide complemen a y o STM2209 (PE2209), and he second eac ion by an oligonucleo ide complemen a y o STM2208 (PE2208). Bo h eac ions yielded ex ension p oduc s wi h iden ical 39ends (Fig. 3), indica ing he exis ence o a single ansc ip ion ini ia ion si e, six nucleo ides ups eam he s a codon o STM2209 p oposed in he XBASE (h p://www.xbase.ac.uk/) and NCBI (h p://www.ncbi.nlm.nih. go /) da abases. A DNA sequence eminiscen o a canonical ibosome-binding si e is howe e missing in his pu a i e mRNA o ganiza ion. Fo his eason, we p opose ha ansla ion o STM2209 may be ac ually ini ia ed a posi ion +25, 10 nucleo ides downs eam a pu a i e ibosome binding si e (59TGTGG 39). This hypo hesis is suppo ed by addi ional e idence: a ansla- ional lac usion cons uc ed ups eam +25 p o ed o be non unc ional: ß-galac osidase ac i i y was no de ec ed in a Dam 2 backg ound (da a no shown). Al oge he , hese obse a ions may indica e ha STM2209 consis s o 34 amino acids and no 40 amino acids as desc ibed in he Salmonella en e ica ATCC 14028 genome anno a ion. In silico analysis o he DNA sequence ups eam o he +1 si e iden i ied DNA sequences wi h ea u es simila o hose o canonical, sigma 70 -dependen p omo e s [42]: (i) a pu a i e 210 module including he mo i 59TAAAAT 39, which shows 5/6 ma ches wi h he consensus sequence [42]; (ii) a pu a i e space , 17 nucleo ides long; and (iii) a 59GTGAAT 39sequence de ining a pu a i e 235 module, wi h 3/6 ma ches wi h he consensus sequence [42]. We p opose ha STM2209 and STM2208 a e co- ansc ibed om his p omo e , an hypo hesis consis en wi h he obse a ion ha he STM2209 and STM2208 p oduc s a e co- exp essed (Fig. 2). Iden i ica ion o OxyR as a egula o o STM2209-STM2208 A gene ic sc een based on he T-POP3 ansposon [43] was used o sea ch o posi i e egula o s o STM2209-STM2208. Fo his pu pose, a Dam 2 s ain ca ying a lac ansla ional usion in STM2208 (SV5683) was used. This s ain o ms deep blue colonies on LB supplemen ed wi h X-gal. Isola es ca ying T-POP3 inse ions we e selec ed on LB + e acycline +kanamycin +X- gal, and whi e colonies we e sough . Only a small whi e colony was ob ained in he sc een. Cloning and sequencing o T-POP3 bounda ies indica ed ha T-POP3 had inse ed in he oxyR gene. OxyR 2 mu an s a e se e ely impai ed o o m colonies on LB pla es [44], hus explaining he small colony size o he isola e. Howe e , he isola e o med la ge colonies on LB +ca alase, a s anda d p ocedu e ha pe mi s colony o ma ion by OxyR 2 mu an s [44]. To con i m ha oxyR loss-o - unc ion abolished STM2209-STM2208 exp ession in a Dam 2 backg ound, he oxyR gene was dis up ed using lambda Red ecombinee ing. The esul ing s ain (SV5925), which ca ies a null oxyR allele, was used in u he expe imen s. Figu e 1. Diag am o he egion con aining STM2209-STM2208 on he Salmonella en e ica ch omosome. The homologous egions o Salmonella bongo i, E. coli, and Shigella lexne i a e also shown. The STM2209-STM2208 ope on is shown in yellow. Black a ows ep esen conse ed genes. Whi e a ows ep esen non conse ed genes. G ey a ows ep esen genes ound a a di e en ch omosome loca ion on he S. en e ica ch omosome. doi:10.1371/jou nal.pone.0036863.g001 Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 5 May 2012 | Volume 7 | Issue 5 | e36863 Analyses o ß-galac osidase ac i i y and Wes e n blo ing showed ha exp ession o STM2209-STM2208 is i ually abolished in an OxyR 2 backg ound (Fig. 4). As abo e (Fig. 2), high le els o ß-galac osidase and o he STM2209-3xFLAG and STM2208-3xFLAG p oduc s we e de ec ed in he Dam 2 back- g ound only. These expe imen s indica e ha OxyR is essen ial o he exp ession o STM2209-STM2208. In e es ingly, pu a i e OxyR binding si es a e ound in he p omo e egion o STM2209-STM2208 (see below). OxyR is a global ansc ip ion ac o ha can sense oxida i e s ess by di ec oxida ion. In he oxidized s a e, OxyR ac i a es he exp ession o oxida i e-s ess- esponding genes [45]. Howe e , OxyR also ac s as a ansc ip ional egula o i espec i e o i s oxida i e s a e. In he absence o oxida i e s ess, OxyR emains mos ly in he educed o m due o he educing en i onmen o he cell [46]. Se e al obse a ions sugges ed ha he oxida i e s a e o OxyR is no ele an o STM2209-STM2208 egula ion. One was ha an H 2 O 2 concen a ion su icien o p omo e he exp ession o genes belonging o he classical OxyR egulon (genes ac i a ed by oxida i e damage) showed no e ec on he exp ession o STM2209-STM2208 (da a no shown). Fu he mo e, he spacing be ween he hal si es in he pu a i e OxyR binding si es desc ibed below is consis en wi h speci ic binding o he educed o m o OxyR [47]. To de e mine he e ec o oxida ion o OxyR upon STM2209-STM2208 exp ession, we cons uc ed a poin mu an e sion o he oxyR gene (s ain SV6397). The esul ing OxyR C199S p o ein is locked in he educed o m as i canno o m he disul ide bond equi ed o oxida ion [46,47]. Dam + and Dam 2 s ains ha bo ing his mu a ion showed le els o STM2209- STM2208 exp ession simila o hose desc ibed abo e o s ains ca ying he wild ype oxyR allele (da a no shown). These obse a ions sugges ha oxida ion o OxyR is no necessa y o STM2209-STM2208 exp ession. STM2209-STM2208 exp ession unde goes phase a ia ion unde he con ol o Dam me hyla ion and OxyR In he cou se o ou expe imen s wi h s ains ca ying STM2209::lac o STM2208::lac usions in a wild ype backg ound, we de ec ed pheno ypic he e ogenei y when cul u e aliquo s we e sp ead on pla es con aining X-gal. These s ains o med whi e colonies ha la e u ned pale blue, indica ing low exp ession o STM2209 and STM2208. Howe e , deep blue colonies we e also Figu e 2. Regula ion o STM2209-STM2208 by Dam me hyla ion. A. Le els o STM2209 and STM2208 mRNAs, measu ed by qRT-PCR (Dam + : whi e his og ams; Dam 2 : black his og ams). Le el o STM2209 mRNA in Dam 2 backg ound is conside ed 100%. Values a e a e ages and s anda d de ia ions om 7 independen expe imen s. B. ß-galac osidase ac i i y o ansc ip ional STM2209::lac and STM2208::lac usions in Dam + and Dam 2 backg ounds (whi e and black his og ams, espec i ely). Values a e a e ages and s anda d de ia ions om 3 independen expe imen s. C. ß- galac osidase ac i i ies o ansla ional STM2209::lac and STM2208::lac usions in Dam + and Dam 2 backg ounds (whi e and black his og ams, espec i ely). Values a e a e ages and s anda d de ia ions om 3 independen expe imen s. D. Wes e n blo analysis o STM2209-3xFLAG and STM2208-3xFLAG p o eins in Dam + and Dam 2 backg ounds. doi:10.1371/jou nal.pone.0036863.g002 Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 6 May 2012 | Volume 7 | Issue 5 | e36863 seen, especially on pla es ha con ained high numbe s o colonies (e. g., $1,000 colonies). Whene e a blue colony was isola ed and s eaked ou o single colonies, a mix u e o whi e and blue colonies was ob ained. This obse a ion sugges ed ha STM2209- STM2208 exp ession migh unde go phase a ia ion, and ha swi ching om OFF o ON migh occu a lowe equencies han swi ching om ON o OFF. Phase a ia ion equencies in he STM2209-STM2208 locus we e calcula ed using he o mula (M/N)/g whe e Mis he numbe o cells ha unde wen a phase ansi ion, N he o al numbe o cells, and g he o al numbe o gene a ions ha ga e ise o he colony [30]. An STM2208::lac ansla ional usion was used o hese expe imen s. The equency o OFFRON ansi ion was es ima ed o be 6.161.7610 25 pe cell and gene a ion. The ONROFF swi ching a e was a ound 1,000- old highe : 3.760.1610 22 pe cell and gene a ion. Phase a ia ion o STM2209-STM2208 exp ession was also una ec ed by he oxida ion s a e o OxyR (da a no shown). Phase a ia ion was abolished in bo h Dam 2 and OxyR 2 mu an s (Fig. 5). Lack o Dam me hyla ion locks STM2209- STM2208 exp ession in he ON s a e, and lack o OxyR locks STM2209-STM2208 exp ession in he OFF s a e. An oxyR mu a ion is epis a ic o e a dam mu a ion, an obse a ion ha may indica e ha ac i a ion o STM2209-STM2208 ansc ip ion by OxyR is Dam-me hyla ion sensi i e. Howe e , bo h Dam me hyla ion and OxyR a e needed o es ablish phase- a iable exp ession o STM2209-STM2208. Si e-di ec ed mu agenesis o GATC si es ups eam he STM2209-STM2208 p omo e abolishes phase a ia ion In silico analysis o he DNA sequence ups eam he STM2209- STM2208 p omo e e ealed he exis ence o 4 GATC si es a anged in a symme ical pa e n (Fig. 6). In addi ion, he egion con ains wo pu a i e OxyR binding si es e y simila o he consensus sequence [46]. These si es o e lap wi h GATC si es numbe 2 and 4 espec i ely (Fig. 6). Because o he pleio opy o dam mu a ions, al e a ion o gene exp ession in Dam 2 mu an s does no necessa ily indica e di ec Dam-dependen con ol [48]. To con i m ha Dam me hyla ion di ec ly con ols STM2209-STM2208 exp ession, he GATC si es p esen in he p omo e egion o STM2209-STM2208 we e elimina ed by si e-di ec ed mu agenesis. I STM2209-STM2208 ep ession by Dam me hyla ion depends di ec ly on me hyla ion o he GATC si es wi hin he STM2209-STM2208 UAS, we easoned, elimina ion o he GATCs should lock STM2209- STM2208 exp ession in he ON s a e. To es his p edic ion, poin mu a ions we e enginee ed o ans o m he STM2209-STM2208 59GATC39sequences o 59CATC39sequences, which a e no a subs a e o Dam me hylase ac i i y (s ain SV6401). Fu he - mo e, he ou base pai subs i u ions in oduced in he STM2209- STM2208 UAS do no des oy known c i ical egions o he OxyR binding sequence [47]. ß-galac osidase ac i i y assays and Wes e n blo ing analysis p o ed ha egula ion by Dam me hyla ion was abolished when he GATC si es we e elimina ed (Fig. 6). Exp ession o STM2209- STM2208 was $2 old highe in he GATC-less mu an (SV7031) han he Dam 2 mu an (SV5683) (Fig. 6), bu STM2209- Figu e 3. Iden i ica ion o he ansc ip ion ini ia ion si e o STM2209-STM2208 by p ime ex ension. Pu a i e -35 and -10 p omo e modules and he +1 si e a e shown in bold ace. The ansc ip ion ini ia ion si e is indica ed by an a ow. doi:10.1371/jou nal.pone.0036863.g003 Figu e 4. Regula ion o STM2209-STM2208 exp ession by Dam me hyla ion and OxyR. A. E ec o an oxyR null mu a ion on he ß- galac osidase ac i i y o ansla ional STM2209::lac and STM2208::lac usions in Dam + and Dam 2 backg ounds (whi e and black his og ams, espec i ely). Values a e a e ages and s anda d de ia ions om 3 independen expe imen s. B. Wes e n blo analysis o he e ec o an oxyR null mu a ion on he le els o STM2209-3xFLAG and STM2208- 3xFLAG p o eins in Dam + and Dam 2 backg ounds. doi:10.1371/jou nal.pone.0036863.g004 Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 7 May 2012 | Volume 7 | Issue 5 | e36863 STM2208 exp ession was locked in he ON s a e in bo h s ains (Fig. 5). Cons uc ion o s ain SV6401 hus pe mi ed o analyze he consequences o STM2209-STM2208 cons i u i e exp ession a oiding he pleio opic e ec s o dam mu a ions (see below). The STM2209 and STM2208 gene p oduc s a e p o eins loca ed in he inne (cy oplasmic) memb ane o Salmonella en e ica The subcellula loca ion o STM2209 and STM2208 was in es iga ed using 3xFLAG- agged a ian s. Elec opho e ic sep- a a ion o cell ac ions (cy osol, cy oplasmic memb ane and ou e memb ane) was pe o med, and Wes e n analysis o he sepa a ed p o ein p epa a ions was ca ied ou wi h a comme cial an i- FLAG an ibody. The esul s unambiguously showed ha STM2209 and STM2208 a e loca ed in he S. en e ica inne (cy oplasmic) memb ane (Fig. 7). E idence o in e ac ion be ween STM2209 and STM2208 in he Salmonella cy oplasmic memb ane STM2209 may ep esen a no el example o a memb ane pep ide, an eme ging class o unc ional molecules [49]. Because ce ain memb ane pep ides ha e been shown o in e ac wi h memb ane p o ein pa ne s, we in es iga ed whe he STM2209 in e ac s wi h he inne -memb ane p o ein STM2208. To es in e ac ion be ween STM2209 and STM2208 in i o, we used he Bac e ial Adenyla e Cyclase Two-Hyb id (BACTH) assay, a p ocedu e ha pe mi s he de ec ion o speci ic in e ac ions be ween inne memb ane p o eins [50]. STM2209 and STM2208 we e independen ly cloned on plasmids pUT18C and pKT25. Fou plasmid cons uc s we e ob ained (pUT18C- STM2209, pKT25-STM2209, pUT18C-STM2208, and pKT25- STM2208), and hei in e ac ion was es ed in an E. coli CyaA 2 mu an (BTH101). Func ional complemen a ion was de e mined by measu ing ß-galac osidase ac i i y. High le els o ß-galac osi- dase ac i i y we e ob ained wi h bo h plasmid pai s, compa ed wi h he basal ac i i ies o he plasmid ec o s o wi h he ac i i y o one usion p o ein only (Fig. 8). These esul s sugges ha STM2209 and STM2208 may in e ac indeed. Cons i u i e exp ession o STM2209-STM2208 educes P22 adso p ion o S. en e ica Du ing s ain cons uc ion expe imen s by P22 HT ansduc- ion, we ob ained educed numbe s o ansduc an s whene e he s ain ha cons i u i ely exp esses STM2209-STM2208 (SV6401) was used as a ecipien . This obse a ion, combined wi h he ac ha STM2209 and STM2208 a e componen s o he cell en elope, aised he possibili y ha cons i u i e syn hesis o STM2209 and STM2208 migh impai adso p ion o bac e io- phage P22. To es his hypo hesis, we compa ed he kine ics o P22 adso p ion o he wild ype s ain, o a s ain ha cons i u i ely exp esses STM2209-STM2208 (SV6401), and o a s ain ha ha bo s a dele ion o STM2209-STM2208 (SV6013). Suspensions o P22 bac e iophage and S. en e ica we e mixed, and samples we e aken e e y wo minu es, and cen i uged. The supe na an was subsequen ly i a ed o moni o he p esence o una ached phages (Fig. 9). Adso p ion o P22 o S. en e ica cells was ound o be se e ely impai ed in he s ain ha cons i u i ely exp essed STM2209-STM2208 (SV6401), which p o ed o be la gely e ac o y o phage P22 a achmen . In con as , P22 adso p ion emained unal e ed in a s ain ca ying a STM2209- STM2208 dele ion (SV6013) ega dless o he p esence o he mu a ed GATCs (SV6976). These expe imen s sugges ha phase a ia ion o STM2209-STM2208 may spli clonal popula ions o S. en e ica in o wo subpopula ions, one o which is P22-sensi i e while he o he is P22- esis an . Cons i u i e exp ession o STM2209-STM2208 al e s chain leng h dis ibu ion in he lipopolysaccha ide O-an igen o S. en e ica Because phage P22 is known o a a ch o he LPS o Salmonella en e ica o ini ia e in ec ion [51], we examined whe he he s ain ha cons i u i ely exp esses STM2209-STM2208 (SV6401) showed LPS al e a ions. Mig a ion o he LPS in polyac ylamide Figu e 5. Visual obse a ion o phase a ia ion on LB + X-gal pla es in s ains ca ying an STM2208 :: lac usion in di e en backg ounds. S ains in he uppe ow a e SV5679 (Dam + OxyR + ), SV5683 (Dam 2 OxyR + ), SV7031 (Dam + OxyR + mu . GATC) and SV7032 (Dam 2 OxyR + mu . GATC). OxyR 2 de i a i es (SV5989, SV5990, SV7232 and SV7233) a e shown in he lowe ow. doi:10.1371/jou nal.pone.0036863.g005 Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 8 May 2012 | Volume 7 | Issue 5 | e36863 gel is known o be a ec ed by he numbe and size o epea ing oligosaccha ide uni s in long-chain LPS, such ha bands in he p o ile ep esen p og essi ely la ge conca eme s o he epea ing oligosaccha ide uni s [52]. Compa ison o he LPS p o iles in s ain SV6401 and he wild ype e ealed d as ic al e a ions in he leng h o O-an igen chains (Fig. 10). Wild ype Salmonella LPS shows a bimodal dis ibu ion ypical o many En e obac e iaceae, wi h highe amoun s o bands wi h 16–35 and .100 epea s [40,53–55]. S ain SV6401 showed a unimodal dis ibu ion, wi h bands concen a ed in he 3–8 epea ange. This sho and homogeneous LPS migh well explain educed phage P22 a achmen . No al e a ion o he LPS p o ile was de ec ed in a s ain ca ying a STM2209-STM2208 dele ion (SV6013), in ag eemen wi h i s abili y o pe mi a no mal le el o P22 adso p ion (Fig. 9). The main conclusion om hese expe imen s was ha exp ession o STM2209-STM2208 al e s O-an igen chain leng h. Cons i u i e exp ession o STM2209-STM2208 educes S. en e ica esis ance o guinea pig se um O-an igen chain leng h has been desc ibed o be c ucial o se um esis ance in Salmonella [54,56–59]. Su i al in se um was analyzed by ea ing exponen ially g owing cells wi h 30% non- immune guinea pig se um. Cons i u i e exp ession o STM2209- STM2208 caused inc eased killing by se um (Fig. 11). This is likely o be complemen -media ed, since hea -inac i a ed se um did no impai g ow h o s ain SV6401 (da a no shown). Cons i u i e exp ession o STM2209-STM2208 educes S. en e ica p oli e a ion in mac ophages Addi ional sc eens and pheno ypic assays we e pe o med in sea ch o unc ions o STM2209-STM2208 phase a ia ion besides he o ma ion o a P22- esis an subpopula ion wi h educed esis ance o se um. The ials included: (i) g ow h in a ious media a di e en empe a u es and di e en osmola i ies; (ii) esis ance o acidic pH, ca ionic pep ides, bile, and hyd ogen pe oxide; (iii) mo ili y; (i ) bio ilm o ma ion; ( ) and in asion o and p oli e a ion in epi helial and mac ophage cell lines. Mos ials did no show di e ences associa ed ei he o loss o cons i u i e exp ession o STM2209-STM2208. A ema kable excep ion was ha cons i u i e exp ession o STM2209-STM2208 impai ed in acellula p oli e a ion wi hin mac ophages (Fig. 12). On he o he hand, a s ain ca ying a STM2209-STM2208 dele ion showed in amac ophage p oli e a ion a a le el simila le el o ha o he wild- ype s ain. These obse a ions sugges ha ep ession o STM2209-STM2208 exp ession may be equi ed o pe mi Salmonella p oli e a ion wi hin mac ophages. Howe e , a nonp oli e a ing S. en e ica popula ion may be also gene a ed by swi ching STM2209-STM2208 o he ON s a e. Discussion STM2209 and STM2208, hi he o anno a ed as pu a i e genes o unknown unc ion in he genome o Salmonella en e ica se o a Typhimu ium, a e absen in Salmonella bongo i and in o he species o en e ic bac e ia (Fig. 1). This asso men , combined wi h G+C con en lowe han he co e Salmonella genome (38% s. 52%, app oxima ely), sugges s acquisi ion by ho izon al ans e . STM2209 and STM2208 a e pa o a single ansc ip ional uni , and a e ansc ibed om a p omo e ups eam STM2209 (Fig. 3). The STM2209 gene p oduc is a small hyd ophobic pep ide (pu a i ely, 34 amino acids) while STM2208 encodes a la ge hyd ophobic p o ein (pu a i ely, 221 amino acids). Bo h Figu e 6. E ec o GATC mu a ions on STM2209-STM2208 exp ession. A. Diag am o he p omo e egion o STM2209- STM2208, showing GATC si es ( ed squa es), pu a i e OxyR-binding- si es (o ange ba s), pu a i e 235 and 210 modules (g een boxes) and he ansc ip ion ini a ion si e (black a ow). B. E ec o elimina ing he 4 GATC si es ups eam he STM2209-STM2208 p omo e on STM2209- STM2208 exp ession, moni o ed by compa ing he ß-galac osidase ac i i y o a ansla ional STM2208::lac usion in Dam + and Dam 2 backg ounds (whi e and black his og ams, espec i ely). Values a e a e ages and s anda d de ia ions om 6 independen expe imen s. C. E ec o elimina ing he 4 GATC si es ups eam he STM2209-STM2208 p omo e on STM2209-STM2208 exp ession, moni o ed by Wes e n blo analysis o STM2208-3xFLAG le els in di e en backg ounds. doi:10.1371/jou nal.pone.0036863.g006 Figu e 7. Dis ibu ion o STM2209 and STM2208 p o eins agged wi h a 3xFLAG epi ope in subcellula ac ions o S. en e ica se o a Typhimu ium. An i-FLAG Wes e n hyb idiza ion is shown o h ee ac ions: cy oplasm, inne memb ane, and ou e memb ane. The olume loaded o all ac ions was no malized o he same numbe o bac e ia (7610 7 c. .u.). doi:10.1371/jou nal.pone.0036863.g007 Phase Va ia ion o STM2209-STM2008 in Salmonella PLoS ONE | www.plosone.o g 9 May 2012 | Volume 7 | Issue 5 | e36863