The indole pulse: A new perspective on indole signalling in Escherichia coli

The Indole Pulse: A New Pe spec i e on Indole Signalling in Esche ichia coli Hannah Gaims e 1 *, Jehangi Cama 2 , Sil ia He na ´ndez-Ainsa 2 , Ul ich F. Keyse 2 , Da id K. Summe s 1 1Depa men o Gene ics, Uni e si y o Camb idge, Camb idge, Uni ed Kingdom, 2Ca endish Labo a o y, Uni e si y o Camb idge, Camb idge, Uni ed Kingdom Abs ac Indole has di e se signalling oles, including modula ion o bio ilm o ma ion, i ulence and s ess esponses. Changes a e induced by indole concen a ions o 0.5–1.0 mM, simila o hose ound in he supe na an o Esche ichia coli s a iona y phase cul u e. He e we desc ibe an al e na i e mode o indole signalling ha p omo es he su i al o E. coli cells du ing long- e m s a iona y phase. A mu an ha has los he abili y o p oduce indole demons a es educed su i al unde hese condi ions. Signi ican ly, he addi ion o 1 mM indole o he cul u e supe na an is insu icien o es o e long- e m su i al o he mu an . We p o ide e idence ha he pe inen signal in his case is no 1 mM indole in he cul u e supe na an bu a ansien pulse o in a-cellula indole a he ansi ion om exponen ial g ow h o s a iona y phase. Du ing his pulse he cell-associa ed indole eaches a maximum o app oxima ely 60 mM. We a gue ha his is su icien o inhibi g ow h and di ision by an ionopho e-based mechanism and causes he cells o en e s a iona y phase be o e esou ces a e exhaus ed. The unused esou ces a e used o epai and main ain cells du ing he ex ended pe iod o s a a ion. Ci a ion: Gaims e H, Cama J, He na ´ndez-Ainsa S, Keyse UF, Summe s DK (2014) The Indole Pulse: A New Pe spec i e on Indole Signalling in Esche ichia coli. PLoS ONE 9(4): e93168. doi:10.1371/jou nal.pone.0093168 Edi o : Finba Hayes, Uni e si y o Manches e , Uni ed Kingdom Recei ed No embe 14, 2013; Accep ed Feb ua y 28, 2014; Published Ap il 2, 2014 Copy igh : ß2014 Gaims e 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: HG was unded by a BBSRC DTG s uden ship, h p://www.bbs c.ac.uk/home/home.aspx, g an numbe PCAG-EJNF. JC, SHA and UFK acknowledge suppo om an ERC s a ing g an , h p://e c.eu opa.eu/, g an numbe 261101 Passmemb ane. JC is also suppo ed by an In e nal G adua e S uden ship om T ini y College, Camb idge, h p://www. in.cam.ac.uk/. 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 Indole is a signalling molecule sec e ed by o e 85 species o bac e ia including Esche ichia coli [1]. I is p oduced by he enzyme yp ophanase (TnaA) ha con e s yp ophan in o indole, py u a e, and ammonia [2]. E. coli makes e y li le indole du ing exponen ial g ow h. Howe e yp ophanase exp ession is s ongly up- egula ed by he s a iona y phase sigma ac o RpoS [3], so indole p oduc ion ises as cells app oach s a iona y phase [4]. The inal concen a ion o indole in s a iona y phase cul u e depends upon he amoun o yp ophan in he g ow h medium. In LB medium ee yp ophan can ange be ween 0.5–1 mM, and supe na an concen a ions o indole also ypically each 0.5– 1 mM [5]. Indole has di e se signalling oles including modula ion o bio ilm o ma ion, i ulence and s ess esponses [6–8]. I can also ac as an in e kingdom signal and has been shown o a ec gene exp ession in human en e ocy es [9] and memb ane po en ial in mi ochond ia [10]. In each o hese cases he sys em is esponding o ela i ely low (,1 mM), pe sis en concen a ions o indole. Recen wo k has e ealed a ole o indole in plasmid s abili y. Dime isa ion o he mul icopy plasmid ColE1 is a well-cha ac- e ised cause o plasmid ins abili y [11]. The accumula ion o plasmid dime s in a cell igge s he syn hesis o a 70n egula o y RNA, Rcd, encoded wi hin he plasmid ce si e [12–13]. Rcd exp ession leads o inhibi ion o cell di ision and i is sugges ed ha he ac i a ion o his checkpoin gi es su icien ime o he dime s o be con e ed o monome s by si e-speci ic ecombina- ion a he plasmid ce si e [11]. The a ge o Rcd is yp ophanase [4] and Rcd binding educes i s K m o yp ophan, s imula ing indole p oduc ion. Since he addi ion o indole o he cul u e medium o g owing cells was shown e e sibly o a es cell di ision [10], i was p oposed ha indole p oduc ion by Rcd-ac i a ed yp ophanase was he mechanism by which he Rcd checkpoin blocks cell di ision. Indole has been shown o a es bac e ial g ow h and cell di ision by ac ing as an ionopho e and making he cy oplasmic memb ane pe meable o hyd ogen ions [10]. The consequen loss o memb ane po en ial p e en s he unc ion o he MinCDE sys em and localiza ion o he F sZ ing ha is equi ed o cell di ision. Howe e , while his e ec is seen a indole concen a ions o 3–5 mM, he maximum concen a ion o indole in a cul u e supe na an cul u e is ypically 0.5–1 mM. Unsu p isingly, he e o e, i has been unclea whe he he e ec s o 3–5 mM indole upon bac e ial cells a e biologically ele an . He e we epo a no el mode o indole signalling du ing E. coli s a iona y phase en y ha is impo an o iabili y in long- e m s a iona y phase. We show ha du ing he ansi ion om exponen ial o s a iona y phase cells ansien ly expe ience a e y high (.50 mM) concen a ion o indole and ha his pulse is necessa y o long- e m s a iona y phase iabili y. This i s example o a ‘‘pulse signalling’’ mechanism expands he epe oi e o indole e ec s on bac e ia o include ionopho e-media ed e ec s on cell di ision and g ow h. We specula e ha i may p o e o be o widesp ead signi icance. PLOS ONE | www.plosone.o g 1 Ap il 2014 | Volume 9 | Issue 4 | e93168 Ma e ials and Me hods S ains and cul u e condi ions E. coli BW25113, W3110, BW25113 D naA (Kanamcyin esis an : Km R ) and W3110 D naA we e ob ained om he Keio collec ion [14]. Cells we e cul u ed ou inely in Lu ia Be ani (LB) medium a 37uC, wi h shaking a 120 pm. O e nigh cul u es we e dilu ed o OD 600 = 0.05 and allowed o g ow o 2 hou s be o e samples we e emo ed o subsequen assays. Whe e equi ed, indole (dissol ed in e hanol) was added; e hanol alone was added o con ols whe e app op ia e. To assess colony o ming uni s (CFU), cul u es we e dilu ed app op ia ely and sp ead on o LA pla es. These we e incuba ed a 37uC and colonies coun ed he ollowing day. Mixed cul u e expe imen O e nigh cul u es o BW25113 and BW25113 D naA (Km R ) cells we e dilu ed in o esh LB medium, in iplica e, o an OD 600 o 0.05 a an ini ial a io o 99:1 (BW25113 cells o BW25113 D naA). These cul u es we e g own a 37uC, wi h shaking, o 24 hou s. A 0, 2, 4, 6, 8, and 24 hou s he samples we e dilu ed app op ia ely and we e pla ed on o bo h LA and LA +kanamycin pla es. This allowed he p opo ion o D naA cells in he popula ion o be de e mined. Ko acs Assay Ko acs assay is a commonly-used echnique in he ield which gi es consis en esul s in di e en labo a o ies [4–5]. The cell associa ed indole concen a ions p esen ed in his epo ( o a maximum o 60 mM) a e much highe han cul u e medium concen a ions measu ed p e iously using he Ko acs assay. Howe e , hese a e calcula ed cell-associa ed alues. The ‘‘ aw’’ assay unde pinning he calcula ion is in he con en ional, linea ange o he assay. To assay indole in cul u e supe na an s, a sample (1 ml) om a g owing cul u e was emo ed, he OD 600 measu ed and cells ha es ed by cen i uga ion a 11337 x g o 15 seconds (Eppendo Minispin mic o uge). The supe na an was emo ed and assayed: 300 ml o Ko acs Reagen (10 g o p-dime hylamino- benzaldehyde dissol ed in a mix u e o 50 ml o HCl and 150 ml o amyl alcohol) was added o he supe na an and incuba ed o 2 minu es. A 50 ml po ion was emo ed and added o 1 ml o HCl- amyl alcohol solu ion (75 ml o HCl and 225 ml o amyl alcohol). The abso bance a 540 nm was measu ed (Gene Quan 1300, GE Spec opho ome e ). The concen a ion o indole in he supe na- an was calcula ed using a calib a ion cu e. To assay indole wi hin he cell pelle , a modi ied me hod was used. A sample (1 ml) om a g owing cul u e was emo ed, he OD 600 measu ed and cells ha es ed by cen i uga ion a 11337 x g o 15 seconds. The supe na an was disca ded and he cell pelle assayed: 300 ml o Ko acs Reagen was added o he cell pelle o 2 minu es. This lysed he cells and allowed he Ko acs eagen o eac wi h indole. This mix u e was pipe ed in o 1 ml LB be o e a 50 ml po ion was emo ed and added o 1 ml o HCl-amyl alcohol solu ion. The abso bance a 540 nm was measu ed (Gene Quan 1300, GE Spec opho ome e ). The concen a ion o indole was calcula ed using a calib a ion cu e. The samples used o gene a e he calib a ion cu e con ained an app op ia e densi y o indole non-p oducing cells as well as indole a a known concen a ion. In addi ion, he blank o he assay was gene a ed om cul u e medium con aining cells bu no added indole. The e o e, any backg ound due o cell deb is is aken in o accoun . Using he concen a ion o indole ob ained om he calib a ion cu e allowed us o de e mine he numbe o moles o indole p esen in he cell pelle (Ip). Using he OD 600 o he cul u e, we can calcula e he o al bac e ial cell olume (Vc) con ained in he pelle [15] and hence he appa en cell associa ed indole concen a ion (C A )as shown in equa ion 1. CA~Ip Vc ð1Þ Calcula ions o he appa en cell associa ed indole concen a- ions based on cell pelle assays may o e es ima e he indole concen a ion. This is due o he inclusion (in he Ip e m) o indole dissol ed in he cul u e supe na an and apped in he cell pelle . The calcula ion ou lined below allows us o co ec o his e o . Vp~VczVs~Vcz1 2Vc~3 2Vcð2Þ Equa ion 2 s a es ha ha he olume o he pelle is equal o he sum o he olume o he apped supe na an and he olume o he cells. Re e ence 16 s a es ha in a cell pelle , app oxima ely 1/3 o he pelle will co espond o apped supe na an . Cp~Ip Vp ~2 3|Ip Vc ~2 3CAð3Þ Cp~Cc|RVczCs|RVs~2 3Ccz1 3Csð4Þ whe e C A is he appa en cell associa ed indole concen a ion in he pelle , I p is he numbe o moles o indole in he pelle , V c is he olume o he cells, V p is he olume o he pelle and V s is he olume o he supe na an , C p is he concen a ion o indole in he pelle , C c is he concen a ion o indole in he cells and C s is he concen a ion o indole in he apped supe na an , R Vp is he olume ac ion o he cells in he pelle (R Vp =2 3) and R Vs is he olume ac ion o he supe na an in he pelle (R Vs =1 3), aken om [16]. Fo example, whe e C A = 60 mM and C s = 4 mM, so by using Equa ions 3 and 4 we can es ima e C c = 58 mM. UV Abso bance Assay o Indole Concen a ion Abso bance measu emen s we e ca ied ou using a Va ian Ca y 300 Bio UV-VIS Spec opho ome e . Unless o he wise s a ed, chemicals and media we e ob ained om Sigma Ald ich UK. W3110 D naA cells we e cul u ed o e nigh a 37uC a 250 pm in shake lasks con aining 30 ml LB medium con aining kanamycin (30 mgml 21 ). Cells we e washed by cen i uging he cul u e o 10 min a app oxima ely 2685 x g (4,000 pm in a Ha ie 18/80 Re ige a ed Cen i uge), emo ing he supe na an and esuspending he pelle in phospha e bu e ed saline (PBS; p epa ed by dissol ing PBS able s in an app op ia e olume o deionized (Milli-Q) wa e ; his was hen au ocla ed and s e ile il e ed (0.22 mm, Millipo e) p io o use). A e washing he cells we e again ha es ed by cen i uga ion and e-suspended in he desi ed amoun o PBS. The OD 600 o he cul u es was de e mined bo h be o e washing (in LB) and a e washing (in PBS). Indole Pulse Signalling PLOS ONE | www.plosone.o g 2 Ap il 2014 | Volume 9 | Issue 4 | e93168 One ml aliquo s o cells in PBS we e ans e ed o Eppendo ubes (Axygen, 1.5 ml MaxyClea Mic o ubes). App op ia e amoun s o 75 mM indole s ock solu ion (in absolu e e hanol) we e added o ob ain he desi ed supe na an concen a ions. A con ol wi hou indole was also p epa ed. A e indole addi ion, he samples we e o exed and he cells we e pelle ed by cen i uga ion o 5 minu es a 3500 g (6,000 pm in a The mo Scien i ic He aeus F esco 17 Cen i uge). The supe na an s ( e e ed o as ‘bac e ial supe na an s’) we e dilu ed 1:20 in PBS and hei abso bance alues measu ed in he 250–300 nm ange. The dilu ion was equi ed o he abso bance alues o lie wi hin he linea ange o he spec opho ome e . The indole peak abso bance a 268 nm was de e mined o all he samples. Backg ound sub ac ion was applied using he con ol. Sepa a ely, abso bance cu es we e measu ed in he 250–300 nm ange o indole solu ions (0–2.5 mM) p epa ed in PBS (a e dilu ing 1:20 in PBS simila o he s eps desc ibed abo e). The indole abso bance peak a 268 nm was measu ed, backg ound sub ac ion was pe o med using he ‘no-indole’ sample and he peak abso bance alues we e used o plo a calib a ion cu e o Abso bance s Ex e nal Indole Concen a ion. Using he abo e calib a ion cu e and he abso bance alue measu ed om he bac e ial supe na an , he indole concen a ion o he bac e ial supe na an was de e mined. Sub ac ing his om he known ex e nal concen a ion allows us o de e mine he numbe o moles o indole p esen in he bac e ial pelle . Using he OD 600 ob ained (in PBS), we can calcula e he o al bac e ial cell olume con ained in he pelle [15] and hence he appa en cell associa ed indole concen a ion. De e mina ion o he E. coli lipids-bu e pa i ion coe icien o indole E.coli o al lipid ex ac (A an i Pola Lipids) dissol ed in chlo o o m was added o a 5 ml ial. The o ganic sol en was emo ed by high- acuum d ying o 3–4 hou s o gi e a 5 mg lipid ilm. Solu ion A (5 mM indole, Sigma Ald ich, in 15 mM phospha e bu e (PB; pH = 7)) was p epa ed by adding he app op ia e olume o a s ock solu ion o indole in e hanol o he 15 mM PB bu e . The lipid ilm was dispe sed by adding 3.5 ml o solu ion A and sonica ing using an ul asonic ba h (G an , MXB6, 160W) o a ound 2 hou s a 25uC. In o de o sepa a e he lipid and aqueous phases, samples we e cen i uged wi h a Beckman Coul e -Op ima max XP Ul acen i uge a 50,000 x g o 45 min a 25uC. A e ul acen i uga ion, lipids appea ed as a pelle a he bo om o he cen i uga ion ubes. The aqueous phase was aken ca e ully wi h a Pas eu pipe e and kep in an Eppendo ube o he spec opho ome ic measu emen s. The E. coli lipids-bu e pa i ion coe icien o indole was de e mined using a Va ian Ca y 300 Bio UV-VIS Spec opho- ome e . A calib a ion cu e o abso bance s indole concen a ion a 268 nm was ini ially ob ained by p epa ing indole solu ions in he 15 mM PB bu e in a ange o concen a ions o 0–0.2 mM (linea ange). The indole concen a ion o he aqueous phase was de e mined by measu ing he abso p ion a 268 nm, no malising he alues wi h espec o he ‘no-indole’ and using he calib a ion cu e o es ima e he concen a ion. No-indole samples con aining he same quan i y o lipids (5 mg) dispe sed in PB bu e bu lacking indole, we e submi ed o he same ul acen i uga ion and sepa a ion p ocess. The concen a ion o indole in he lipid phase was calcula ed by sub ac ing he amoun o indole in he aqueous phase om he o al amoun p esen in he ini ial solu ion A. The pa i ion coe icien (P) was hen calcula ed using equa ion [5] [17] whe e C i is he concen a ion o indole in solu ion A, C w is he concen a ion o indole in he aqueous phase, w w is he weigh o he aqueous phase (3500 mg) and w l he weigh o he lipid phase (5 mg) in he samples. P~Ci{Cw Cw |ww wl ð5Þ Es ima ion o he olume o he lipid memb ane in E. coli The olume o he lipid memb ane in E.coli (0.04 mm 3 ) has been de e mined using equa ion [6] and applying simple geome ical calcula ions. In hese calcula ions, E. coli is assumed o ha e a cylind ical shape. lis he leng h o E.coli (1.6 mm) [15], is he es ima ed adius o he cylinde (0.55 mm) and dis he hickness o he lipid bilaye (a ound 5nm) [18]. Volume lipid memb ane~2pl ðÞz2p 2  |dð6Þ The olume o he en i e E. coli is 1.5 mm 3 , so he lipid memb ane ep esen s 1/40 h o he o al cell olume. Es ima ion o he molecula a io indole:lipid and he weigh pe cen age indole:lipid The es ima ion o he molecula a io indole:lipid (0.460.1) has been made using Equa ion [8] (simpli ied om Equa ion [7]) whe e dis he densi y o he lipids (1000 g L 21 ), V lipid is he olume o he lipid memb ane in a single E. coli (0.04 mm 3 ), M lipid is he molecula weigh o E. coli o al ex ac lipids (811.5 g mol 21 ), N A is he A ogad o cons an (6.023610 23 ), C indole is he concen a ion o indole added o he supe na an (0.005 mol L 21 ) and P is he E. coli lipids-bu e pa i ion coe icien (92.9624.5). Numbe indole molecules Numbe lipid molecules ~P|Cindole|Vlipid |NA d|Vlipid |1 Mlipid  NA ð7Þ Numbe indole molecules Numbe lipid molecules ~P|Cindole|Mlipid dð8Þ The pe cen age in weigh indole:lipid has been calcula ed mul iplying Equa ion [4] by he a io o molecula weigh s o indole:lipid, namely (117.2:811.5). Resul s Indole p oduc ion inc eases apidly du ing en y in o s a iona y phase The concen a ion o indole in he supe na an o an L-b o h cul u e o E. coli BW25113 was measu ed using he Ko acs assay (Fig. 1). Consis en wi h epo s elsewhe e [5], he supe na an concen a ion eached a maximum o 0.7–0.8 mM in s a iona y phase. The concen a ion was low in la e exponen ial phase bu ose apidly when he cul u e eached an op ical densi y (OD 600 ) Indole Pulse Signalling PLOS ONE | www.plosone.o g 3 Ap il 2014 | Volume 9 | Issue 4 | e93168 o 1.0–1.5. This co esponded o he pe iod o ansi ion be ween exponen ial g ow h and s a iona y phase. The apid accumula ion o indole in he supe na an (5- old inc ease o e 30 min) e lec ed an inc eased p oduc ion a e pe cell a he han simply an inc ease in cell numbe s, since he OD 600 o he cul u e inc eased only 1.4- old du ing his pe iod. Indole p oduc ion is equi ed o long- e m s a iona y phase iabili y In o de o assess he unc ional signi icance o indole p oduc ion du ing he onse o s a iona y phase, we compa ed he g ow h and iabili y o BW25113 and BW25113 D naA (a yp ophanase knock-ou ha p oduces no indole) o e 10 days a 37uC. Fo he i s h ee days he densi y o BW25113 cul u es was signi ican ly lowe han BW25113 D naA, implying ha indole p oduc ion inhibi s g ow h du ing s a iona y phase en y. How- e e o e he subsequen 7 days he OD 600 o he mu an cul u e declined, while he densi y o he wild- ype con inued slowly o inc ease (Fig. 2A). Consequen ly, a e 10 days in s a iona y phase, i was he wild- ype cul u e ha had he highe op ical densi y. Op ical densi y measu emen s p o ide an es ima e o he numbe o cells in a cul u e bu no whe he hese cells a e ali e o dead. To compa e he iabili ies o cells in TnaA + and TnaA 2 cul u es, samples we e dilu ed and sp ead on L-aga pla es o es ima e iable coun s (Fig. 2B). We obse ed ha o e 9 days in s a iona y phase he colony o ming uni s (CFU) in he mu an cul u e dec eased by app oxima ely 75%, compa ed o he wild- ype cul u e whe e CFU dec eased by less han 30%. One possible in e p e a ion o hese da a is ha indole p oduc ion causes g ow h o he wild- ype cul u e o slow be o e esou ces a e exhaus ed and he a ailabili y o unused esou ce assis s long- e m su i al in s a iona y phase. In an a emp o es o e he s a iona y phase iabili y o he D naA mu an s ain, indole (1 mM) was added o he cul u e medium when he OD 600 eached 1.5. This mimicked he apid accumula ion o indole in he cul u e supe na an o wild- ype cells a his poin . Howe e , he addi ion o 1 mM indole had no e ec on ei he he cul u e densi y o he long- e m iabili y o he mu an (Fig. 2). The ailu e o 1 mM indole o es o e he long- e m iabili y o he D naA mu an was ini ially su p ising. Howe e , indole p oduc ion by wild- ype cells du ing s a iona y phase en y appea s o slow hei g ow h (Fig. 2A), and indole concen a ions o below 2–3 mM a e known o ha e li le o no e ec on g ow h [4]. A signi ican ly highe (4–5 mM) indole concen a ion is equi ed o inhibi g ow h o cell di ision. Because indole p oduc ion du ing s a iona y phase en y is e y apid, i is possible ha a highe concen a ion o indole exis s inside he cells du ing he p oduc ion pe iod. I a su icien ly high concen a ion we e eached, his migh explain he slowing o g ow h obse ed o he wild- ype s ain. To es he p oposal ha i is he in e nal a he han he ex e nal concen a ion o indole which de e mines he kine ics o en y in o s a iona y phase, a mixed cul u e expe imen was pe o med. The cul u e con ained bo h wild- ype and D naA mu an cells, a an ini ial a io 99:1. This allowed us o obse e he beha iou o D naA cells in he p esence o le els o indole ha occu in he wild- ype cul u e supe na an . The e is a clea p edic ion ha i he kine ics o s a iona y phase en y a e a ec ed by he ex e nal le el o indole, hen all he cells in he mixed cul u e should en e s a iona y phase a he same ime and he p opo ion o he D naA mu an cells should emain cons an . Howe e i an ele a ed in e nal le el o indole in he wild- ype p oduce cells egula es s a iona y phase en y hen he wild- ype cells will en e s a iona y phase ea lie and he D naA mu an cells will inc ease hei p opo ion in he popula ion. Samples we e aken om he mixed cul u e a egula in e als and he p opo ion o mu an cells was measu ed. The p opo ion inc eased om 0.760.4% when he cul u es we e se up, o 4.460.2% a 24 hou s. This co esponds o a 6- old inc ease in he p opo ion o mu an cells in 24 hou s. Fu he mo e, he g ea es inc ease in he p opo ion o mu an cells (a 4- old inc ease) occu ed be ween 6 and 8 hou s and was he e o e a e indole was p oduced by he wild- ype cells. This esul sugges s ha esou ces unused by wild- ype cells emained in he supe na an whe e hey we e a ailable o use by he mu an s. Cell associa ed indole inc eases apidly be o e s a iona y phase To es he possibili y ha a high in acellula indole concen- a ion exis s du ing s a iona y phase en y, we used he Ko acs assay o measu e appa en cell-associa ed indole. Cul u e samples we e cen i uged, he supe na an emo ed and he cell pelle lysed by he addi ion o Ko acs eagen . The concen a ion o indole in he lysa e was measu ed by he Ko acs assay and was con e ed o he appa en cell-associa ed concen a ion using an es ima e o E. coli cell olume [15]. We use he e m ‘appa en ’ o acknowledge ha he concen a ion has no , a his s age, been co ec ed o any con ibu ion om indole in he cul u e supe na an ha is apped in he cell pelle . The esul showed ha appa en cell-associa ed indole inc eased sha ply bu ansien ly du ing s a iona y phase en y, eaching a maximum o 60 mM (Fig. 3). Also signi ican was he obse a ion ha he appa en cell-associa ed indole concen- a ion ne e d opped below 15 mM and was always subs an ially highe han he supe na an concen a ion (Fig. 1). The e ec s o de ined concen a ions o indole added o he cul u e medium on he g ow h and di ision o E. coli a e well es ablished bu his is no ue o cell-associa ed indole. To help Figu e 1. Indole is p oduced apidly o e a 30 minu e pe iod du ing he onse o s a iona y phase. A cul u e o g owing BW25113 cells was sampled egula ly. The OD 600 was measu ed and he samples we e cen i uged o emo e cells, and he supe na an was assayed o indole using Ko acs assay. Da a shown a e he mean alues 6s anda d de ia ion o h ee independen epea s. doi:10.1371/jou nal.pone.0093168.g001 Indole Pulse Signalling PLOS ONE | www.plosone.o g 4 Ap il 2014 | Volume 9 | Issue 4 | e93168 in e p e he da a o Fig. 3, known concen a ions o indole (0– 5 mM) we e added o s a iona y phase cul u es o a D naA mu an s ain. Cells we e ha es ed and he pelle assayed o appa en cell-associa ed indole (Fig. 4). A supe na an concen a ion o 0.75 mM ( ypical o s a iona y phase cul u es o wild- ype cells) ga e an appa en cell-associa ed concen a ion o app ox. 20 mM, while an appa en cell associa ed concen a ion o 60 mM ( he maximum de ec ed du ing s a iona y phase en y) esul ed om a supe na an concen a ion o app ox. 4 mM. The appa en cell associa ed indole concen a ion can be co ec ed o he con ibu ion o indole om he cul u e supe na an . I has p e iously been es ima ed ha a cell pelle is likely o con ain app oxima ely 30% cul u e supe na an in addi ion o he cells [16]. We ha e desc ibed in he ma e ial and me hods a calcula ion o compensa e o he con ibu ion o he ex acellula indole in he pelle . In Fig. 4, we see ha 4 mM indole in he cul u e medium ga e an appa en cell associa ed indole o 60 mM. Applying he co ec ion, he ac ual cell associa ed indole concen a ion is shown o be 58 mM a he han 60 mM. Thus, he con ibu ion o indole om apped supe na an o ou appa en cell associa ed indole concen a ions is negligible and well wi hin he expe imen al e o s o ou assay. To con i m ha he high concen a ion o cell-associa ed indole was no an a e ac o he Ko acs assay, he ela ionship be ween cell-associa ed and supe na an indole was in es iga ed by an independen echnique. Indole non-p oducing (TnaA 2 ) cells we e suspended in medium con aining a known concen a ion o indole and hen ha es ed by cen i uga ion. The amoun o indole emo ed om he medium by he cells was es ima ed by using an UV abso bance assay o measu e he indole concen a ion in he medium be o e and a e ha es ing (see Ma e ials and Me hods o de ails). When he indole concen a ion in he medium was 1 mM, a cell-associa ed concen a ion o 763 mM was calcula ed (8 epea s). This compa es wi h a alue o 1763 mM when cell-associa ed indole was measu ed using he Ko acs assay (Fig. 4). The di e ence be ween he wo alues may be a sys ema ic e o due o p ocedu al di e ences be ween he assays, howe e hey a e s ill wi hin an o de o magni ude o each o he and hey help con i m ha he high cell associa ed alues a e no simply an a e ac o he Ko acs assay. Indole dis ibu ion wi hin cells Appa en cell-associa ed indole is an a e age o he indole concen a ions in he cy oplasm and in he lipid memb anes. To de e mine he ela i e indole a ini y o hese wo cell componen s we measu ed he pa i ion coe icien (P) o indole be ween wa e and E. coli o al lipid. We ob ained a alue o log (P) = 1.9560.12 (n = 9), which is simila o he calcula ed alue o 2.17 o oc anol- wa e pa i ioning epo ed in [19]. Thus indole has an app ox. 90- old highe a ini y o lipid han wa e , due o he hyd opho- bici y o he a oma ic ing o he molecule. Discussion The e ec s o low (0.5 –1.0 mM) concen a ions o indole on bac e ial physiology a e well es ablished [1]. In addi ion, ecen s udies ha e shown ha a highe (4–5 mM) concen a ions, indole can egula e bac e ial g ow h and di ision h ough i s ac ion as a p o on ionopho e [10]. Howe e , he biological ele ance o hese e ec s has been open o ques ion since concen a ions abo e 1 mM a e no seen in cul u e supe na an s. In his epo we desc ibe a mode o indole signalling ha is independen o he supe na an concen a ion, bu is d i en by a ansien , high concen a ion o indole inside he cell. A cul u e o wild- ype E. coli makes he ansi ion om exponen ial o s a iona y phase ea lie han a cul u e o an indole non-p oducing mu an . The wild- ype also displays highe iabili y in long- e m s a iona y phase, and a likely explana ion is ha Figu e 2. Non indole p oducing mu an s ini ially g ow o a highe densi y and a e mo e iable han indole p oducing coun e pa s, bu a e signi ican ly less iable in he long e m. The densi y (OD 600 : A) and iabili y (CFU 610 26 / ml :B) o wild- ype, mu an and mu an wi h 1 mM indole added we e assessed o e 10 days. Da a shown a e he mean alues 6s anda d de ia ion o h ee independen epea s. doi:10.1371/jou nal.pone.0093168.g002 Indole Pulse Signalling PLOS ONE | www.plosone.o g 5 Ap il 2014 | Volume 9 | Issue 4 | e93168 esou ces unused o g ow h emain a ailable o he wild- ype o epai and main enance du ing he pe iod o s a a ion as sugges ed by he mixed cul u e esul . We we e unable o es o e wild- ype beha iou o he mu an by supplemen ing he cul u e medium wi h 1 mM indole. This is pe haps unsu p ising when one emembe s ha 1 mM indole has no de ec able e ec upon ei he g ow h o di ision o E. coli [4]. The indole supplemen a ion expe imen was designed so ha indole was added o he cul u e a he same ime, and wi h he same kine ics, as i appea s in wild- ype cul u e (Fig. 1). The only di e ence be ween he indole- supplemen ed mu an cells and wild- ype cells was ha he wild- ype cells expe ienced a ansien cell-associa ed indole concen- a ion o 60 mM (Fig. 3). We he e o e conclude ha he subsequen di e ence in g ow h and iabili y o he wo cul u es was due o he p esence o absence o his pulse o cell-associa ed indole. This dis inc ion be ween he e ec s o ex e nal and cell- associa ed indole is u he demons a ed in he mixed cul u e expe imen . He e, mu an cells ha did no expe ience he indole pulse en e ed s a iona y phase la e han co-cul u ed wild- ype cells. The 6- old inc ease in indole non-p oducing cells in he mixed cul u e expe imen also sugges s ha esou ces unused by wild- ype cells emain in he cul u e medium and a e a ailable o use by o he bac e ia. This seems a li le su p ising since he ese e is hen a ailable o use by ‘‘chea s’’ ha con inue o g ow un il he esou ce is exhaus ed. I his is ue, we would p edic ha du ing long- e m s a iona y phase he mu an cells would main ain hei ele a ed p opo ion in he mixed cul u e bu ha bo h mu an and wild- ype cells would die a he highe a e exhibi ed by mu an cells in pu e cul u e. The pulse is well de ined and las s app ox. 20 min (Fig. 3). We sugges ha his co esponds o a pe iod when indole is being p oduced as e han i can escape om he cell by di usion h ough he memb ane. The s a o he pulse can hen be explained by he RpoS-induced up- egula ion o yp ophanase exp ession as s a iona y phase app oaches [3]. The end o he pulse is mo e di icul o explain since yp ophanase is p esumably s ill p esen . Howe e he o al amoun o indole p oduced by a cul u e is limi ed by he amoun o ee yp ophan in he g ow h medium [5], so he end o he pulse p obably e lec s he ime when all a ailable yp ophan has been con e ed o indole. I is impo an o no e ha he pulse du a ion o 20 min is an a e age o all cells in he cul u e. I he e is signi ican popula ion he e ogenei y in he ime o pulsing, he pulse du a ion in indi idual cells may be sho e and he maximum concen a ion highe han he alues we p esen he e. In e p e a ion o ou da a is complica ed by he ac ha E. coli cells, by i ue o hei lipid memb anes (and possibly o he cell componen s), ha e a g ea e a ini y o indole han hei aqueous su oundings. This is why cell-associa ed concen a ions a e always highe han supe na an concen a ions, no jus du ing he indole pulse. Since in p e ious epo s he e ec s o indole on he g ow h and di ision o E. coli ha e been de e mined expe imen ally by he addi ion o indole o he g ow h medium (4, 20), i is help ul o con e appa en cell-associa ed indole o ‘‘supe na an -equi alen concen a ions’’ using he da a o Fig. 4. Thus when he appa en cell-associa ed concen a ion is 20 mM in la e s a iona y phase, he supe na an -equi alen concen a ion is app ox. 0.75 mM and his is insu icien o ha e any e ec on g ow h. Howe e , when he appa en cell-associa ed concen a- ion is 60 mM a he peak o he pulse, he supe na an -equi alen concen a ion is app oxima ely 4 mM (Fig. 4) and high enough o cause signi ican inhibi ion o g ow h [4]. Thus ou da a sugges ha he magni ude o he indole pulse du ing s a iona y phase en y is su icien o egula e g ow h and di ision by he ionopho e mechanism desc ibed by Chime el e al. [10]. I has p e iously been epo ed ha a he onse o s a iona y phase, E. coli cells become smalle [21] due o con inued elonga ion and di ision. Figu e 3. Appa en cell associa ed indole accumula es apidly a he onse o s a iona y phase. A cul u e o g owing BW25113 cells was sampled egula ly The OD 600 was measu ed and he samples we e cen i uged o pelle cells. The esul an cell pelle was assayed o indole using Ko acs assay. Da a shown a e he mean alues 6s anda d de ia ion o h ee independen epea s. doi:10.1371/jou nal.pone.0093168.g003 Figu e 4. The ela ionship be ween appa en cell associa ed concen a ions o indole and supe na an concen a ions o indole. Known concen a ions o indole we e added o he supe na- an o s a iona y phase BW25113 D naA cells, he mix u e was o exed and cen i uged, and he esul an pelle assayed o indole using Ko acs assay. Do ed lines indica es he appa en cell associa ed indole a he peak o he pulse (60 mM) and he co esponding supe na an concen a ion and indica es he appa en cell associa ed indole when cells a e in s a iona y phase and he co esponding supe na an concen a ion (0.75 mM). Da a shown a e he mean alues 6s anda d de ia ion o a leas h ee independen epea s. doi:10.1371/jou nal.pone.0093168.g004 Indole Pulse Signalling PLOS ONE | www.plosone.o g 6 Ap il 2014 | Volume 9 | Issue 4 | e93168 Cell size depends on he ela i e con ibu ions o cell g ow h and cell di ision. Indole has been shown o a ec bo h hese p ocesses and i g ow h is inhibi ed ea lie , o mo e s ongly, han cell di ision by he indole pulse hen smalle cells would esul . I is clea om ou da a ha he indole pulse is a non- equilib ium phenomenon. A he peak o he pulse, he concen a ion o appa en cell-associa ed indole (60 mM; Fig. 3) is 150- old highe han he supe na an concen a ion (0.4 mM; Fig. 1). As he sys em e u ns owa ds equilib ium in s a iona y phase, he appa en cell-associa ed concen a ion (20 mM) exceeds he supe na an concen a ion (0.75 mM) by less han 30- old. The indole:wa e pa i ion coe icien (log (P) = 1.95) indica es ha he equilib ium concen a ion o indole in he memb ane i sel (as opposed o he o al cell-associa ed concen a ion) will also be subs an ially highe han he concen a ion in he su ounding medium. I he lipid memb ane accoun s o 1/40 h o he o al cell olume, he e will be app oxima ely wice as much indole in he memb anes as in he cy oplasm. We es ima e he mola indole:lipid a io as 0.4 (60.1):1 when 5 mM indole is added o he g ow h medium (see Ma e ials and Me hods o de ails). Howe e , he molecula weigh o E. coli lipids is conside ably g ea e han he molecula weigh o indole, so i is use ul also o exp ess he a io o indole:lipid as 0.05:1 by mass. Ei he way, we en isage ha he ema kably high concen a ion o indole in he lipid memb ane du ing he pulse may ha e a signi ican di ec e ec on he p ope ies o memb ane p o eins, in addi ion o i s e ec on ionic pe meabili y. Does he a ini y o E. coli lipids o indole explain comple ely he high cell-associa ed indole concen a ions ha we measu ed unde equilib ium condi ions (Fig. 4)? To answe his we need o assume ha all cell componen s, o he han lipid, ha e he same a ini y o indole as wa e . We can hen calcula e he expec ed cell-associa ed concen a ion o any gi en ex e nal indole concen a ion, based on he lipid:wa e pa i ion coe icien and he p opo ion o a cell ha is lipid. This calcula ion p edic s ha he cell-associa ed concen a ion should be app oxima ely 3 imes highe han he concen a ion in he su ounding medium. This is signi ican ly di e en om he expe imen al esul s shown in Fig. 4, whe e he cell-associa ed concen a ion is app oxima ely 15 imes highe han he concen a ion in he g ow h medium. This seems o imply ha some addi ional componen in he cell may ha e a high a ini y o indole. Al e na i ely, i is possible ha he pa i ion coe icien measu ed in i o may no en i ely e lec he si ua ion in i o whe e he lipid composi ion may a y and he lipid solubili y o indole may be in luenced by he memb ane cha ge. Au ho Con ibu ions Concei ed and designed he expe imen s: HG JC SH-A UFK DKS. Pe o med he expe imen s: HG JC SH-A. Analyzed he da a: HG JC SH- A UFK DKS. Con ibu ed eagen s/ma e ials/analysis ools: HG JC SH- A. W o e he pape : HG JC SH-A UFK DKS. Re e ences 1. Lee JH, Lee J (2010) Indole as an in e cellula signal in mic obial communi ies. 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Vazquez JL, Me ino S, Domenech O, Be langa M, Vinas M, e al. (2001) De e mina ion o he pa i ion coe icien s o a homologous se ies o cip o loxacin: in luence o he N-4 pipe azinyl alkyla ion on he an imic obial ac i i y. In e na ional Jou nal o Pha maceu ics 220: 53–62. 18. Albe s B (2005) Molecula Biology o he Cell: Ga land Science. 19. Kama aju K, Smi h J, Wang J, Roy V, Sin im HO, e al. (2011) E ec s on Memb ane La e al P essu e Sugges Pe mea ion Mechanisms o Bac e ial Quo um Signaling Molecules. Biochemis y 50: 6983–6993. 20. Pine o-Fe nandez S, Chime el C, Keyse UF, Summe s DK (2011) Indole T anspo ac oss Esche ichia coli Memb anes. Jou nal o Bac e iology 193: 1793– 1798. 21. Ake lund T, No ds om K, Be nande R (1995) Analysis o cell-size and DNA con en in exponen ially g owing and s a iona y-phase ba ch cul u es o Esche ichia coli. Jou nal o Bac e iology 177: 6791–6797. Indole Pulse Signalling PLOS ONE | www.plosone.o g 7 Ap il 2014 | Volume 9 | Issue 4 | e93168