Functional dissection and evidence for intercellular transfer of the heterocyst-differentiation PatS morphogen

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Corrales-Guerrero, L., Mariscal, V., Flores, E. and Herrero, A. (2013), Functional dissection of PatS. Molecular Microbiology, 88: 1093-1105., which has been published in final form at https://doi.org/10.1111/mmi.12244 . This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." ! 1! Functional*Dissection*and*Evidence*for*Intercellular*Transfer*of*the* 1 ! Heterocyst-Differentiation*PatS*Morphogen! 2 ! Laura* Corrales-Guerrero,* Vicente* Mariscal,* Enrique* Flores* and* Antonia* 3 ! Herrero** 4 ! Instituto( de( Bioquímica( Vegetal( y(Fotosíntesis,(Consejo(Superior(de( Investigaciones( 5 ! Científicas(and(Universidad(de(Sevilla,(Américo(Vespucio(49,(E-41092(Seville,(Spain! 6 ! *Corresponding!author.!E-mail:![email protected];!Tel.:!34!95!448!9522;!Fax:!34! 7 ! 95!446!0065.! 8 ! ! 9 ! Running!Title:!Functional!dissection!of!PatS( 10 ! ( 11 ! Key!words:!Anabaena;!Bacterial!development;!Cyanobacteria;!Intercellular! 12 ! signaling! 13 ! ! 14 ! 15 ! ! 2! Summary* 16 ! The! formation! of! a! diazotrophic! cyanobacterial! filament! represents! a! simple! 17 ! example! of! biological! development.! In! Anabaena,! a! non-random! pattern! of! one! 18 ! nitrogen-fixing! heterocyst! separated! by! about! 10! photosynthetic! vegetative! cells! 19 ! results!from!lateral!inhibition!elicited!by!the!cells!differentiating!into!heterocysts.! 20 ! Key!to!this!process!is!the!patS!gene,!which!has!been!shown!to!produce!an!inhibitor! 21 ! of! heterocyst! differentiation! that! involves! the! C-terminal! RGSGR! pentapeptide.! 22 ! Complementation! of! a! ∆patS( Anabaena( mutant! with! different! versions! of! PatS,! 23 ! including!point!mutations!or!tag!fusions,!showed!that!patS!is!translated!into!a!17- 24 ! amino! acid! polypeptide.! Alterations! in! the! N-terminal! part! of! PatS! produced! 25 ! inhibition! of! heterocyst! differentiation,! thus! this! part! of! the! peptide! appears! 26 ! necessary! for! proper! processing!and! self-immunity! in! the! producing! cells.! 27 ! Alterations! in! the! C-terminal! part! of! PatS! led! to! over-differentiation,! thus! 28 ! supporting! its! role! in! inhibition! of! heterocyst! differentiation.! A! polypeptide,! 29 ! produced!in!proheterocysts,!consisting!of!a!methionine!followed!by!the!eight,!but! 30 ! not!the!five,!terminal!amino!acids!of!PatS!recreated!the!full!activity!of!the!native! 31 ! peptide.!Immunofluorescence!detection!showed!that!an!RGSGR-containing!peptide! 32 ! accumulated! in! the! cells! adjacent! to! the! producing! proheterocysts,! illustrating! 33 ! intercellular!transfer!of!a!morphogen!in!the!cyanobacterial!filaments.! 34 ! * 35 ! Introduction* 36 ! In!the!living!world,!multicellularity!appears!to!have!arisen!several!times!during!the! 37 ! course!of!evolution!(Bonner,!1998).!Communal!behaviors!that!provide!benefit!to!a! 38 ! ! 3! group! involving! communication,! cooperative! behavior! and! molecular! exchange! 39 ! between!cells!are!frequent!in!bacteria!(Dunny,!2008;!Manteca!and!Sánchez,!2009).! 40 ! Nevertheless,! filamentous! heterocyst-forming! cyanobacteria! represent! a! further! 41 ! step!in!multicellularity!based!on!a!structured!supracellular!organization!including! 42 ! different!interdependent!cell!types,!each!specialized!in!a!different!nutritional!task,! 43 ! that! contribute! to! the! growth! of! the! filament! as! the! organismic! unit! (Flores! and! 44 ! Herrero,!2010;!Kumar!et(al.,!2010).!This!organization!can!be!traced!back!at!least!2! 45 ! billion!years,!before!multicellularity!appeared!in!eukaryotes!(see!Tomitani!et( al.,! 46 ! 2006;!Schirrmeister!et(al.,!2011).! 47 ! Heterocyst! differentiation! represents! a! remarkable! solution! for! the! co- 48 ! occurrence! of! the! incompatible! processes! of! oxygenic! photosynthesis! and! N2! 49 ! fixation.!Cyanobacteria!ancestors!developed!oxygenic!photosynthesis!and!played!a! 50 ! crucial!role!in!the!evolution!of!the!biosphere,!including!the!oxidation!of!the!Earth’s! 51 ! atmosphere! 2.22-2.45! billion! years! ago! and! the! establishment! of! symbiotic! 52 ! associations!leading!to!the!development!of!algal!and!plant!plastids!(Knoll,!2008).! 53 ! While!endorsing!the!cyanobacterial!ancestors!with!a!unique!trait!of!using!sunlight! 54 ! and! water! to! generate! energy! for! living,! the! development! of! oxygenic! 55 ! photosynthesis!represented!a!burden!for!the!use!of!atmospheric!N2!as!a!source!of! 56 ! nitrogen! for! growth! due! to! the! general! sensitivity! of! the! nitrogenase! system! to! 57 ! oxygen.! In! some! filamentous! cyanobacteria! some! of! the! cells! in! the! filament! 58 ! differentiate!into!specialized!cells,!called!heterocysts,!where!the!N2-fixation!system! 59 ! is!confined,!so!that!the! vegetative! cells!perform!oxygenic!photosynthesis!driving! 60 ! photosynthetic! CO2! fixation,! whereas! heterocysts! rely! on! a! mainly! heterotrophic! 61 ! metabolism! to! carry! out! the! fixation! of! atmospheric! N2.! Vegetative! cells! provide! 62 ! heterocysts!with!reduced!C!to!serve!as!source!of!energy!and!reductant,!whereas! 63 ! ! 4! heterocysts!transfer!combined!nitrogen!to!the!vegetative!cells!(Flores!and!Herrero,! 64 ! 2010;!Wolk!et(al.,!1994).!!! 65 ! Heterocyst! differentiation! requires! the! action! of! a! number! of! regulators,! 66 ! being!pivotal!the!N-control!transcription!factor!NtcA,!a!member!of!the!CRP-family! 67 ! of! regulators,! and! the!differentiation-specific! protein! HetR,! which! is! also! a! 68 ! transcription!factor!(Flores!and!Herrero,!2010;!Kumar!et(al.,!2010;!Kim!et(al.,!2011;! 69 ! Herrero!et(al.,!2013).!!Heterocysts!are!found!in!a!non-random!distribution!along! 70 ! the!filament!(Wolk!et(al.,!1994).!In!the!case!of!strains!of!the!genera(Anabaena(or! 71 ! Nostoc,!heterocysts!are!distributed!at!semi-regular!intervals!separated!by!ca.!10- 72 ! 15!vegetative!cells.!Nutritionally,!this!pattern!appears!to!respond!to!the!constraints! 73 ! imposed! by! the! limited! number! of! vegetative! cells! that! can! be! fed! by! each! 74 ! heterocyst!and!the!limited!number!of!cells!that!can!be!dispensed!of!contributing!to! 75 ! CO2! fixation.! When! filaments! growing! with! combined! nitrogen! are! subjected! to! 76 ! nitrogen! step-down,! that! pattern! is! established! early,! much! before! heterocyst! 77 ! maturation! has! been! completed! (Yoon! and! Golden,! 2001).! Thus,! during! this! 78 ! developmental!process,!positional!information!is!generated!before!N2!fixation!has! 79 ! started.! 80 ! A!product!of!the!patS(gene!has!been!implicated!in!establishing!the!spatial! 81 ! pattern! of! heterocyst! distribution! after! nitrogen! deprivation! (Yoon! and! Golden,! 82 ! 1998,! 2001).! The! gene! is! induced! early! in! the! differentiating! cells! and! its! 83 ! inactivation!leads!to!a!Mch!(Multiple!contiguous!heterocysts)!phenotype,!whereas! 84 ! its!over-expression!abolishes!differentiation.!In!Anabaena(sp.!strain!PCC!7120!the! 85 ! patS!gene! would! encode! a! 17-! or! 13-amino! acid! peptide! of! which! a! subset! 86 ! consisting! of! the! 5! C-terminal! amino! acids! (RGSGR,! a! peptide! called! PatS-5)! 87 ! ! 5! inhibits! differentiation! when! added! to! the! external! medium,! although! it! fails! to! 88 ! restore!a!wild-type!heterocyst!pattern!on!a!patS!mutant!(Yoon!and!Golden,!1998).! 89 ! In! vitro,! PatS-5! has! been! shown! to! interact! with! HetR! and! to! inhibit! binding! of! 90 ! HetR! to! DNA! upstream! from! several! regulated! genes! (Huang! et( al.,! 2004;! Risser! 91 ! and!Callahan,!2007;!Du!et(al.,!2012;!Feldmann!et(al.,!2011).!It!is!considered!that! 92 ! PatS,!or!a!processed!derivative!of!it,!is!exported!from!the!differentiating!cells!to! 93 ! inhibit! the! differentiation! of! its! neighbors! likely! by! acting! on! the! transcription! 94 ! factor! HetR! (Yoon! and! Golden,! 1998;! Khudyakov! and! Golden,! 2004;! Risser! and! 95 ! Callahan,! 2009).! However,! information! about! the! identity! of! the! primary! gene! 96 ! product! and! of! the! molecule! actually! acting! as! a! trans-cellular! inhibitor! of! 97 ! differentiation,! thus! behaving! as! a! morphogen,! as! well! as! about! the! in! vivo! 98 ! processing,!is!lacking.!! 99 ! In!this!work!we!present!evidence!for!the!synthesis!of!a!17-amino!acid!PatS! 100 ! in! (pro)heterocysts.! Adhering! to! definitions! by! C.! P.! Wolk,! in! Anabaena,! a! 101 ! proheterocyst!is!an!intermediate,!between!a!vegetative!cell!and!a!heterocyst,!that! 102 ! differs!in!shape!and!granulity!from!vegetative!cells,!and!a!mature!heterocyst!is!a! 103 ! cell!capable!of!aerobic!fixation!of!N2((see!Wolk!et(al.,!1994).!We!also!show!that!the! 104 ! C-terminal! octapeptide,! but! not! the! C-terminal! heptapeptide,! hexapeptide! or! 105 ! pentapeptide,!of!PatS!is!sufficient!for!regulation!of!heterocyst!patterning!along!the! 106 ! filament.! Specific! residues! in! the! N-terminal! part! of! PatS! are! crucial! for! self- 107 ! immunity! in! the! producing! cells,! likely! reflecting! their! role! in! PatS! processing! 108 ! and/or!export,!whereas!specific!C-terminal!amino!acids!determine!the!heterocyst! 109 ! differentiation! inhibitory! activity! of! the! peptide.! Additionally,! using! an! 110 ! immunofluorescence!approach,!we!have!detected!the!in!vivo!location!of!PatS.! 111 ! ! 6! Results* 112 ! To!better!mimic!the!physiological!conditions,!we!have!chosen!to!express!a!number! 113 ! of! versions! of! the! patS! gene! from! the! native! patS! promoter! in! the! chromosome! 114 ! (Fig.!1).!In!support!of!this!approach,!a!recent!report!documents!an!extensive!copy- 115 ! number!variability!of!heterologous!replicons!commonly!used!in!strain!PCC!7120! 116 ! (Yang!et(al.,!2012).!The!engineered!patS!gene!was!transferred!by!conjugation!from! 117 ! Escherichia(coli!to!Anabaena!sp.!strain!CSVT20,!a!derivative!of!strain!PCC!7120!in! 118 ! which!the!wild-type!patS(gene!has!been!deleted.!In!this!way,!strains!in!which!only! 119 ! the! altered! version! of! patS( is! present! were! generated! (Table! 1).! Because! strain! 120 ! CSVT20,!like!other!patS!deletion!strains!(Yoon!and!Golden,!1998),!produces!a!Mch! 121 ! phenotype,! which! correlates! with! a! disinhibition! of! heterocyst! formation,! this! 122 ! approach! permitted! a! simple! test! of! phenotypic! complementation.! As! shown! 123 ! below,!three!basic!phenotypes!were!observed!with!the!altered!patS(genes:!lack!of! 124 ! complementation!retaining!the!∆patS!Mch!phenotype,!complementation!resulting! 125 ! in! a! wild-type! phenotype,! and! complete! or! partial! inhibition! of! heterocyst! 126 ! differentiation.! 127 ! Expression(of(PatS-17( 128 ! As!mentioned!above,!the!patS(ORF!could!encode!a!17-!or!13-amino!acid!peptide,! 129 ! which! depends! on! whether! translation! starts! at! the! first! or! the! second! possible! 130 ! Met-encoding! codon.! We! have! studied! translation! initiation! of! the! patS!gene! 131 ! monitoring!fluorescence!emission!from!strains!bearing!the!gfp-mut2!gene!(lacking! 132 ! its! start! codon)! inserted! in! phase! after! the! 1st!(strain! CSL21)! or! the! 5th!(strain! 133 ! CSL19)!triplet!of!the!patS!ORF.!As!a!reference!of!the!time-course!activation!of!the! 134 ! patS!promoter,!we!monitored!GFP!fluorescence!from!strain!CSVM17!(Mariscal!et( 135 ! ! 7! al.,!2007),!a!derivative!of!strain!PCC!7120!bearing!a!transcriptional!PpatS-gfp!fusion! 136 ! inserted!in!a!neutral!region!of!the!Anabaena!a!megaplasmid,!which!has!the!same! 137 ! copy! number! as! the! chromosome! (Lee! et# al.,! 2003).! The! GFP! fluorescence! was! 138 ! monitored! in! filaments! grown! with! ammonium! and! subjected! to! combined- 139 ! nitrogen!deprivation.!Strains!CSL21!and! CSL19!both!produced!GFP!fluorescence,! 140 ! localized!to!specific!cells!(Fig.!2),!indicating!that!the!first!Met!codon!can!be!used!as! 141 ! a!translation!start!of!the!patS!gene.! 142 ! !Phenotype#of#strains#bearing#GFP-#and#6His-tag-fusions# 143 ! In!addition!to!the!strains!producing!PatS-GFP!fusions,!we!studied!a!strain!derived! 144 ! from! the! patS# mutant! CSVT20! that! bears! a! modified! patS!gene! encoding! a! PatS! 145 ! version! with! a! 6His! sequence! inserted! between! M5!and! L6!of! the! polypeptide! 146 ! (strain!CSL22).!Phenotypic!characteristics!of!strains!CSL19,!CSL21,!and!CSL22!with! 147 ! regard! to! the! frequency! and! distribution! of! proheterocysts! plus! heterocysts,! 148 ! expression! of! nitrogenase! activity! and! ability! to! grow! diazotrophically! (Table! 2)! 149 ! were! studied! using! strains! PCC! 7120! and! CSVT20! as! controls.! In! all! the! mutant! 150 ! strains!generated!in!this!work,!proheterocysts!and!heterocysts!were!visualized!by! 151 ! Alcian! Blue! staining! that! colors! the! Hep! layer! of! the! heterocyst! envelope! (see! 152 ! López-Igual!et#al.,!2012b).!The!data!of!percentage!of!total!heterocysts,!with!regard! 153 ! to!total!cells,!and!of!contiguous!heterocysts,!as!well!as!the!distribution!of!sizes!of! 154 ! vegetative! cell! intervals! between! heterocysts,! are! shown! in! Figure! 3! and! 155 ! summarized!in!Table!1.* 156 ! Strain!CSL21!(GFP!inserted!after!M1)!exhibited!a!(pro)heterocyst!frequency! 157 ! of!2.94%!of!the!total!cells!(the!wild!type!has!10.31%!and!the!control!strain!CSL48,! 158 ! bearing! the! wild-type! patS!sequence! construct,! has! 12.39%)! (Table! 1),! very! low! 159 ! ! 8! nitrogenase! activity! and! impaired! diazotrophic! growth! (Table! 2).! Strain! CSL19! 160 ! (GFP!inserted!after!M5)!exhibited!a!very!low!(pro)heterocyst!frequency!(0.54%!of! 161 ! the! total! cells)! and,! consistent! with! a! very! low! nitrogenase! activity,! a! very! low! 162 ! diazotrophic!growth!rate.!Strain!CSL22!(6His!sequence!inserted!after!M5)!exhibited! 163 ! a! (pro)heterocyst! frequency! of! 5.31%! of! total! cells.! In! this! strain,! nitrogenase! 164 ! activity! and! diazotrophic! growth! rate! were! also! lower! than! in! the! wild! type,! 165 ! although! the! impairment! in! diazotrophic! growth! was! less! pronounced! than! in! 166 ! CSL21.!Thus,!insertion!of!GFP!after!M1!(strain!CSL21)!or!of!6His!(strain!CSL22)!or! 167 ! GFP! (strain! CSL19)! after! M5! leads! to! impairment! in! heterocyst! differentiation,! 168 ! which!is!most!severe!in!the!latter!case.!These!results!are!consistent!with!the!idea! 169 ! that! the! modified! forms! of! PatS! expressed! in! strains! CSL19,! CSL21! and! CSL22! 170 ! inhibit!differentiation!of!the!producing!cells.! 171 ! Point#amino#acid#mutations#of#patS## 172 ! Strains! expressing! versions! of! PatS! with! point! amino! acid! substitutions! in! each! 173 ! position! of! the! peptide! were! also! investigated! (Table! 1! and! Fig.! 3).! Whereas! 174 ! substitution! M5A! (present! in! strain! CSL49)! had! little! effect! on! (pro)heterocyst! 175 ! frequency!or!distribution!with!respect!to!the!wild!type!or!the!CSL48!control!strain,! 176 ! substitution! M1A! (in! strain! CSL44)! produced! a! patS# phenotype.! These! results! 177 ! indicate!that!M1!is!necessary!and!sufficient!as!a!translation!start!of!patS.! 178 ! In!strain!CSL90!(K2A)!(pro)heterocyst!frequency!was!4.47%!of!total!cells,! 179 ! whereas! in! strains! CSL91! (A3Q)! and! CSL92! (I4Q)! it! was! 2.10! and! 1.97%,! 180 ! respectively.! Substitution! L6Q! (in! strain! CSL50)! had! almost! no! effect! on! 181 ! (pro)heterocyst!frequency!or!interval-size!distribution.!Substitution!V7Q!(in!strain! 182 ! CSL51)! abolished! differentiation! whereas! V7A! (strain! CSL93)! led! to! a! 183 ! ! 15! its!substitution!by!A!(in!strain!CSL93)!impairs!differentiation,!and!its!substitution! 330 ! by!Q!(in!CSL51)!abolishes!differentiation,!although!this!can!be!compensated!in!part! 331 ! by! introducing! a! V! residue! in! the! 5th!position! of! the! peptide! (in! CSL62).! These! 332 ! results!imply!that!the!modified!versions!of!PatS!produced!in!strains!CSL90,!CSL91,! 333 ! CSL92,!CSL93!and!CSL51,!as!is!also!the!case!for!those!produced!in!CSL19,!CSL21! 334 ! and! CSL22,! inhibit! the! differentiation! of! the! producing! cells! into! heterocysts.! 335 ! Although!to!a!lesser!extent,! also! the!changes!in!N8!(strain!CSL52)!and!F9!(strain! 336 ! CSL53)!inhibit!differentiation.!Whatever!the!mechanism!the!wild!type!uses!for!self- 337 ! immunity!of!the!producing!cells!against!PatS,!it!appears!impaired!in!these!mutant! 338 ! strains.!In!contrast,!the!PatS! inhibitory! activity!is!retained!in!them.!Thus,! the!9-,! 339 ! and!specially!the!7-residue!N-terminal!part!of!PatS!is!required!for!self-immunity!of! 340 ! the!producing!cells!but!not!for!PatS!activity.! 341 ! !In! the! C-terminal! region! of! PatS,! whereas! C10,! E12,! G14!and! R17!appear! 342 ! important,!R13,!S15!and!G16!are!essential!for!the!inhibitory!function!of!PatS!in!cells! 343 ! adjacent!to!the!producing!cells!(Fig.!5).!The!hetN!gene!is!also!needed!for!wild-type! 344 ! heterocyst! patterning! in! Anabaena# sp.! strain! PCC! 7120,! although! its! function! is! 345 ! evident!in!filaments!growing!steadily!on!N2!rather!than!in!filaments!subjected!to! 346 ! nitrogen!deprivation (Callahan and Buikema, 2001). A HetN-related product would be 347 ! produced by the mature heterocysts to inhibit neighboring cells from differentiating. 348 ! The 287-residue HetN polypeptide bears an internal ERGSGR sequence, of which 349 ! RGSGR has been shown to be essential for heterocyst patterning, and in which the two 350 ! R residues are essential to suppress heterocyst differentiation (Higa et al., 2012). Thus, 351 ! some similarities may exist between the mechanism of action of PatS and that of HetN 352 ! (Khudyakov and Golden, 2004). 353 ! ! 16! As! mentioned! above,! it! has! been! proposed! that! a! PatS-related! product! 354 ! moves!from!the!producing!proheterocyst!to!the!adjacent!cells.!We!have!detected! 355 ! by! immunofluorescence! PatS-5! showing! an! accumulation! of! a! PatS-5! containing! 356 ! product! in! the! cells! adjacent! to!proheterocysts! (Fig.! 4).! This! represents! a! direct! 357 ! evidence!for!intercellular!transfer!of!a!PatS!peptide.!In!other!bacteria,!peptides!that! 358 ! have!a!role!in!the!control!of!developmental!processes!have!been!identified,!and!in! 359 ! some!cases!the!biological!function!depends!on!events!of!peptide!transport!in!and! 360 ! out! of! the! cells.! A! number! of! examples! have! been! reported! for! Bacillus!peptides! 361 ! involved!in!the!regulation!of!sporulation,!quorum!sensing!and!virulence,!such!as! 362 ! PhrA! and! CSF! pentapeptides! that! are! produced! by! processing! of! precursor! 363 ! molecules! and! secretion! (Lazazzera! et# al.,! 1997).! The! processed! forms! of! both! 364 ! peptides!are!transported!into!the!cell!by!the!oligopeptide!permease!SpoOK!(OPP)! 365 ! (Solomon!et#al.,!2011;!Perego!and!Hoch,!1996).!PhrA!is!synthesized!as!a!44-amino! 366 ! acid! molecule! with! an! N-terminal! hydrophobic! domain! and! a! C-terminal! 367 ! hydrophilic!region!separated!by!a!potential!signal!peptidase!I!cleavage!site!(which! 368 ! would!correspond!to! a!V,!H!or! A!residue),!implying!that! the!carboxyl!half!of!the! 369 ! protein!is!secreted!from!the!cell!(Perego!and!Hoch,!1996).!In!the!case!of!CSF,!an! 370 ! important! role! of! the! region! comprising! the! 5! amino! acids! preceding! the! active! 371 ! pentapeptide! in! directing! cleavage! of! the! precursor! protein! has! been! reported! 372 ! (Laningan-Gerdes#et#al.,!2008).!In!the!case!of!PatS,!consistent!with!its!small!size,!no! 373 ! signal! peptide! could! be! recognized! using! different! prediction! programs! (e.g.! 374 ! SignalP!4.0;!Petersen!et#al.,!2011),!and!no!obvious!matches!to!the!pattern!of!signal! 375 ! peptidase!I!(Paetzel!et#al.,!2002)!could!be!recognized!either.!Thus,!PatS!would!be! 376 ! exported! from! the! producing! proheterocysts! by! a! novel! mechanism,! which! may! 377 ! involve!direction!to!the!periplasm!from!which!it!could!be!imported!by!neighboring! 378 ! ! 17! vegetative! cells,! or! direct! cell-to-cell! transfer! through! channels! located! in! the! 379 ! intercellular!septa!(Flores!and!Herrero,!2010).! 380 ! The!possibility!that!PatS!were!cleaved!to!generate!an!active!smaller!peptide! 381 ! was!consistent!with!all!available!data,!but!not!finally!proved.!Previously,!patS4!to! 382 ! patS8!minigenes!were!expressed!in!Anabaena!from!promoters!with!different!cell! 383 ! specificity!(Wu!et#al.,!2004).!When!patS5#was!expressed!in!proheterocysts!in!the! 384 ! patS! background,! from! either! the! patS! promoter! or! the! (pro)heterocyst-specific! 385 ! hepA!promoter,!no!or!only!a!weak!inhibition!could!be!detected.!These!results!are! 386 ! consistent!with!those!obtained!with!strain!CSL45!described!above,!confirming!that! 387 ! PatS-5! expressed! in! proheterocysts! is! able! to! produce! neither! inhibition! in! the! 388 ! producing!cells!nor!cell-cell!signaling.!The!fact!that!similar!results!were!obtained! 389 ! when!the!PpatS-patS5#construct!was!placed!either!in!the!chromosome!(this!work)!or! 390 ! in!a!multicopy!plasmid!(Wu!et#al.,!2004)!makes%a!low!peptide!dosage!an!unlikely! 391 ! reason!to!explain!lack!of!activity!of!the!produced!PatS-5.!Moreover,!we!have!shown! 392 ! that!in!strain!CSL45!PatS-5!is!accumulated!in!the!producing!proheterocysts!(Fig.! 393 ! 4).!!In!contrast,!PatS-8!(in!strain!CSL47),!which!includes!all!the!residues!that!our! 394 ! results! have! implicated! in! the! inhibitory! activity! of! PatS,! appears! to! produce! a! 395 ! regulation!similar!to!that!produced!by!wild-type!PatS.!When!expressed!from!the! 396 ! copper-inducible!petE!promoter,!which!is!active!mainly!in!vegetative!cells,!patS8,! 397 ! patS7,! patS6!and! patS5,! but! not! patS4,! produced! inhibition! of! heterocyst! 398 ! differentiation! both! in! the! wild! type! and! a! patS-null! mutant! (Wu! et# al.,! 2004).! 399 ! Expression! of! patS!or!patS5! from! the! rbcL! promoter,! which! directs! strong! 400 ! expression!only!in!vegetative!cells,!completely!inhibits!heterocyst!formation!both! 401 ! in!the!wild!type!and!the!patS!mutant!(Wu!et#al.,!2004).!Additionally,!PatS-5!added! 402 ! to!the!culture!medium!inhibits!heterocyst!differentiation!(Yoon!and!Golden,!1998;! 403 ! ! 18! Wu!et#al.,!2004).!Thus!PatS-5,!which!is!not!active!in!the!proheterocysts!and!would! 404 ! not!be!exported!from!these!cells,!inhibits!heterocyst!differentiation!when!placed!in! 405 ! the!vegetative!cells.!It!appears!that,!in!contrast!to!the!receptor!vegetative!cells,!the! 406 ! producing!proheterocysts!are!insensitive!to!the!small!processed!peptides!whereas,! 407 ! as! deduced! from! the! phenotype! of! strains! expressing! peptides! with! point! 408 ! substitutions! in! the! N-terminal! 9-amino! acid! stretch,! they! are! sensitive! to! the! 409 ! unprocessed!PatS.!In!this!regard,!whether!the!processed!peptides!are!accumulated! 410 ! at!different!steps!of!the!differentiation!process!in!the!producing!and!receptor!cells,! 411 ! and! whether! the! mechanism! of! inhibition! by! the! unprocessed! and! processed! 412 ! peptides!is!different!will!merit!future!investigation.! 413 ! Several!reports!have!shown!in!vitro!activity!of!synthetic!PatS-5!at!binding!to! 414 ! HetR! and! inhibition! of! HetR-binding! to! DNA! (Huang! et# al.,! 2004;! Risser! and! 415 ! Callahan,!2007;!Du!et#al.,!2012;!Feldmann!et#al.,!2011).!Binding!to!HetR!was!tighter! 416 ! for!PatS-6!(ERGSGR)!than!for!PatS-5,!and!binding!was!tighter!for!PatS-5!than!for! 417 ! PatS-7! (DERGSGR),! whereas! no! binding! was! detected! for! PatS-8! (CDERGSGR)! 418 ! (Feldmann! et# al.,! 2012).! Thus,! some! differences! could! be! found! between! the! 419 ! relative!affinities!of!the!different!peptides!for!in!vitro!binding!to!HetR!and!for!their! 420 ! activity! of! inhibition! of! heterocyst! differentiation.! The! most! striking! of! these! 421 ! differences!is!that!found!with!PatS-8,!which!does!not!appreciably!bind!to!HetR!in! 422 ! vitro,! but! appears! to! be! fully! active! in! strain! CSL47.! It! is! possible! that:! (i)! the! 423 ! affinity! for! a! molecular! target! (HetR)! is! influenced! by! additional! conditions! or! 424 ! factors!in!vivo;!(ii)!the!molecule!exhibiting!a!tighter!binding,!or!even!a!higher!in! 425 ! vivo! activity! when! over-produced! in! the! cyanobacterium! (e.g.! specifically! in! 426 ! vegetative! cells),! is! not! the! best! suited! for! a! regulatory! effect!in! vivo;! (iii)! the! 427 ! presence! of! a! M! residue! in! the! in! vivo-produced,! but! not! in! the! in! vitro! used,! 428 ! ! 19! peptides! influences! the! studied! parameters;! (iv)! in! addition! to! HetR,! other! 429 ! molecular! targets! of! PatS! influence! its! role! as! inhibitor! of! heterocyst! 430 ! differentiation.!Although!with!the!present!data!it!is!not!possible!to!exclude!that!the! 431 ! exported!peptide!could!be!further!cleaved!during!the!transfer!or!in!the!receptor! 432 ! cells! to! render! PatS-5,! it! is! also! possible! that! this! is! not! the! physiologically! 433 ! significant!inhibitory!peptide.! 434 ! In!conclusion,!our!results!are!consistent!with!the!model!illustrated!in!Figure! 435 ! 5,! in! which!PatS-17! is! the! product! of! the! patS!gene! in! the! proheterocysts.! This! 436 ! polypeptide,! which! if! accumulated! appears! to! inhibit! differentiation! of! the! 437 ! producing!cells,!is!not!exported!as!such,!but!it!is!susceptible!of!cleavage!to!render!a! 438 ! smaller!peptide!derived!from!its!C-terminal!part!and!likely!consisting!of!8!amino! 439 ! acids,!which!would!be!the!morphogen!transferred!to!the!neighboring!cells.!In!PatS,! 440 ! K2,! A3,! and! I4! appear! to! be! very! important! and! V7!essential! for! PatS! 441 ! processing/export,!but!the!fact!that!expression!of!M-PatS-8!in!proheterocysts!(in! 442 ! strain!CSL47)!leads!to!a!wild-type!heterocyst!distribution!implies!that!export!from! 443 ! proheterocysts!to!vegetative!cells!could!also!take!place!unlinked!from!processing.! 444 ! Additionally,!C10,!E12,!G14!and!R17!appear!important!and!R13,!S15!and!G16!essential! 445 ! for!PatS!activity!of!inhibition!of!differentiation.!! 446 ! ! 447 ! Experimental*Procedures* 448 ! Strains#and#growth#conditions# 449 ! Growth! conditions! of! Anabaena!sp.! (also! known! as! Nostoc!sp.)! strain! PCC! 7120! 450 ! were!as!described!(López-Igual!et#al.,!2012a).!Except!for!strain!CSVT20,!which!was! 451 ! grown!without!antibiotics,!all!the!mutants!listed!in!Table!1!were!grown!in!medium! 452 ! ! 20! supplemented!with!streptomycin!(Sm)!and!spectinomycin!(Sp)!at!5!µg!ml-1!each!in! 453 ! solid! media! and! 2! µg! ml-1! each! in! liquid! media.! Growth! rate! constant! 454 ! determinations!were!performed!as!in!Valladares!et#al.!(2011).!Nitrogenase!activity! 455 ! was!determined!under!oxic!conditions!in!filaments!incubated!for!24!h!in!bubbled! 456 ! cultures!without!nitrogen!and!without!antibiotics!(López-Igual!et#al.,!2012b).! 457 ! Construction#of#mutants# 458 ! To! delete! the! patS! gene,! two! DNA! fragments,! one! encompassing! sequences! 459 ! upstream!of!the!gene!and!the!other!including!sequences!downstream!of!the!gene,! 460 ! were!amplified!by!PCR!using!DNA!from!Anabaena!sp.!strain!PCC!7120!as!template! 461 ! and! the! primer! pairs! asl2301-8/asl2301-9! and! asl2301-10/asl2301-11,! 462 ! respectively!(all!oligodeoxynucleotide!primers!are!listed!in!Table!3).!The!upstream! 463 ! and!downstream!DNA!fragments!were!cloned!in!pMBL-T!(Dominion!MBL,!Spain),! 464 ! sequenced!and!inserted!together,!in!direct!orientation,!into!pCSRO.!This!plasmid,! 465 ! which! is! derived! from! pRL500! (Elhai! and! Wolk,! 1988),! can! be! mobilized! by! 466 ! conjugation! and! bears! an! SmR/SpR! determinant! and! the! sacB!gene! for! positive! 467 ! selection.!The!plasmid!produced,!pCSVT51,!bears!cloned!Anabaena!DNA!from!the! 468 ! asl2301!locus! with! a! deletion! of! 180! bp,! which! includes! the! whole! patS!gene.! 469 ! Plasmid!pCSVT51!was!transferred!to!Anabaena!sp.!strain!PCC!7120!by!conjugation,! 470 ! which!was!performed!as!described!(Elhai!et#al.,!1997).!Exconjugants!were!selected! 471 ! by!their!resistance!to!Sm!and!Sp!and!double!recombinants!were!then!selected!by! 472 ! their! resistance! to! sucrose.! The! clone! isolated,! CSVT20,! lacked! those! 180!bp! 473 ! including!the!patS#gene.! 474 ! For! construction! of!fusions! of! the! gfp-mut2!gene! to! patS,! two! DNA! 475 ! fragments!were!amplified,!one!using!Anabaena!DNA!as!template!and!primer!pairs! 476 ! ! 21! asl2301-9/als2301-15! (for! strain! CSL19)! and! asl2301-9/asl2301-17! (for! strain! 477 ! CSL21),! and! the! other! using! pCSAM135! (Flores! et# al.,! 2007)! as! template! and! 478 ! primer! pairs! gfp-asl2301-1/gfp-asl2301-2! (for! strain! CSL19)! and! gfp-asl2301- 479 ! 5/gfp-asl2301-6!(for!strain!CSL21).!PCR!products!were!cloned!together!in!pMBL-T! 480 ! producing!pCSL29*and!pCSL33,!respectively.!KpnI!fragments!from!these!plasmids! 481 ! bearing! the! gfp-mut2! gene! were! inserted! into! mobilizable! vector! pCSV3! 482 ! (Valladares! et# al.,! 2011)! digested! with! the! same! enzyme,! producing! pCSL30! and! 483 ! pCSL34,!respectively.! 484 ! !For!construction!of!a!PatS-6His!fusion!(strain!CSL22),!a!DNA!fragment!was! 485 ! amplified!using!Anabaena!DNA!as!template!and!primer!pair!asl2301-9/als2301-18,! 486 ! and! cloned! in! pMBL-T! producing! pCSL3.! A! KpnI! fragment! from! this! plasmid! 487 ! carrying! the! patS-6His! fusion! was! inserted! into! pCSV3! digested! with! the! same! 488 ! enzyme,!producing!pCSL36.! 489 ! !! For!construction!of!PatS!point!mutants!and!minigenes,!a!DNA!fragment!was! 490 ! amplified!using!Anabaena!DNA!as!template!and!primer!pairs!asl2301-9/asl2301- 491 ! 32!to!asl2301-49!(for!strains!CSL44!to!CSL62),!asl2301-9/asl2301-77!(for!strain! 492 ! CSL94)! or! asl2301-69/asl2301-71! or! asl2301-72! (for! strains! CSL91! and! CSL92,! 493 ! respectively)!and!finally!cloned!into!pCSV3!producing!pCSL82!to!pCSL95,!pCSL110,! 494 ! pCSL104,!and!pCSL105,!respectively.!For!construction!of!strains!CSL90!and!CSL93,! 495 ! DNA! fragments,! from! 2771000! to! 2771734! (coordinates! of! the! strain! PCC! 7120! 496 ! genomic!sequence;!Kaneko!et#al.,!2001),!including!nucleotide!changes!to!produce! 497 ! K2A!and!V7A!substitutions,!respectively,!were!synthesized!(GeneArtÒ,!Invitrogen)! 498 ! and!cloned!into!pCSV3!producing!plasmids!pCSL103!and!pCSL106,!respectively.! 499 ! Plasmids! pCSL30,! pCSL34,! pCSL36,! pCSL38,! pCSL82! to! pCSL95,! pCSL110,! 500 ! pCSL104,! pCSL105,! pCSL103! and! pCSL106! were! transferred! to! the! patS# mutant,! 501 ! ! 22! strain!CSVT20,!by!conjugation! (Elhai!et#al.,!1997).!Clones!resistant!to! Sm+and!Sp! 502 ! were!selected,!and!their!genomic!structure!in!the!patS!region!was!tested!by!PCR! 503 ! with! primers! asl2301-74! (which! lies! outside! of! the! construct,! 5’! from! it)! and! 504 ! asl2301-75!(which!lies!inside!the!region!deleted!in!strain!CSVT20)!or!asl2301-76! 505 ! (which! lies! outside! of! the! construct,! 3’! from! it)! to! check! recombination! in! the! 506 ! correct! place! and! segregation! of! the! patS-mutant! chromosomes.! Additionally,! 507 ! except! for! CSL19! and! CSL21,! the! patS!ORF! was! sequenced! in! all! the! Anabaena! 508 ! strains!generated!in!this!work.! 509 ! Microscopy# 510 ! For! light! microscopy,! filaments! grown! in! BG110! +! ammonium! medium! (in! the! 511 ! presence!of!Sm!and!Sp!for!the!mutants)!were!harvested,!washed!with!nitrogen-free! 512 ! (BG110)!medium!and!incubated!for!24!h!in!bubbled!cultures!at!30ºC!in!the!light.!At! 513 ! least! 300! cells! or! 100! intervals! were! counted! for! each! strain! in! each! of! three! 514 ! independent! experiments.! Dividing! cells! were! counted! as! two! cells.! For! staining! 515 ! (pro)heterocysts,! cell! suspensions! were! mixed! (1:10)! with! a! 1%! Alcian! Blue! 516 ! (Sigma)! solution! (López-Igual! et# al.,! 2012b).**For! confocal! microscopy,! samples! 517 ! from!cultures!of!Anabaena!sp.!set!atop!solidified!medium!(BG110!+!ammonium!or! 518 ! BG110)! were! visualized! using! a! Leica! HCX! PLAN-APO! 63X! 1.4! NA! oil! immersion! 519 ! objective!attached!to!a!Leica!TCS!SP2!confocal!laser-scanning!microscope.!GFP!was! 520 ! excited!using!488-nm!irradiation!from!an!argon!ion!laser.!Fluorescence!from!GFP! 521 ! was!monitored!by!collection!across!windows!of!500-540!nm.* 522 ! For!immunofluorescence!detection!of!the!PatS-5!sequence,!300!µl!of!liquid! 523 ! cultures!(containing!2!to!3!µg!of!chlorophyll!a/ml)!were!placed!atop!poly-L-lysine! 524 ! precoated!microscope!slides!and!covered!with!a!45-µm!pore-size!Millipore!filter.! 525 ! ! 23! After!that,!the!filter!was!removed!and!the!slide!was!let!to!dry!at!room!temperature! 526 ! and,!then,!immersed!in!70%!ethanol!during!30!min!at!-20!ºC!and!dried!10!min!in! 527 ! an!oven!at!80!ºC.!The!cells!were!then!washed!twice!by!covering!the!slide!with!PBS- 528 ! T! buffer! (140! mM! NaCl,! 1.5! mM! KH2PO4,! 2.7! mM! KCl! [pH! 7.4],! Tween-20! [0.05! 529 ! p/v])!(2!min!each!time,!room!temperature)!and!treated!with!blocking!buffer!(5%! 530 ! milk!powder!in!PBS-T,!15!min).!The!cells!on!the!slides!were!then!incubated!with!a! 531 ! primary! anti-PatS-5! antibody! solution! (rabbit! polyclonal! antibody! raised! against! 532 ! synthetic! RGSGR,! Cambridge! Research! Biochemicals,! purified! by! affinity! 533 ! chromatography!and!1:100!diluted!in!blocking!buffer)!for!90!min,!washed!three! 534 ! times! with! PBS-T,! incubated! 45! min! in! the! dark! with! secondary! anti-rabbit! 535 ! antibody!conjugated!to!fluorescein!isithiocyanate!(FITC)!(Sigma,!1:500!dilution!in! 536 ! PBS-T)!and!washed!three!times!with!PBS-T.!After!dried,!several!drops!of!FluorSave! 537 ! (Carl!Biochem)!were!added!atop,!covered!with!a!cover!slip,!sealed!with!nail!lack! 538 ! and! let! dry! overnight! at! 4! ºC.! Fluorescence! was! imaged! using! a! Leica! DM6000B! 539 ! fluorescence!microscope!and!an!ORCA-ER!camera!(Hamamatsu).!Fluorescence!was! 540 ! monitored! using! a! FITC! L5! filter! (excitation,! band-pass! [BP]! 480/40! filter;! 541 ! emission,!BP!527/30!filter)!and!quantified!analyzing!in!each!cell!“the!mean!gray! 542 ! value”!of!the!ImageJ!software!(http://imagej.nih.gov/ij).! 543 ! ! 544 ! Acknowledgements 545 ! We thank Victoria Merino-Puerto (Instituto de Bioquímica Vegetal y Fotosíntesis, 546 ! CSIC) for making strain CSVT20, and Jeff! Elhai! (Virginia! Commonwealth! 547 ! University)! for! a! bioinformatic! search! of! PatS.! Work was supported by grants 548 ! BFU2007-60457 and BFU2010-17980 from the Spanish Government, co-financed by 549 ! FEDER. L.C.-G. was the recipient of a JAE-predoc fellowship from CSIC.* 550 ! ! 24! References* 551 ! Bonner, J.T. (1998) The origins of multicellularity. Integr Biol 1: 27-36. 552 ! Callahan, S.M., and Buikema, W.J. (2001) The role of HetN in maintenance of the 553 ! heterocyst pattern in Anabaena sp. PCC 7120. Mol Microbiol 40: 941–950. 554 ! Du, Y., Cai, Y., Hou, S., and Xu, X. (2012) Identification of the HetR recognition 555 ! sequence upstream of hetZ in Anabaena sp. PCC 7120. J Bacteriol 194: 2297- 556 ! 2306. 557 ! Dunny, G.M. (2008) Multicellular behavior in bacteria: communication, cooperation, 558 ! competing and cheating. BioEssays 30: 296-298. 559 ! Elhai, J., and Wolk, C.P. (1988) A versatile class of positive-selection vectors based on 560 ! the nonviability of palindrome-containing plasmids that allows cloning into long 561 ! polylinkers. Gene 68: 119-138. 562 ! Elhai, J., Vepritskiy, A., Muro-Pastor, A.M., Flores, E., and Wolk, C.P. (1997) 563 ! Reduction of conjugal transfer efficiency by three restriction activities of Anabaena 564 ! sp. strain PCC 7120. J Bacteriol 179: 1998-2005. 565 ! Flores, E., and Herrero, A. (2010) Compartmentalized function through cell 566 ! differentiation in filamentous cyanobacteria. Nat Rev Microbiol 8: 39-50. 567 ! Flores, E., Pernil, R., Muro-Pastor, A.M., Mariscal, V., Maldener, I., et al. (2007) 568 ! Septum-localized protein required for filament integrity and diazotrophy in the 569 ! heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 570 ! 189: 3884-3890. 571 ! Feldmann, E.A., Ni, S., Sahu, I.D., Mishler, C.H., Risser, D.D., et al (2011) Evidence 572 ! for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5. 573 ! Biochemistry 50: 9212-9224. 574 ! Feldmann, E.A., Ni, S., Sahu, I.D., Mishler, C.H., Levengood, J.D., et al (2012) 575 ! Differential binding between PatS C-terminal peptide fragments and HetR from 576 ! Anabaena sp. PCC 7120. Biochemistry 51: 2436-2442. 577 ! Herrero, A., Picossi, S., and Flores, E. (2013) Gene expression during heterocyst 578 ! differentiation. In: Genomics of cyanobacteria. Chauvat, F., and Cassier-Chauvat, 579 ! C. (ed.) Elsevier Ltd. pp. 281-329. 580 ! Higa, K.C., Rajagopalan, R., Risser, D.D., Rivers, O.S., Tom, S.K., et al. (2012) The 581 ! RGSGR amino acid motif of the intercellular signaling protein, HetN, is required 582 ! for patterning of heterocysts in Anabaena sp. strain PCC 7120. Mol Microbiol 83: 583 ! ! 31! 715 ! 716 ! 717 ! 718 ! 719 ! 720 ! 721 ! 722 ! 723 ! 724 ! 725 ! 726 ! 727 ! 728 ! 729 ! ! 32! 730 ! 731 ! 732 ! ! 33! 733 ! 734 ! 735 ! 736 ! 737 ! 738 ! 739 ! 740 ! 741 ! 742 ! 743 ! 744 ! 745 ! 746 ! 747 ! 748 ! 749 ! 750 ! 751 ! 752 ! 753 ! 754 ! 755 ! 756 ! 757 ! ! 34! 758 ! 759 ! 760 ! 761 ! 762 ! 763 ! 764 ! 765 ! 766 ! Corrales(Guerrero+et#al.#+Figure+1! patS PCC 7120 CSVT20 CSL Δ patS patS* patS* SmR/SpR SmR/SpR Corrales(Guerrero+et#al.#+Figure+2! CSL21 CSVM17 CSL19 M★KAIMLVNFCDERGSGR MKAIM★LVNFCDERGSGR PpatS::gfpmut2 (α-plasmid) 0 h 5 h 8 h 27.5 h Percentage of counted intervals Corrales(Guerrero+et#al.#+Figure+3! Interval between heterocysts (number of cells) 19.00 ± 2.12 CSL48 12.39 ± 0.70 CSL47 9.02 ± 0.85 CSL45 20.29 ± 1.58 CSL22 5.31 ± 0.76 CSL44 18.67 ± 0.69 CSL49 10.56 ± 1.30 CSL50 10.09 ± 0.76 CSL52 8.35 ± 1.08 CSL53 7.45 ± 1.65 CSL54 10.24 ± 0.71 CSL55 12.02 ± 2.05 CSL56 11.46 ± 0.92 CSL57 22.75 ± 2.33 CSL59 15.30 ± 2.38 CSL60 16.92 ± 1.52 CSL61 16.03 ± 1.56 CSL62 6.01 ± 1.82 A CSL46 13.79 ± 1.12 CSL94 10.01 ± 0.61 CSL58 24.62 ± 3.79 10.31 ± 0.64 PCC!7120! A Q V Q E A Q A A A A A A Q CSVT20 CSL45 Corrales-Guerrero et al. Figure 4 PCC 7120 A B -50 0 50 100 150 200 250 300 350 h n1 n2 n3 n4 n5 n6 n7 h n1 n2 n3 n4 n5 n6 n7 Fluorescence (arbitrary units) PCC 7120 CSL45 h n1 n2 n3 n4 n5 n6 n7 Cell position Fluorescence (a.u.) 350 300 250 200 150 100 50 0 h n1 n2 n3 n4 n5 n6 n7 M1# A3# I4# M5# L6# V7# N8# F9# C10#D11#E12# R13# G14#S15#G16# R17#K2# Corrales-Guerrero et al. Figure 5