The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7

1" " The$ sRNA$ NsiR4$ is# involved# in# nitrogen( assimilation!control' in# cyanobacteria!by# targeting!glutamine* synthetase* inactivating! factor'IF7! " Stephan" Klähn1," Christoph" Schaal1," Jens" Georg1," Desirée" Baumgartner1," Gernot" Knippen1," Martin"Hagemann2,"Alicia"M."MuroAPastor3"and"Wolfgang"R."Hess1*" " 1Genetics"&"Experimental"Bioinformatics,"Faculty"of"Biology,"University"of"Freiburg,"Germany" 2Plant" Physiology" Department," Institute" of" Biological" Sciences," University" of" Rostock," Germany" 3Instituto" de" Bioquímica" Vegetal" y" Fotosíntesis," Consejo" Superior" de" Investigaciones" Científicas"and"Universidad"de"Sevilla,"Spain" " *Corresponding" author:" Wolfgang" R." Hess," University" of" Freiburg," Faculty" of" Biology," Schänzlestr."1,"DA79104"Freiburg,"Germany;"Tel:"+49A(0)761A203A2796;"Fax:"+49A(0)761A203A 2745" EAmail:"[email protected]freiburg.de" " Running!title:"Regulation"by"cyanobacterial"sRNA"NsiR4 Classification:"Biological"Sciences"(Plant"Biology"and"Microbiology)"" "" 2" " Abstract! Glutamine"synthetase"(GS),"a"key"enzyme"in"biological"nitrogen"assimilation,"is"regulated"in" multiple" ways" in"response" to" varying" nitrogen" sources" and" levels." Here" we" show" a" small" regulatory"RNA,"NsiR4"(nitrogen"stress"induced"RNA"4),"which"plays"an"important"role"in"the" regulation"of"GS"in"cyanobacteria."NsiR4"expression"in"the"unicellular"Synechocystis+sp."PCC" 6803"and"in"the"filamentous,"nitrogenAfixing"Anabaena"sp."PCC"7120"is"stimulated"through" nitrogenAlimitation" via" NtcA," the" global" transcriptional" regulator" of" genes" involved" in" nitrogen" metabolism." NsiR4" is" widely"conserved" throughout" the" cyanobacterial" phylum," suggesting" a" conserved" function." In+ silico" target" prediction," transcriptome" profiling" upon" pulse" overexpression" and" siteAdirected" mutagenesis" experiments" using" a" heterologous" reporter"system"showed"that"NsiR4"interacts"with"the"5’UTR"of"gifA+mRNA,"which"encodes" glutamine" synthetase" inactivating" factor" IF7." In" Synechocystis," we" observed" an" inverse" relationship" between" the" levels" of" NsiR4" and" the" accumulation" of" IF7" in+ vivo." This" NsiR4A dependent"modulation"of"gifA"(IF7)"mRNA"accumulation"influenced"the"glutamine"pool"and" thus"NH4+"assimilation"via"glutamine"synthetase."As"a"second"target,"we"identified"ssr1528,"a" hitherto" uncharacterized" nitrogenAregulated" gene." Competition" experiments" between" wild" type" and" an" NsiR4" knockAout" mutant" showed" that" the" lack" of" NsiR4" led" to" decreased" acclimation" capabilities" of" Synechocystis" towards" oscillating" nitrogen" levels." These" results" suggest"a"role"for"NsiR4"in"the"regulation"of"nitrogen"metabolism"in"cyanobacteria,"especially" for"the"adaptation"to"rapid"changes"in"available"nitrogen"sources"and"concentrations."NsiR4" is"the"first"identified"bacterial"sRNA"regulating"the"primary"assimilation"of"a"macronutrient." " Key! words:" regulatory" RNA," Synechocystis," nitrogen" assimilation," glutamine" synthetase" inactivating"factors" " " " 3" " Significance!Statement! Inorganic"nitrogen"assimilation"is"primarily"performed"through"phototrophic"organisms"that" donate" organic" nitrogen" to" heterotrophic" organisms." A" key" enzyme" in" this" process," glutamine" synthetase," is" the" target" of" multiple" regulatory" mechanisms." Here" we" describe" NsiR4," a" small" regulatory" RNA" that" reduces" the" expression" of" IF7," an" inhibitory" factor" of" glutamine" synthetase" in" cyanobacteria." The" expression" of" NsiR4" is" under" positive" control" through"the"transcription"factor"NtcA."NtcA"also"induces"the"transcription"of"the"glutamine" synthetase"gene"and"represses"the"gene"encoding"IF7."Therefore,"NsiR4"is"a"new"player"in" the"NtcAAmediated"regulation"of"nitrogen"assimilation,"which"is"important"for"adaptations"to" rapid"changes"in"available"nitrogen"sources"and"concentrations."" " " 4" " \body!! Introduction! Cyanobacteria"originated"oxygenic"photosynthesis"and"are"of"substantial"morphological"and" physiological"diversity."In"addition"to"the"photosynthetic"fixation"of"inorganic"carbon,"some" cyanobacteria" also" fix" atmospheric" dinitrogen," hence" injecting" combined" nitrogen" sources" into" the" biogeochemical" cycles." Therefore," cyanobacteria" play" an" important" role" in" global" carbon" and" nitrogen" cycles" (1," 2)." Unicellular" strains," such" as" the" nonAdiazotrophic" Synechocystis+ sp." PCC" 6803" (hereafter" Synechocystis"6803)," and" multicellular" strains" with" differentiated"cells,"such"as"the"diazotrophic"Anabaena"sp."PCC"7120"(hereafter"Anabaena" 7120),"have"been"established"as"widely"accepted"model"organisms."" Nitrogen! Assimilation! in! Cyanobacteria." Cyanobacteria" utilize" different" inorganic" and" organic"sources"of"combined"nitrogen,"such"as"ammonium"(NH4+),"nitrate"(NO3A),"nitrite"(NO2A )"and"urea,"or"some"amino"acids"(3–6)."Intracellularly,"NO3A,"NO2A"and"urea"are"converted"to" NH4+."The"availability" of"NH4+," the" energetically" most" favorable" nitrogen" source," represses" the"uptake"of"other"nitrogen"sources"(7)."As"in"eukaryotic"algae"and"plants,"the"key"enzyme" of"nitrogen"assimilation"is"glutamine"synthetase"(GS,"encoded"by"glnA),"which"incorporates" NH4+"into"metabolism"via"the"amidation"of"glutamate"to"glutamine."Subsequently,"glutamate" synthase" (glutamine" oxoglutarate" amidotransferase," GOGAT)" catalyzes" the" transfer" of" the" amide" group"from" glutamine" to" 2Aoxoglutarate" (2AOG)," producing" two" molecules" of" glutamate."The"postAtranslational"inactivation"of"GS"is"a"major"regulatory"mechanism"to"limit" nitrogen"assimilation"and"maintain"C/N"balance."Whereas"in"E.+coli"and"most"other"GramA negative" bacteria," GS" activity" is" modulated" through" adenylation/deadenylation" (8)," cyanobacteria"have"evolved"a"unique"mechanism"of"GS"inactivation"mediated"through"the" small"inhibitory"proteins"(inactivating"factors)"IF7"and"IF17,"encoded"by"the"genes"gifA"and" gifB,"respectively"(9)."In"addition"to"its"function"as"substrate"of"the"GSAGOGAT"cycle,"2AOG"is" also"a"metabolite"of"the"TCA"cycle,"connecting"nitrogen"and"carbon"metabolism"at"a"central" point."Depending"on"the"nitrogen"or"carbon"availability,"the"level"of"2AOG"varies,"making"this" metabolite"an"excellent"indicator"of"nitrogen"status"and"the"C/N"ratio"(10,"11)." The!Nitrogen!Regulatory!Network!in!Cyanobacteria."At"the"molecular"level,"2AOG"regulates" the" activity" of" the" main" transcriptional" regulators" of" nitrogen" assimilation" (NtcA," (12," 13))" 5" " and"carbon"metabolism"(NdhR/CcmR,"(14))."Moreover,"2AOG"also"modulates"the"activity"of" two"other"regulators"of"cyanobacterial"nitrogen"metabolism:"the"signal"transduction"protein" PII"and"the"regulatory"factor"PipX."Depending"on"the"2AOG"level,"which"reflects"the"nitrogen" status," PipX" interacts" with" either" NtcA" or" PII"(15," 16)." For" a" schematic" overview" of" the" nitrogen"regulatory"network,"see"Appendix,"Figure!S1." The" nitrogen" control" transcription" factor" NtcA" binds" as" a" homodimer" to" the" conserved" palindromic" sequence"GTAAN8ATAC"(17–20)." Under" nitrogen" excess" (e.g.," in" presence" of" NH4+),"the"2AOG"level"is"low"and"NtcA"is"present"in"an"inactive"form,"which"has"low"affinity"to" its"target"promoters"(21)."Under"nitrogen"depletion,"however,"the"2AOG"level"increases"and" stimulates"the"complex"formation"between"PipX"and"NtcA"and"the"affinity"of"the"binding"of" this"complex"to"target"promoters"(22,"23)."Depending"on"the"location"of"the"binding"motif" relative"to"the"transcriptional"start"site"(TSS),"NtcA"can"act"as"either"an"activator"or"repressor" (24)."NtcA"activates" the" expression" of" genes"that"are"important" when" nitrogen" is" limiting," such"as"nitrogen"uptake"systems"(e.g.,"amt1)"and"components"for"NO3A"(e.g.,"nirA)"and"NH4+" assimilation"(e.g.,"glnA)"(18)."In"contrast,"upon"nitrogen"depletion,"NtcA"acts"as"a"repressor" of"gifA"and"gifB,"which"encode"the"GS"IFs"(24)."" Bacterial!Small! RNAs! with! Regulatory! Potential." Bacterial" small" RNAs" (sRNAs)" can" postA transcriptionally" activate" or" repress" gene" expression" (25)." GenomeAwide" mapping" of" TSSs" revealed"a"high"number"of"potentially"regulatory"sRNAs"in"cyanobacteria"(19,"26,"27)."One"of" sRNAs," the" lightAregulated" and" widely" conserved" sRNA" PsrR1," is" a" key" regulator" of" photosynthesis"(28)."" The" expression"of" some" sRNAs" increases" in" nitrogenAlimited" cells" of" Anabaena"7120" or" Synechocystis"6803,"qualifying"these"molecules" as" potential" regulators"within"the"nitrogen" regulatory" network." Accordingly," these" sRNAs" were" named" nitrogen" stress" induced" RNAs" (NsiR)." The" expression" of" NsiR1+is" induced" upon" nitrogen" depletion," specifically" in" cells" of" Anabaena"7120" that" are" differentiating" as" nitrogenAfixing" heterocysts" (29," 30)." NsiR2" and" NsiR3"have"also"been"identified"in"Anabaena"7120"(19),"but"the"functions"of"these"molecules" have"remained"enigmatic."NsiR4"was"first"computationally"predicted"in"Synechocystis"6803" (31)" and" subsequently" verified" as" an" expressed" sRNA" (called" SyR12)" in" the" genomeAwide" mapping" of" TSSs" (26)." NsiR4" is"a"70" nt" transcript" that" becomes" strongly" induced" under" nitrogen"depletion"in"Synechocystis"6803"(27)."" 6" " Here" we" elucidated" the" molecular" functions" of" NsiR4" through" the" identification" of" two" different" targets," the" mRNAs" of" gifA" and" ssr1528," the" latter" encoding" a" conserved" but" uncharacterized"protein."We"present"evidence"for"an"additional"layer"of"GS"regulation"via"the" direct" interaction" of" NsiR4" with" the" 5’UTR" of" the" mRNA" of" the" GS" inactivating" factor" IF7," affecting" the" expression" of" this" molecule" and" thereby" impacting" GS" activity" and" NH4+" assimilation." Thus," NsiR4" is" the" first" example" of" an" sRNA" controlling" the" assimilation" of" a" macronutrient." Results! NsiR4!is!a!Widely!Conserved!sRNA!in!Cyanobacteria."The"phylogenetic"conservation"of"an" sRNA" is" an" indicator" of" functionality." Therefore," we" investigated" whether" NsiR4" homologs" are" also" detected" within" the" more" than" 200" cyanobacterial" genomes" publicly" available" through"the"Joint"Genome"Institute"(JGI)"database"in"January"2015."Using"BlastN,"putative" nsiR4"homologs" were" identified" in" at" least" 38" additional" genomes," including" the" closely" related"Synechocystis"sp." PCC" 6714" (hereafter" referred" to" as" Synechocystis"6714)" (32,"33)," more"distantly"related"strains,"such"as"Synechococcus"sp."PCC"7002,"or"several"Anabaena"and" Nostoc"species" (Fig.! 1A)." Homologs" of" NsiR4" were" not" found" in" αAcyanobacteria" (mainly" marine"Prochlorococcus"and"Synechococcus),"Thermosynechococcus,"Gloeobacter"as"well"as" some"Oscillatoria+(Fig.!S2)."We"concluded"that"nsiR4"homologs"exist"in"representatives"from" all" five" morphological" subsections" but" not" in" all" cyanobacteria" (34)," suggesting" a" widely" conserved"function."" In" addition" to" Synechocystis"6803," the" expression" of" nsiR4" was" observed" in" the" transcriptomic"datasets"available"for"Synechocystis"6714,"Anabaena"7120"and"Synechococcus+ sp." PCC" 7002"(19," 35," 36)." The" genomic" information" and" mapped" TSSs" indicated" that" two" distinct"NsiR4"forms"of"different"lengths"exist"in"different"cyanobacteria."NsiR4"in"strains"with" the"longer"form"(e.g.,"Synechocystis,"Microcystis,+Cyanothece)"possesses"an"additional"19A20" nt"domain"at"the"5’"end,"capable"to"form"a"short"hairpin"(Fig.!1A,B)."Otherwise,"the"predicted" secondary"structures"appeared"similar"for"both"NsiR4"types,"suggesting"a"potential"role"for" the"16"nt"singleAstranded"region,"which"is"nearly"identical"in"all"NsiR4"sequences"(Fig.!1A,B)." Such"regions"facilitate"interactions"with"the"respective"target"mRNAs."" 7" " NsiR4!Expression!is!Associated!with!the!Nitrogen!Status!and!Positively!Regulated!through! NtcA." NsiR4" is" one" of" the" most" highly" induced" sRNAs" upon" nitrogen" depletion" in" Synechocystis"6803"(27)"and"is"the"secondAmost"abundant"sRNA"in"the"primary"transcriptome" (37)."To"determine"the"time"course"of"induction"and"potential"effects"of"different"nitrogen" sources,"we"measured"NsiR4"expression"in"wildAtype"(WT)"Synechocystis"6803"grown"under" standard"conditions"(nitrogenAreplete,"17.6"mM"NO3A)"and"after"transfer"into"nitrogenAfree" medium."NsiR4"was"detected"at"a"low"level"in"cells"grown"in"the"presence"of"NO3A,"started"to" accumulate"at"a" higher" level" 12" h"after"removing"NO3A."After"48A72" h," the" expression" was" approximately" 10Afold" higher" than" at" the" start" of" the" experiment" (Fig.! 1C,!left" panel)." To" examine"the"influence"of"the"nitrogen"source,"three"WT"cultures"were"initially"grown"under" standard" conditions" and" then" transferred" to" nitrogenAfree" medium." Into" two" of" these" cultures,"we"added"10"mM"NH4+"or"17.6"mM"NO3A,"cultivation"was"continued"for"24"h,"and" the"relative"NsiR4"abundance"was"analyzed."Approximately"10Afold"higher"NsiR4"expression" was"observed"in"nitrogenAdepleted"cells"compared"with"cells"grown"in"the"presence"of"NO3A." In"contrast,"in"the"presence"of"NH4+,"NsiR4"levels"remained"below"the"detection"limit"(Figure! 1C,"right" panel)." We"concluded"that" the" expression"of"NsiR4" is"influenced"by"the" nitrogen" status" of" the" cell." Moreover," NsiR4" expression" is" also" regulated" through" nitrogen" in" Synechocystis"6714"and"Anabaena+7120"(19,"35),"suggesting"a"conserved"function"associated" with"nitrogen"metabolism." To" examine" whether" the" nitrogenAregulated" expression" of" NsiR4" is" under" transcriptional" control," we" conducted" reporter" gene" assays." The" upstream" sequence" of"nsiR4+ from" Synechocystis+ 6803" was" fused" to" luxAB"genes" encoding" luciferase," and" expression" was" measured"as"bioluminescence"in+vivo."Indeed,"the"promoter"activity"showed"similar"kinetics" in"response"to"nitrogen"depletion,"as"observed"for"the"sRNA"accumulation"(Fig.!2A)."In"NtcAA activated"promoters,"NtcAAbinding"sites"frequently"overlap"the"A35"region"and"are"centered" close" to" position" A41.5" with" respect" to" the" TSS" (18," 19)." Indeed," the" sequence" 5’A GTCAAATAGGCTACA3’,"similar"to"the"consensus"for"NtcA"binding"sites"(17),"is"located"48A35" nt"upstream" the"TSS"of"nsiR4"(at" pos."1289326"on" the"complementary"strand)." Moreover," potential" NtcA" binding" sites" exist" upstream" of" the" putative" nsiR4"homologs" in" all" other" strains" (Figure! 2C)."We" examined" the" functionality" of" this" site" in" a" reporter" gene" assay" comparing" the" WT" promoter" with" a" mutated" version" in" which" three" nucleotides" were" substituted" (GTCAAATAGGCTAC" to" CTCAAATAGGCATC)." Indeed," the" activation" of" luxAB" 8" " expression"through"nitrogen"depletion"was"lost"in"the"strain"carrying"the"mutated"promoter" (Fig.!2B)."Moreover,"NsiR4"expression"could"not"be"activated"in"the"nitrogenAdepleted"cells" of" an" Anabaena"7120"ntcA" mutant" (Fig.! 2D)." These" data" demonstrate" that" the" NsiR4" promoter"is"under"NtcA"control"in"both"Synechocystis+6803"and"Anabaena"7120."" Advanced!Target!Prediction!and!Transcriptome!Analyses!Suggest!gifA!and!ssr1528!as!NsiR4! Targets."To"predict"potential"targets"of"NsiR4,"we"used"the"CopraRNA"algorithm,"considering" the"folding,"hybridization"and"conservation"of"a"particular"sRNA"(38)."The"highest"interaction" probability"was"predicted"for"the"gifA"(ssl1911)"mRNA"(Table!1)."This"gene"encodes"the"GS" inactivating"factor"IF7"and"is"also,"but"negatively,"regulated"through"NtcA."" To"identify"potential"target"genes"experimentally,"we"generated"Synechocystis"strains"with" altered" NsiR4" expression," i.e.," overexpression" (NsiR4oex)," knockout"(∆nsiR4)" and" compensatory" (∆nsiR4::oex)" strains." To" achieve" inducible"expression," we" fused" the" NsiR4" sequence"to"the"petE"promoter,"which"is"specifically"activated"in"the"presence"of"Cu2+."The" absence"of"NsiR4"in"the"knockout"strain"and"the"Cu2+Adependent"inducibility"of"NsiR4"in"the" other"two"strains"were"experimentally"verified"(Fig.!S3)." The" targetAsRNA" interaction" frequently" reduces" mRNA" stability" (39)." Thus," a" microarray" experiment" was" performed" with" RNA" extracted" from"cells" in" which" NsiR4" was" pulseA expressed"through"the"addition"of"Cu2+"for"12"h."Compared"with"WT,"in"NsiR4oex"reduced" gifA+ mRNA" abundance" was" observed," for" which" an" interaction" was" also" predicted" by" the" CopraRNA" algorithm" (compare" Table! 1"&" Table! S1)." Additionally," gifA"expression" was" increased" in" ∆nsiR4,"but" decreased" upon" introduction" of" the" compensatory" construct" (∆nsiR4::oex)"(Table!S1)."Another"gene,"ssr1528,"exhibited"a"similar"pattern,"suggesting"that" this"gene"is"a"second"target"of"NsiR4."The"product"of"ssr1528+is"a"hypothetical"protein,"with"a" domain" (DUF4090)" widely" conserved" among" cyanobacteria" (Fig.! S2)." Moreover," similar" to" gifA,"the"expression"of"this"protein"is"repressed"under"nitrogen"depletion"(40),"suggesting"a" function"related"to"the"cellular"nitrogen"status." We"speculated"that"the"modulation"of"NsiR4"expression"and"its"potential"impact"on"nitrogenA associated"target"genes"might"also"affect"growth"or"the"tolerance"to"high"concentrations"of" NH4+." In" shortAterm" growth" experiments" strains" with" altered" levels" of" NsiR4" showed" no" noticeable" phenotype" regarding" NH4+" tolerance" (Fig.! S4)." However," in" longAterm" growth" competition" experiments," especially" when" the" availability" of" nitrogen" is" fluctuating," the" 9" " ΔnsiR4"mutant"strain"showed"reduced"growth"rates"compared"to"WT"(Fig.!3)."These"findings" directly" support" the" physiological" importance" of" NsiR4" in" addition" to" its" evolutionary" conservation," suggesting" that" the" presence" of" NsiR4" has" been" under" positive" selection" in" cyanobacteria."" The! ssr1528!Gene! is! Directly! and! PostOTranscriptionally! Regulated! through! NsiR4." The" changes"observed"in"the"microarray"experiment"were"verified"through"a"detailed"expression" analysis."The"expression"of"ssr1528+was"detected"in"WT"under"standard"conditions"(17.6"mM" NO3A)," which" was" not" significantly" affected" by" the" addition" of" Cu2+" (Fig.! 4A)." However," in" strain" NsiR4oex" carrying" an" additional," PpetEAregulated" nsiR4"copy," ssr1528" mRNA" abundance" was" diminished" upon" NsiR4" induction." Consistently," ssr1528" mRNA" abundance" increased"in"strain"∆nsiR4"compared"to"WT."In"response"to"nitrogen"depletion,"the"ssr1528" mRNA" levels" decreased" in" WT" and" NsiR4oex," confirming" the" NAregulated" expression." However," whereas" the" ssr1528" mRNA" disappeared" in" WT" after" 12" h" N" depletion," the" expression" of" this" gene" remained" at" a" high" level" in" ∆nsiR4" (Fig.! 4A)." Moreover," ssr1528" expression"did"not"increase"when"10"mM"NH4+"was"added"to"the"medium"(Fig.!S5),"indicating" that"the"decrease"under"N"depletion"is"not"directly"NtcAAdependent"and"might"be"exclusively" regulated"through"NsiR4"at"the"postAtranscriptional"level." To" examine" whether" a" direct" NsiR4:ssr1528" interaction" caused" the" observed" changes" in" mRNA" expression," the" ssr1528" 5’UTR" was" fused" to" the" gene" for" the" superfolder" green" fluorescent" protein" (sgfp)" and" coAexpressed" with" NsiR4" in" E.+ coli." GFP" fluorescence" was" measured" in" strains" carrying" various" combinations" of" plasmids" (41)." Compared" with" the" control"(pXGA0+pJV300),"the"strain"carrying"the"ssr1528Ksgfp"fusion"showed"significant"GFP" fluorescence," demonstrating" that" the" translation" initiation" from" the" ssr1528"5’UTR"of" Synechocystis"is"also"functional"in"E.+coli"(Fig.!4B)."In"the"presence"of"the"NsiR4Aexpressing" plasmid," the" GFP" fluorescence" decreased" approximately" 2Afold," indicating" a" direct" interaction"between"NsiR4"and"the"ssr1528A5’UTR,"which" affects"translation." To" verify" the" interaction"at"the"predicted"site,"point"mutations"were"introduced"in"ssr1528"(pos."+22"and" +23:"UA>AU,"+1"="TSS)"or"NsiR4"(pos."+16"and"+17:"UA>AU,"Fig.!4D)."Indeed,"the"mutation"of" either"one"of"these"sequences"diminished"the"interaction,"indicated"as"reduced"repression" (Fig.!4C)."However,"the"combination"of"both"mutations"restored"repression"(both"mutations" 16" " reporter" strains" and" bioluminescence" measurements" were" performed" as" described" (52)." Target" prediction" was" performed" using" CopraRNA" (38," 42)" with" the" NsiR4" homologs" of" Synechocystis"6803," Microcystis+ aeruginosa" NIESA843," Cyanothece"sp." ATCC" 51142," Cyanothece"sp." PCC" 7424," Cyanothece"sp." PCC" 7822," Synechococcus"sp." PCC" 7002," Pleurocapsa"sp."PCC"7327"and"Stanieria+cyanosphaera"PCC"7437"and"default"settings." RNA!extraction,!microarrays!and!Northern!blots."The"collection"of"Synechocystis"cells,"the" RNA"extraction,"DNase"treatment,"labeling"and"hybridization"to"microarrays"was"performed+ as"previously"described"(55,"56,"57)."For"Northern"hybridization,"Synechocystis"6803"RNA"was" separated" on" denaturing" agarose" gels," transferred" to" HybondAN+" membranes" (Amersham," Germany)"and"hybridized"as"previously"described"(55)."" Protein! extraction! and! immunoblots." For" analysis" of" IF" accumulation," 2" µM" CuSO4" was" added"to"induce"the"petE"promoter"eight"hours"before"addition"of"10"mM"NH4Cl"and"20"mM" TESANaOH"(pH"7.5)."Extracts"were"prepared"using"glass"beads"as"previously"described"(56)." Proteins" were" fractionated" by"15%" SDSAPAGE" and" immunoblotted" with" antiAIF7" (1:2000)," antiAIF17" (1:2000)" or" antiATrxA" (1:3000)." AntiAIF7," antiAIF17" and" antiATrxA" antisera" were" obtained"from"M.I."MuroAPastor"and"F.J."Florencio"and"used"as"described"(57,"58)."" Reporter! assays! for! the! in, vivo!verification! of! targets." We" used" the" reporter" system" described"before"(59)"with"sGFP"plasmid"pXGA10ASF"(41)."The"primers"used"for"cloning"and" the" resulting" plasmids" are" given" in" Tables! S2! and! S3." Further" details" of" the" generation" of" tested"constructs"can"be"found"in"the"SI!Appendix."" Quantification!of!amino!acids."Cells"were"grown"in"100"ml"BG11"containing"17.6"mM"NO3A." After"reaching"an"OD750"of"ca."0.8,"2"µM"CuSO4" and"after"further"24"h"10"mM"NH4Cl"were" added." Samples" of" 2" ml" were" taken" prior" to" and" in" a" narrow" time" series" after" adding" ammonia."Samples"were"shortly"centrifuged"and"cell"pellets"frozen"in"liquid"nitrogen."Free" amino"acids"were"extracted"from"frozen"cyanobacterial"cells"with"80%"ethanol"at"65°C"for"3" h."After"centrifugation,"the"extraction"was"repeated"for"the"remaining"cell"pellet."Afterwards," both"supernatants"were"combined,"dried"in"a"vacuum"centrifuge"and"reAdissolved"in"sample" buffer."Individual"amino"acids"were"quantified"via"HPLC"(60)." " 17" " Author!contributions! S.K.," J.G.," A.M.M.AP." &" W.R.H." designed" and" supervised" the" study." J.G." &" C.S." performed" genome" analysis" and" target" prediction." S.K." &" J.G." generated" the" Synechocystis"nsiR4" mutants." S.K.," D.B." &" G.K." performed" promoter" analysis." C.S." &" A.M.M.AP." performed" northern" blots." A.M.M.AP." performed" western" blot" experiments." C.S." performed" target" verification" experiments." M.H." contributed" the" amino" acid" data." S.K." performed" the" microarray"data"analysis,"growth"competition"assay"and"prepared"all"figures."S.K.,"A.M.M.AP." &"W.R.H."evaluated"and"interpreted"the"data"and"drafted"the"manuscript"with"contributions" from"all"authors."All"authors"proofread"the"manuscript." Acknowledgments! This" work" was" supported" through" grants" from" the" Federal" Ministry" of" Education" and" Research"“e:bio"CYANOSYSII”"0316183"(to"W.R.H."and"M.H.)"and"the"Ministerio"de"Economía" y"Competitividad,"Spain"(grant"BFU2013A48282AC2A1AP"to"A.M.M.AP,"cofinanced"through"the" European"Regional"Development"Fund)."The"authors"thank"Gudrun"Krüger"and"Klaudia"Michl" for"technical"assistance."We"thank"M.I."MuroAPastor"and"F.J."Florencio"for"providing"antisera."" " " 18" " References! 1."" Montoya" JP," et" al." (2004)" High" rates" of" N2" fixation" by" unicellular" diazotrophs" in" the" oligotrophic"Pacific"Ocean."Nature"430(7003):1027–1032." 2."" LaRoche" J," Breitbarth" E" (2005)" Importance" of" the" diazotrophs" as" a" source" of" new" nitrogen"in"the"ocean."J+Sea+Res+53(1–2):67–91." 3."" Omata" T," Andriesse" X," Hirano" A" (1993)" Identification" and" characterization" of" a" gene" cluster"involved"in"nitrate"transport"in"the"cyanobacterium"Synechococcus"sp."PCC7942." Mol+Gen+Genet"236(2A3):193–202." 4."" Montesinos" ML," Herrero" A," Flores" E" (1997)" Amino" acid" transport" in" taxonomically" diverse"cyanobacteria"and"identification"of"two"genes"encoding"elements"of"a"neutral" amino" acid" permease" putatively" involved" in" recapture" of" leaked" hydrophobic" amino" acids."J+Bacteriol"179(3):853–862." 5."" Montesinos" ML," MuroAPastor" AM," Herrero" A," Flores" E" (1998)" Ammonium/methylammonium" permeases" of" a" cyanobacterium." Identification" and" analysis" of" three" nitrogenAregulated" amt"genes" in" Synechocystis"sp." PCC" 6803." J+ Biol+ Chem"273(47):31463–31470." 6."" Valladares"A,"Montesinos"ML,"Herrero"A,"Flores"E"(2002)"An"ABCAtype,"highAaffinity"urea" permease"identified"in"cyanobacteria."Mol+Microbiol"43(3):703–715." 7."" Flores"E,"Herrero"A"(2005)"Nitrogen"assimilation"and"nitrogen"control"in"cyanobacteria." Biochem+Soc+Trans"33(Pt"1):164–167." 8."" Merrick"MJ,"Edwards"RA"(1995)"Nitrogen"control"in"bacteria."Microbiol+Rev"59(4):604– 622." 9."" GarcíaADomínguez"M,"Reyes"JC,"Florencio"FJ"(1999)"Glutamine"synthetase"inactivation" by"protein–protein"interaction."Proc+Natl+Acad+Sci+USA"96(13):7161–7166." 10."" MuroAPastor"MI,"Reyes"JC,"Florencio"FJ"(2001)"Cyanobacteria"perceive"nitrogen"status" by"sensing"intracellular"2Aoxoglutarate"levels."J+Biol+Chem"276(41):38320–38328." 11."" Forchhammer" K" (2004)" Global" carbon/nitrogen" control" by" PII" signal" transduction" in" cyanobacteria:"from"signals"to"targets."FEMS+Microbiol+Rev"28(3):319–333." 12."" VázquezABermúdez"MF,"Herrero"A,"Flores"E"(2002)"2AOxoglutarate"increases"the"binding" affinity"of"the"NtcA"(nitrogen"control)"transcription"factor"for"the"Synechococcus+glnA" promoter."FEBS+Lett"512(1A3):71–74." 13."" Tanigawa" R," et" al." (2002)" Transcriptional" activation" of" NtcAAdependent" promoters" of" Synechococcus"sp." PCC" 7942" by" 2Aoxoglutarate" in+ vitro." Proc+ Natl+ Acad+ Sci+ USA" 99(7):4251–4255." 19" " 14."" Daley"SME,"Kappell"AD,"Carrick"MJ,"Burnap"RL"(2012)"Regulation"of"the"cyanobacterial" CO2Aconcentrating"mechanism" involves" internal"sensing"of" NADP+" and"αAketogutarate" levels"by"transcription"factor"CcmR."PLoS+ONE"7(7):e41286." 15."" Llácer" JL," et" al." (2010)" Structural" basis" for" the" regulation" of" NtcAAdependent" transcription"by"proteins"PipX"and"PII."Proc+Natl+Acad+Sci+USA"107(35):15397–15402." 16."" Espinosa" J," et" al." (2014)" PipX," the" coactivator" of" NtcA," is" a" global" regulator" in" cyanobacteria."Proc+Natl+Acad+Sci+USA"111(23):E2423–2430." 17."" Luque"I,"Flores"E,"Herrero"A"(1994)"Molecular"mechanism"for"the"operation"of"nitrogen" control"in"cyanobacteria."EMBO+J"13(12):2862–2869." 18."" Herrero" A," MuroAPastor" AM," Flores" E" (2001)" Nitrogen" control" in" cyanobacteria." J+ Bacteriol"183(2):411–425." 19."" Mitschke" J," Vioque" A," Haas" F," Hess" WR," MuroAPastor" AM" (2011)" Dynamics" of" transcriptional"start"site"selection"during"nitrogen"stressAinduced"cell"differentiation"in" Anabaena"sp."PCC7120."Proc+Natl+Acad+Sci+USA"108(50):20130–20135." 20."" Picossi" S," Flores" E," Herrero" A" (2014)" ChIP" analysis" unravels" an" exceptionally" wide" distribution" of" DNA" binding" sites" for" the" NtcA" transcription" factor" in" a" heterocystA forming"cyanobacterium."BMC+Genomics"15:22." 21."" Luque"I,"VázquezABermúdez"MF,"PazAYepes"J,"Flores"E,"Herrero"A"(2004)"In+vivo"activity" of"the"nitrogen"control"transcription"factor"NtcA"is"subjected"to"metabolic"regulation"in" Synechococcus"sp."strain"PCC"7942."FEMS+Microbiol+Lett"236(1):47–52." 22."" Espinosa" J," Forchhammer" K," Burillo" S," Contreras" A" (2006)" Interaction" network" in" cyanobacterial" nitrogen" regulation:" PipX," a" protein" that" interacts" in" a" 2Aoxoglutarate" dependent"manner"with"PII"and"NtcA."Mol+Microbiol"61(2):457–469." 23."" ForcadaANadal"A,"Forchhammer"K,"Rubio"V"(2014)"SPR"analysis"of"promoter"binding"of" Synechocystis"PCC6803" transcription" factors" NtcA" and" CRP" suggests" crossAtalk" and" sheds"light"on"regulation"by"effector"molecules."FEBS+Lett"588(14):2270–2276." 24."" GarcíaADomínguez"M,"Reyes"JC,"Florencio"FJ"(2000)"NtcA"represses"transcription"of"gifA" and" gifB," genes" that" encode" inhibitors" of" glutamine" synthetase" type" I" from" Synechocystis"sp."PCC"6803."Mol+Microbiol"35(5):1192–1201." 25."" Waters"LS,"Storz"G"(2009)"Regulatory"RNAs"in"bacteria."Cell"136(4):615–628." 26."" Mitschke"J,"et"al."(2011)"An"experimentally"anchored"map"of"transcriptional"start"sites" in" the" model" cyanobacterium" Synechocystis"sp." PCC6803." Proc+ Natl+ Acad+ Sci+ USA" 108(5):2124–2129." 27."" Kopf" M," et" al." (2014)" Comparative" analysis" of" the" primary" transcriptome" of" Synechocystis"sp."PCC"6803."DNA+Res"21(5):527–539." 20" " 28."" Georg" J," et" al." (2014)" The" small" regulatory" RNA" SyR1/PsrR1" controls" photosynthetic" functions"in"cyanobacteria."Plant+Cell"26(9):3661–3679." 29."" Ionescu" D," Voss" B," Oren" A," Hess" WR," MuroAPastor" AM" (2010)" HeterocystAspecific" transcription"of"NsiR1,"a"nonAcoding"RNA"encoded"in"a"tandem"array"of"direct"repeats"in" cyanobacteria."J+Mol+Biol"398(2):177–188." 30."" MuroAPastor"AM"(2014)"The"heterocystAspecific"NsiR1"small"RNA"is"an"early"marker"of" cell"differentiation"in"cyanobacterial"filaments."MBio"5(3):e01079–01014." 31."" Voss"B,"Georg"J,"Schön"V,"Ude"S,"Hess"WR"(2009)"Biocomputational"prediction"of"nonA coding"RNAs"in"model"cyanobacteria."BMC+Genomics"10:123." 32."" Kopf"M,"et"al."(2014)"Comparative"genome"analysis"of"the"closely"related"Synechocystis" strains"PCC"6714"and"PCC"6803."DNA+Res"21(3):255–266." 33."" Kopf" M," et" al." (2014)" Finished" Genome" Sequence" of" the" Unicellular" Cyanobacterium" Synechocystis" sp." Strain" PCC" 6714." Genome+ Announc"2(4)." doi:10.1128/genomeA.00757A14." 34."" Rippka" R," Deruelles" J," Waterbury" JB," Herdman" M," Stanier" RY" (1979)" Generic" assignments," strain" histories" and" properties" of" pure" cultures" of" cyanobacteria." J+ Gen+ Microbiol"111(1):1–61." 35."" Kopf" M," Klähn" S," Scholz" I," Hess" WR," Voss" B" (2015)" Variations" in" the" nonAcoding" transcriptome" as" a" driver" of" interAstrain" divergence" and" physiological" adaptation" in" bacteria."Scientific+Reports"5:9560." 36."" Ludwig" M," Bryant" DA" (2012)" Acclimation" of" the" global" transcriptome" of" the" cyanobacterium"Synechococcus"sp."strain"PCC"7002"to"nutrient"limitations"and"different" nitrogen"sources."Front+Microbiol"3:145." 37."" Kopf" M," Hess" WR" (2015)" Regulatory" RNAs" in" photosynthetic" cyanobacteria." FEMS+ Microbiol+Rev"39(3):301–315." 38."" Wright" PR," et" al." (2013)" Comparative" genomics" boosts" target" prediction" for" bacterial" small"RNAs."Proc+Natl+Acad+Sci+USA"110(37):E3487–3496." 39."" Vogel"J,"Wagner"EGH"(2007)"Target"identification"of"small"noncoding"RNAs"in"bacteria." Curr+Opin+Microbiol"10(3):262–270." 40."" Krasikov"V,"Aguirre"von"Wobeser"E,"Dekker"HL,"Huisman"J,"Matthijs"HCP"(2012)"TimeA series"resolution"of"gradual"nitrogen"starvation"and"its"impact"on"photosynthesis"in"the" cyanobacterium"Synechocystis"PCC"6803."Physiol+Plantarum"145(3):426–439." 41."" Corcoran"CP,"et"al."(2012)"Superfolder"GFP"reporters"validate"diverse"new"mRNA"targets" of"the"classic"porin"regulator,"MicF"RNA."Mol+Microbiol"84(3):428–445." 42."" Wright"PR,"et"al."(2014)"CopraRNA"and"IntaRNA:"predicting"small"RNA"targets,"networks" and"interaction"domains."Nucleic+Acids+Res"42:W119–123." 21" " 43."" Wenner" N," Maes" A," CotadoASampayo" M," Lapouge" K" (2014)" NrsZ:" a" novel," processed," nitrogenAdependent," small" nonAcoding" RNA" that" regulates" Pseudomonas+ aeruginosa" PAO1"virulence."Environ+Microbiol"16(4):1053–1068." 44."" Sharma" CM," et" al." (2011)" Pervasive" postAtranscriptional" control" of" genes" involved" in" amino" acid" metabolism" by" the" HfqAdependent" GcvB" small" RNA." Mol+ Microbiol" 81(5):1144–1165." 45."" Brauer"MJ,"et"al."(2006)"Conservation"of"the"metabolomic"response"to"starvation"across" two"divergent"microbes."Proc+Natl+Acad+Sci+USA"103(51):19302–19307." 46."" Yuan"J,"et"al."(2009)"MetabolomicsAdriven"quantitative"analysis"of"ammonia"assimilation" in"E.+coli."Mol+Syst+Biol"5:302." 47."" Saelices"L,"RoblesARengel"R,"Florencio"FJ,"MuroAPastor"MI"(2015)"A"core"of"three"amino" acids"at"the"carboxylAterminal"region"of"glutamine"synthetase"defines"its"regulation"in" cyanobacteria."Mol+Microbiol."96(3):483A496." 48."" Maheswaran"M,"Urbanke"C,"Forchhammer"K"(2004)"Complex"Formation"and"Catalytic" Activation" by" the" PII" Signaling" Protein" of" NAAcetylAlAglutamate" Kinase" from" Synechococcus+elongatus"Strain"PCC"7942."J+Biol+Chem"279(53):55202–55210." 49."" Mangan" S," Alon" U" (2003)" Structure" and" function" of" the" feedAforward" loop" network" motif."Proc+Natl+Acad+Sci+USA"100(21):11980–11985." 50."" Beisel" CL," Storz" G" (2011)" The" baseApairing" RNA" spot" 42" participates" in" a" multioutput" feedforward" loop" to" help" enact" catabolite" repression" in" Escherichia+ coli." Mol+ Cell" 41(3):286–297." 51."" Frías"JE,"Flores"E,"Herrero"A"(1994)"Requirement"of"the"regulatory"protein"NtcA"for"the" expression" of" nitrogen" assimilation" and" heterocyst" development" genes" in" the" cyanobacterium"Anabaena"sp."PCC"7120."Mol+Microbiol"14(4):823–832." 52."" Klähn" S," et" al." (2014)" Alkane" biosynthesis" genes" in" cyanobacteria" and" their" transcriptional"organization."Front+Bioeng+Biotechnol"2:24." 53."" Hein"S,"Scholz"I,"Voß"B,"Hess"WR"(2013)"Adaptation"and"modification"of"three"CRISPR" loci"in"two"closely"related"cyanobacteria."RNA+Biol"10(5):852–864." 54.""Georg" J," et" al." (2009)" Evidence" for" a" major" role" of" antisense" RNAs" in" cyanobacterial" gene"regulation."Mol+Syst+Biol"5:305." 55."" Steglich"C,"et"al."(2008)"The"challenge"of"regulation"in"a"minimal"photoautotroph:"nonA coding"RNAs"in"Prochlorococcus."PLoS+Genet"4(8):e1000173." 56."" Reyes"JC,"Florencio"FJ"(1995)"A"novel"mechanism"of"glutamine"synthetase"inactivation" by"ammonium"in"the"cyanobacterium"Synechocystis" sp."PCC" 6803."Involvement"of"an" inactivating"protein."FEBS+Lett"367(1):45–48." 22" " 57."" Navarro" F," MartínAFigueroa" E," Florencio" FJ" (2000)" Electron" transport" controls" transcription" of" the" thioredoxin" gene" (trxA)" in" the" cyanobacterium" Synechocystis"sp." PCC"6803."Plant+Mol+Biol"43(1):23–32." 58."" Galmozzi" CV," FernándezAAvila" MJ," Reyes" JC," Florencio" FJ," MuroAPastor" MI" (2007)" The" ammoniumAinactivated"cyanobacterial"glutamine"synthetase"I"is"reactivated"in+vivo"by"a" mechanism" involving" proteolytic" removal" of" its" inactivating" factors." Mol+ Microbiol" 65(1):166–179." 59."" Urban"JH,"Vogel"J"(2007)"Translational"control"and"target"recognition"by"Escherichia+coli" small"RNAs"in+vivo."Nucleic+Acids+Res"35(3):1018–1037." 60."" Hagemann" M," Vinnemeier" J," Oberpichler" I," Boldt" R," Bauwe" H" (2005)" The" glycine" decarboxylase"complex"is"not"essential"for"the"cyanobacterium"Synechocystis"sp."strain" PCC"6803."Plant+Biol+(Stuttg)"7(1):15–22." 61."" Smith" C," Heyne" S," Richter" AS," Will" S," Backofen" R" (2010)" Freiburg" RNA" Tools:" a" web" server"integrating"INTARNA,"EXPARNA"and"LOCARNA."Nucleic+Acids+Res"38i:W373–377." "" " 23" " Figure!Legends! " Fig.!1:!The!nitrogenOstress!induced!RNA!4!(NsiR4)!is!broadly!conserved!in!cyanobacteria." A:!Alignment"of"genome"loci"putatively"encoding"NsiR4."Verified"TSSs"are"boxed"in"red"(19," 26," 35)." Roman" Numerals" indicate" the" respective" morphological" subsections" (34)." B:! Conservation"of"the"NsiR4"structure."The"consensus"structure"was"predicted"using"RNAalifold" (61)" and" 30" randomly" selected" NsiR4" homologs" of" both" types." The" short" hairpin" was" predicted" for" the" 5’" extension" of" long" NsiR4" forms." The" color" indicates" the" number" of" different"interacting"pairs"(CAG,"GAC,"AAU,"UAA,"GAU"or"UAG)"and"thereby"conservation"of"these" base" pairs." The" saturation" increases" with" the" number" of" compatible" base" pairs," thus" indicating" the" structural" conservation." C:! NitrogenAresponsive" expression" of" NsiR4" in" Synechocystis"6803.!Cells"were"transferred"from"standard"conditions"(17.6"mM"NO3A)"to"NO3A Afree" BG11," total" RNA" was" separated," blotted" and" hybridized" with" 32PAlabeled," singleA stranded"RNA" probes."Left"panel:" Expression" kinetics"of"NsiR4" in"response"to" NAdepletion." Right" panel:" SteadyAstate" levels" in" response" to" the" nitrogen" status" mediated" through" different"nitrogen"sources"or"the"depletion"of"N."Cells"grown"in"the"presence"of"NO3A"were" washed"and"resuspended"in"media"containing"17.6"mM"NO3A,"10"mM"NH4+"or"no"nitrogen" source."The"cells"were"grown"for"an"additional"24"hours"before"RNA"extraction." " Fig.!2.!NsiR4!expression!is!mediated!through!an!NtcAOactivated!promoter.! A:!Bioluminescence"of"a"Synechocystis"reporter"strain"harboring"a"transcriptional"fusion"of" PnsiR4"(A130"to"+49,"TSS"at"+1)"and"luxAB+genes"in"response"to"N"depletion."Initially,"cells" were"grown"under"standard"conditions"(17.6"mM"NO3A)"and"subsequently"transferred"to"NO3A Afree" BG11" medium." B:! Bioluminescence" of" a" Synechocystis" PnsiR4::luxAB" reporter" strain" bearing"a"mutated"NtcA"motif"in"the"presence"of"17.6"mM"NO3A"(+N)"or"under"NAdepletion" for"24"h"(AN)."To"improve"and"compare"bioluminescence"mediated"through"both"promoters" under"+N,"10" mM" glucose" was" added" to"the"cultures."Thus,"the" values" are" higher" than" in" panel" A." Bioluminescence" data" are" presented" as" the" means" ±" SD" of" n" independent" measurements"in"at"least"two"independent"experiments,"including"two"biological"replicates" (="independent"transformants)."In"each"experiment,"a"strain"carrying"a"promoterless"luxAB" 24" " was"used"as"a"negative"control"(in"each"case"measured"in"two"independent"cultures,"n=2)."C:! The"sequences"upstream"of"nsiR4"from"different"cyanobacteria."Putative"NtcA"binding"sites" and"A10"elements"are"highlighted."The"verified"TSSs"are"shown"in"red."D:!Verification"of"NA regulated" and" NtcAAdependent" NsiR4" expression" in" Anabaena" 7120" WT" and" an" ntcA" insertion"mutant"through"northern"blot"analysis." " Fig.! 3.!Competitive! growth! analysis! of! WT! and! ΔnsiR4.! A:! Experimental" setup." Three" independent,"exponentially"growing"cultures"of"WT"and"ΔnsiR4"were"diluted"to"an"OD750"of" 0.1"and"mixed"1:1."B:"Growth"performance"during"longAterm"cultivation."Cultures"were"reA diluted"every"3"or"4"days"to"an"OD750"of"0.1"and"25"µl"of"a"1:1000"dilution"were"dropped"on" BG11"agar"plates"with"and"without"40"µg/ml"kanamycin."Data"are"the"mean"±"SD"of"WT"and" ΔnsiR4+as"well"as"three"mixed"WT/ΔnsiR4"coAcultures."Due"to"the"lowered"NO3A"content"(1" mM"instead"of"17.6"mM"NO3A),"a"strong"fluctuation"in"the"cellular"N"status"could"be"assumed," which"was"also"indicated"by"the"cultures"switching"between"green"or"bleached"appearance" at"lower"(0.1A0.8)"and"higher"(>0.8)"ODs"(see"also"C)."C:!Whole"cell"absorption"spectra"of"the" WT" culture" indicating" cellular" bleaching" was" due" to" pigment" degradation" as" response" to" initiated"internal"N"limitation."D:!Number"of"generations"that"were"observed"over"50"days"at" average" growth" rates" of" µ" =" 0.751" ±" 0.065." E:! Evolution" of" the" ratio" between" kanamycinA resistant" (ΔnsiR4)" and" total" (WT" +" ΔnsiR4)" colony" forming" units" (CFU)" during" consecutive" cultivation"and"reAdilution."F:!Photographs"of"CFUs"at"the"beginning"of"the"experiment"and" after"33"and"48"generations"(g)."G:!Verification"of"the!ΔnsiR4"mutant"allele"reduction"(upper" band)"compared"to"the"WT"allele"(lower"band)"by"PCR."*To"one"of"the"WT/ΔnsiR4"coAcultures" once"per"day"100"µM"NH4Cl"(f.c.)"were"added"to"intensify"the"fluctuation"in"the"N"availability." However," no" change" in" growth" performance" compared" to" the" other" two" coAcultures" was" observed."Thus,"all"three"coAcultures"were"averaged"as"shown"in"panel"E." " Fig.!4.! Verification!of!the! postOtranscriptional!regulation!of!ssr1528!through!direct! NsiR4! interaction.! A:! Changes" in" the" mRNA" abundance" of" ssr1528" in" response" to" altered" NsiR4" expression" detected" by" hybridization" with" a" singleAstranded" 32PAlabeled" transcript" probe." Nitrogen" 25" " depletion"was"induced"in"cultures"grown"in"the"presence"of"2"µM"Cu2+"for"48"h"(="0"h"AN)."A" representative"5S"rRNA"loading"control"hybridization"(for"WT)"is"shown.!WT"A"Synechocystis" 6803" wild" type," NsiR4oex" –" strain" carrying" pVZ322APpetE::nsiR4::oop" plasmid" (overexpressor)," ∆nsiR4" A" deletion" mutant," ∆nsiR4::oex" A" deletion" strain" in" which" NsiR4" expression" was" restored" by" the" pVZ322APpetE::nsiR4::oop" plasmid." B:! GFP" fluorescence" measurements" of" E.+ coli+ TOP10+strains" with" various" combinations" of" plasmids" expressing" NsiR4" or" ssr1528AsgfpAfusions." The" plasmids" pXGA0" (encoding" luciferase)" and" pJV300" (encoding"a"control"RNA)"were"used"as"negative"controls"(for"experimental"details"see"(41)).! C:! Repression" was" calculated" by" dividing" the" values" measured" for" ssr1528Asgfp" in" the" presence" of" pJV300" with" the" corresponding" value" when" NsiR4" was" present." Autofluorescence" measured" for" negative" control" cells" was" subtracted" from" every" measurement" prior" to" the" calculation." The" data" are" presented" as" the" means" ±" SD" of" 6" independent" colonies." D:! Predicted" interaction" site" between" NsiR4" and" ssr1528,+ and" the" respective" hybridization" energies" of" the" native" and" mutated" sequences" at" 30°C" (the" respective"nucleotides"are"boxed"in"red)."The"gray"box"highlights"the"ssr1528"start"codon."In" the" RNA" sequences," the" numbers" refer" to" the" TSS" at" position" +1." Predictions" were" made" using"IntaRNA""(42).!! ! Fig.!5.!Changes!in!the!mRNA!abundance!of!gifA!in!response!to!altered!NsiR4!expression.! A:! Expression" kinetics" was" measured" after" Cu2+" addition" and" nitrogen" depletion." Nitrogen" depletion"was" induced"in"cultures" grown"in"presence"of"2" µM"Cu2+" for"48" h"(=" 0"h"AN)." B:" Expression"of"gifA"after"adding"10"mM"NH4+."Prior"to"NH4+"addition,"the"strains"were"preA cultivated"for"6"h"in"presence"of"2"µM"Cu2+."The"order"of"blots"is"the"same"as"in"A.!For"clarity," only" a" representative" 5S" rRNA" loading" control" hybridization" for" NsiR4oex" is" shown." WT" A" Synechocystis" 6803" wild" type," NsiR4oex" –" WT" strain" carrying" pVZ322APpetE::nsiR4::oop" plasmid" (overexpression" strain)," ∆nsiR4" A" deletion" mutant," ∆nsiR4::oex" A" deletion" strain" in" which" NsiR4" expression" was" restored" by" the" pVZ322APpetE::NsiR4::oop" plasmid." C:! Verification" of" the" direct" interaction" between" NsiR4" and" gifA" using" an" in+ vivo+ reporter" system."GFP"fluorescence"measurements"of"E.+coli+TOP10+strains"with"various"combinations" of"plasmids"expressing"NsiR4"or"gifAAsgfpAfusions."The"plasmids"pXGA0"and"pJV300"were"used" as"negative"controls"(for"experimental"details"see"(41).!D:!Repression"calculated"by"dividing" Fig. 5 Fig. 6 Fig. 7 Fig. 8 1 The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7 Supplementary Material Stephan Klähn1, Christoph Schaal1, Jens Georg1, Desirée Baumgartner1, Gernot Knippen1, Martin Hagemann2, Alicia M. Muro-Pastor3 and Wolfgang R. Hess1* 1Genetics & Experimental Bioinformatics, Faculty of Biology, University of Freiburg, Germany 2Plant Physiology Department, Institute of Biological Sciences, University of Rostock, Germany 3Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Spain *Corresponding author: Wolfgang R. Hess, University of Freiburg, Faculty of Biology, Schänzlestr. 1, D-79104 Freiburg, Germany; Tel: +49-(0)761-203-2796; Fax: +49-(0)761-203- 2745; E-mail: [email protected]freiburg.de 2 Supplementary Materials and Methods Strains and growth conditions. For the generation of nsiR4 mutants, we used the glucosetolerant strain Synechocystis 6803 (GT-Kazusa) provided from N. Murata (National Institute for Basic Biology, Okazaki, Japan). Cultivation was performed in Cu2+-free BG11 medium (1) buffered with 20 mM TES, pH 8.0 at 30°C under continuous white light illumination of 50–80 μmol quanta m−2 s−1 and gentle agitation. Mutant strains were grown in presence of the corresponding antibiotics. To induce nitrogen deficiency, the cells from liquid cultures were harvested through centrifugation (for 5 min at 4.000 rpm and room temperature), resuspended in NO3--free BG11 and cultivated further. To re-establish N-replete conditions, NH4Cl or NaNO3 were added to the respective experiments. To induce the ectopic expression of NsiR4, 2 µM CuSO4 was added to the cultures. Anabaena 7120 WT and the mutant strain CSE2 (carrying a streptomycin resistance cartridge within the ntcA coding region; (2)) were grown with bubbling (CO2-enriched air, 1% vol/vol) in BG11 without NO3- and supplemented with 6 mM NH4Cl, 12 mM TES-NaOH (pH 7.5) and 10 mM NaHCO3. For nitrogen step-down experiments, the Anabaena cells were collected through filtration, washed and re-suspended in nitrogen-free medium (BG11 without NO3-). For long-term growth competition experiments, three independent, exponentially growing cultures of Synechocystis 6803 WT and ΔnsiR4 were diluted to an OD750 of 0.1 and mixed in equal numbers. Cultures were grown in 20 ml BG11 in 100 ml Erlenmeyer flasks with 1 mM NO3- (instead of the usual 17.6 mM). After 3 or 4 days cultures were re-diluted to an OD750 of 0.1 and 25 µl of a 1:1000 dilution were dropped on BG11 agar plates with and without 40 µg/ml kanamycin. Amplification of the nsiR4 locus was made using primers SyR12_ko_seg_for/rev and genomic DNA of WT and nsiR4 cultures and a representative, mixed culture after 3, 24 and 45 days. Genome and promoter analysis. The 70 nt of Synechocystis NsiR4 (pos. 1289326 – 1289257, complementary strand) was used as a reference for BlastN searches in the JGI database. However, the identification of an sRNA gene based on sequence alone is not straightforward due to the short length and little sequence conservation. Therefore, the sequences of the resulting hits were extended 100 bp in both directions and further analyzed in multiple alignments using ClustalW (3). The presence of nsiR4 in a given genome was regarded as positive when the sequence aligned to the corresponding sequence from Synechocystis and contained a potential terminator hairpin. Initially, the MEME search tool (4) was used for the comparative analysis of sequences identified upstream of the identified nsiR4 homologous genes. To verify the activity of the nsiR4 promoter in Synechocystis in vivo a sequence spanning the range between -130 to +49 (respective to the TSS at +1) was fused to luxAB reporter genes. The fragment was amplified from the gDNA of Synechocystis (for oligonucleotides see Table S2), followed by restriction digestion with KpnI and cloning into the promoter-probe vector pILA (5). For mutagenesis of the NtcA motif, the corresponding plasmid was re-amplified using the primers prNtc_mut_fw/rev (Table S2). The resulting plasmids, containing either the native or a mutated NsiR4 promoter, were used to transform a Synechocystis host strain carrying the luxCDE operon, which encodes enzymes for the synthesis of decanal, the substrate for the 3 luciferase reaction. The selection of the reporter strains and bioluminescence measurements were performed as described (6). Generation of NsiR4 mutant strains. A schematic presentation of the cloning strategies is shown in Supplementary Figure S6. To generate the NsiR4 knockout strain (∆nsiR4), two fragments covering the adjacent genes sll1697 and sll1698 were amplified from gDNA using the primer combinations 5'SyR12_for/5'SyR12_BsrGI_rev and 3'SyR12_PstI_for/3'SyR12_rev (Supplementary Table S2). The PCR products were digested with the restriction endonucleases BsrGI and PstI, respectively. A kanamycin resistance cartridge (KmR) was amplified from the vector pVZ322 using the primers Kan_PstI_for and Kan_BsrGI_rev, and subsequently digested with BsrGI and PstI and ligated to the compatible ends of both fragments using T4 DNA ligase (Thermo Scientific). The resulting construct comprising a KmR flanked by sequences homologous to the genes sll1697 and sll1698 was re-amplified using the primers 5'SyR12_for and 3'SyR12_rev and introduced into the cloning vector pJET1.2. This plasmid was used to transform WT Synechocystis. The mutant cells were initially selected on BG11 agar plates (0.9% Kobe I agar, Roth, Germany) supplemented with 10 µg ml-1 kanamycin and subsequently grown in the presence of 50 µg ml-1 in liquid cultures. To establish the ectopic expression of nsiR4, a self-replicating plasmid carrying the nsiR4 gene under control of the petE promoter, which mediates Cu2+-regulated transcription in Synechocystis (7), was prepared. The genomic sequence of nsiR4 was amplified from Synechocystis gDNA using the primers SyR12_EcoRI_for and SyR12_EcoRI_rev. The product was digested with EcoRI and introduced into a vector as previously described (8). This plasmid is based on pJET1.2 and contains an EcoRI site between the petE promoter from Synechocystis (ranging from nucleotide -235 to -1 with respect to the TSS at +1), (9) and the oop-terminator. The entire construct was integrated into the Synechocystis chromosome via homologous recombination into the spkA locus, which is a neutral site in the WT strain used here (8). However, divergent from the initial idea of chromosomal integration we cloned the cassette PpetE::nsiR4::oop into the replicative broad-host vector pVZ322. The construct was reamplified using the primers spk_km_hindIII_for and spk_km_xhoI_rev, subsequently digested with HindIII and XhoI and introduced into the plasmid pVZ322, digested with the same enzymes (note that the KmR of pVZ322 was deleted after HindIII/XhoI treatment). The resulting plasmid was transferred into WT Synechocystis and ∆nsiR4 via conjugal transfer from E. coli (10), resulting in the strains NsiR4oex (in WT) and ∆nsiR4::oex (in ∆nsiR4), respectively. The recombinant strains were selected on BG11 agar containing 1 µg ml-1 gentamycin and also grown in presence of the same concentration in liquid cultures. RNA extraction, microarrays and Northern blots. The collection of Synechocystis cells and RNA extraction was performed as previously described (6, 11). Prior to the microarray analysis, 10 µg of total RNA were treated with Turbo DNase (Invitrogen) according to the manufacturer's protocol and precipitated with ethanol/sodium acetate. Labeling and hybridization were performed as previously described (12), using 3 µg of RNA for the labeling reaction and 1.65 µg of labeled RNA for the hybridization. For Northern hybridization, 4 Synechocystis 6803 RNA was separated on denaturing agarose gels and transferred to Hybond-N+ membranes (Amersham, Germany) through capillary blotting with 20x SSC buffer. The membranes were hybridized with [α-32P]-UTP incorporated single-stranded RNA probes generated through in vitro transcription as previously described (13). The signals were detected using a Personal Molecular Imager system (Pharos FX, BIO-RAD, Germany) and analyzed using Quantity One software (BIO-RAD, Germany). RNA from Anabaena was isolated using hot phenol (14). Total RNA was separated on urea-acrylamide gels and transferred to Hybond N+ membranes with 1x TBE buffer in a semi-dry blotter. The membranes were hybridized with oligonucleotides labeled with γ-32P-dATP and polynucleotide kinase (probe for NsiR4) or with probes labeled with γ-32P-dCTP and Ready-to-go DNA labeling kit (Amersham) (probe for 5S rRNA). Protein extraction and immunoblots. Synechocystis and derivative strains were grown in NO3- -containing, Cu2+-free medium. For analysis of IF accumulation, 2 μM CuSO4 was added to induce transcription from the petE promoter eight hours before addition of 10 mM NH4Cl and 20 mM TES-NaOH (pH 7.5). Samples were taken prior to and in a narrow time series after NH4+ addition. Cells from different time points were collected by centrifugation and frozen until protein extraction. Extracts were prepared using glass beads as previously described (15) in 50 mM Hepes-NaOH buffer (pH 7.0), 50 mM KCl, 1 mM EDTA. For Western blot analysis proteins were fractionated on 15% SDS-PAGE and immunoblotted with anti-IF7 (1:2000), anti- IF17 (1:2000) or anti-TrxA (1:3000). Anti-IF7, anti-IF17 and anti-TrxA antisera were obtained from M.I. Muro-Pastor and F.J. Florencio and used as described (16, 17). The ECL Plus immunoblotting system (GE Healthcare) was used to detect the different antigens with antirabbit secondary antibodies. Densitometric evaluation was performed with Quantity One software. Reporter assays for the in vivo verification of targets. For the experimental target verification, we used the reporter system described by (18) and the sGFP plasmid pXG-10-SF introduced by (19). The primers used for cloning and the resulting plasmids are given in Tables S2 and S3. The entire 5’UTR containing the predicted NsiR4 interaction sequence and a part of the coding region were amplified from gDNA using the primer combinations tv_ssl1911_gifA_for/tv_ssl1911_gifA_rev or tv_ssr1528_fw/tv_ssr1528_rev and which covered ranges from +1 to +123 (gifA) or +1 to +119 (ssr1528) with respect to the TSS at position +1. The first nucleotide of the gifA start codon is at +52, for ssr1528 at +30. For gifA the information about the TSS has been taken from (20), for ssr1528 it was extracted from (9). The corresponding PCR product was cloned into the vector pXG-10-SF via the endonuclease sites NsiI/NheI resulting in a translational fusion of the sGFP with a truncated IF7 or Ssr1528 protein. The transcription is mediated by the constitutive promoter PLtetO-1. For the preparation of the plasmid establishing PLlacO-1-mediated sRNA expression in E. coli the nsiR4 gene was amplified from gDNA using the primers 5_SyR12_long_phos/3_SyR12_xbal, digested with XbaI and fused to a plasmid backbone which was amplified from pZE12-luc (by using the primer combination PLlacoB/PLacoD) and also digested with XbaI. 5 For the mutagenesis of NsiR4 and the 5’UTRs of gifA and ssr1528, the plasmids harboring the native versions were re-amplified using the primers SyR12_1911_mut_fwd/ SyR12_1911_mut_rev or SyR12_1528_mut_fwd/ SyR12_1528_mut_rev (for mutating NsiR4 parts interacting with gifA and ssr1528, respectively) and PXG10_1911_mut_fwd/ PXG10_1911_mut_rev (gifA) or PXG10_1528_mut_fwd/ PXG10_1528_mut_rev (ssr1528) (for the respective 5’UTRs) and introduced into E. coli. Positions for mutations were selected on the basis of lowered hybridization energies predicted by IntaRNA (21) while keeping the secondary structures as calculated with RNApdist (22). For testing various combinations of both plasmids, these were introduced into E. coli TOP10 (Invitrogen): e.g. pXG0 + pJV300, pXG10-gifA + pJV300/pZE12-NsiR4. The plasmids pJV300 and pXG-0 were used as negative control plasmids. The fluorescence measurement was done as described previously (23). 6 Supplementary Figures Fig. S1: The nitrogen regulatory network in cyanobacteria. The scheme was prepared based on references (20, 24–27). 13 Table S2: List of oligonucleotides. Name of Oligonucleotide Sequence (in 5’ – 3’ direction) Application Generation of nsiR4 mutant strains in Synechocystis sp. PCC 6803 5'SyR12_for CTCCGGTCCCAATCCTACGAAGC Amplification of sequence flanking nsiR4 upstream region 5'SyR12_BsrGI_rev GAATGTACAGGCCGGATCGGTAGGCTTTATGTA G 3'SyR12_PstI_for GAACTGCAGCCCATTGCTTCAGTGGCGGCTTTC Amplification of sequence flanking nsiR4 downstream region 3'SyR12_rev GCCGTCAGACCAACGCAGACC Kan_PstI_for GAACTGCAGAATAAAAAACGCCCGGCGGCAAC CGAGCGAATCCCGTCAAGTCAGCGTAATGCTC Amplification kanamycin resistance cassette from pVZ322 Kan_BsrGI_rev GAATGTACACAAAGCCACGTTGTGTCTCAAAAT CTCTG SyR12_ko_seg_for CGTCCCAAATCGAGCAGTGCATG Verification of nsiR4 knockout mutants SyR12_ko_seg_rev CTAGGGTGTTGCGTTCCACGTTC SyR12_EcoRI_for GAAGAATTCAAGACATAAAGTCAATATCACCCT CCGATTGC Amplification of nsiR4 from Synechocystis sp. PCC 6803 for generating an NsiR4 expressing plasmid SyR12_EcoRI_rev GAAGAATTCGCATGGCAGCTTCTAAAGGACTAA TAAACTC spk_km_hindIII_for GAAAAGCTTCATTTCCGACACCGAGAAAACC Amplification of insert (PpetE- NsiR4-oop) from shuttle vector pJET_spkA::km_PpetE_ ecoRI_oop for ligation into pVZ322 spk_km_xhoI_rev GAACTCGAGTGGATGATGGGGCGATTCAG Oligonucleotides used for Northern blots (T7 promoters are underlined) fw_pro_ssr1528 GATCGCCGCTGGCATTGATTTTGATGGC Amplification of PCR template used for in vitro transcription generating probes for ssr1528 rev_pro_ssr1528 TAATACGACTCACTATAGGGGCGGGAGCGCAT GGTATTACTGACCCC rev_pro_ssl1911 ATGTCTACTCAACAACAGGCTCGCGCT Amplification of PCR template used for in vitro transcription generating probes for gifA (ssl1911) fw_pro_ssl1911 TAATACGACTCACTATAGGGAGCGGCAGCGCG GGACAACATGGA 5sRNA_for TAATACGACTCACTATAGGAGAAAGAGGAACTT GGCATCGGAC Amplification of PCR template used for in vitro transcription generating probes for 5S rRNA 5sRNA_rev GTCATGGAACCACTCCGATCCC Oligo probe for NsiR4 (Anabaena 7120) GGTCTGGTTAAGCAATCGGAGGGTAAT Northern blot for NsiR4 detection in Anabaena 7120 7120-rrn5Sa-1 AGTTTTCCTGGTGCCTATG PCR fragment probe for detection of 5S rRNA in Anabaena 7120 7120-rrn5Sa-2 ACCTGGCACCGAGCGATTG LuxAB reporter assays Syr12-KpnI_fw GGTACCCCACGTTCAAACACTTTTACATTCG Amplification of the nsiR4 promoter from Synechocystis 6803 for cloning into pILA reporter-plasmid Syr12-KpnI_rev GGTACCGCAATGGGCGACCTCTAGC prNtc_mut_fw CTCAAATAGGCATCATAAAGCCTACCGATC Mutation of the putative NtcA motif upstream of nsiR4 prNtc_mut_rev GATGCCTATTTGAGGAAAGTTCCCGTAAC Target verification in E. coli using a Gfp Reporter system (nucleotides underlined and shown in bold refer to the introduced point mutations) tv_ssr1528_fw ATGCATAGTAAAATAACTCGAGGGTAATATTGA TCATGG Amplification of putative target gene-sequence containing the predicted interaction-region with tv_ssr1528_rev GCTAGCCTTGGCTTCGGGAATGGCACTG 14 tv_ssl1911_gifA_for ATGCATAGAGGGTAATTAACCAAAACTTTTTTCA G NsiR4. The sequence consists of the respective 5´UTR and part of the coding region tv_ssl1911_gifA_rev GCTAGCGGATTGTTGACGGTTTTTGATGAATTG PLlacoB CGCACTGACCGAATTCATTAA Amplification of fragment from plasmid pZE12_luc PLlacoD GTGCTCAGTATCTTGTTATCCG 5_SyR12_long_phos AAGACATAAAGTCAATATCACCCTCC Amplification of nsiR4 (long version) for ligation into pZE12- luc; phosphorylated 3_SyR12_xbal GTTTTTTCTAGATAAAGGACTAATAAACTCTAAA AAGAAAGCC Reverse primer for the amplification of nsiR4 for ligation into pZE12-luc PXG10_1911_mut_rev TGACGATTACTGAAAAAAGTTTTGGTTAATTAC Introduction of a point mutation into gifA sequence PXG10_1911_mut_fw d TTCAGTAATCGTCAAGAGGTATTAACTAT PXG10_1528_mut_rev CCATGATCAAATTTACCCTCGAGTTATTTTA Introduction of a point mutation into ssr1528 sequence PXG10_1528_mut_fw d GTAAATTTGATCATGGCTAATACAACTAAAGGA SyR12_1911_mut_rev CGACCTCTAGTAATCGGAGGGTGATATTG Introduction of compensatory mutation into nsiR4 sequence (gifA mRNA as target) SyR12_1911_mut_fwd CGATTACTAGAGGTCGCCCATTGCTT SyR12_1528_mut_rev TGAATTTGACTTTATGTCTTGTGCTCAGT Introduction compensatory mutation into nsiR4 sequence (ssr1528 mRNA as target) SyR12_1528_mut_fwd ATAAAGTCAAATTCACCCTCCGATTGCTA 15 Table S3: List of plasmids. Plasmid Plasmid backbone Description Marker Reference pJET_spkA::km_PpetE_ ecoRI_oop pJET1.2 Shuttle vector for insertion of nsiR4-sequence. Generation of NsiR4 expressing plasmid KmR This study pJET-spkA::km_PpetE_ nsiR4_oop pJET1.2 Shuttle vector for reamplification of cassette PpetE::nsiR4::oop KmR This study pVZ322-PpetE_NsiR4_ oop_km pVZ322 Plasmid for copper-inducible NsiR4 expression in Synechocystis sp. PCC 6803 GenR, KmR This study pJET- ssl1697::km::ssl1698 pJET1.2 Generation of nsiR4 knockout strain KmR This study pILA Promoter probe vector harbouring the promoterless luxAB genes encoding luciferase, contains recombinations sites for integration into the Synechocystis chromosome KmR, AmpR (5) pILA-PnsiR4 pILA pILA harbouring luxAB under control of the nsiR4 promoter (range between -130 to +49 with respect to the transcriptional start siteTSS at +1 was used) KmR, AmpR This study pILA-PnsiR4_mut pILA same as pILA-PnsiR4 but carrying a mutated NtcA binding motif KmR, AmpR This study pZE12-luc General expression plasmid AmpR (29) pJV300 pZE12-luc Control plasmid, expressing a ~50 nt nonsense transcript derived from rrnB terminator AmpR (30) pXG0 pZA31- luc Plasmid expressing luciferase used as a negative control; cell autofluorescence CmR (18) pXG10 pXG0 Plasmid for synthesis of translational sfGFP fusions CmR (19) pXG10_ssl1911 pXG10-SF GFP reporter plasmid containing the gifA 5’UTR plus the initial part of the coding region CmR this study pXG10_ssr1528 pXG10-SF GFP reporter plasmid containing the ssr1528 5’UTR plus initial part of the coding region CmR this study pXG10_ssl1911_mut pXG10-SF GFP fusion plasmid containing the gifA 5’UTR with mutation in the interacting region with NsiR4 CmR this study pXG10_ssr1528_mut pXG10-SF GFP fusion plasmid containing the ssr1528 5’UTR with mutation in the interacting region with NsiR4 CmR this study pZE12_NsiR4 pZE12-luc Plasmid expressing NsiR4 AmpR this study pZE12_NsiR4_mut1911 pZE12-luc Plasmid expressing NsiR4 with a compensatory mutation for the mutation in gifA AmpR this study 16 pZE12_NsiR4_mut1528 pZE12-luc Plasmid expressing NsiR4 with a compensatory mutation for the mutation in ssr1528 AmpR this study Supplementary References 1. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(1):1–61. 2. Frías JE, Flores E, Herrero A (1994) Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 14(4):823–832. 3. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. 4. Bailey TL, et al. (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37(Web Server issue):W202–208. 5. Kunert A, Hagemann M, Erdmann N (2000) Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB. J Microbiol Meth 41(3):185–194. 6. Klähn S, et al. (2014) Alkane biosynthesis genes in cyanobacteria and their transcriptional organization. Front Bioeng Biotechnol 2:24. 7. Zhang L, McSpadden B, Pakrasi HB, Whitmarsh J (1992) Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803. J Biol Chem 267(27):19054–19059. 8. Eisenhut M, et al. (2012) The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply. J Biol Chem 287(40):33153–33162. 9. Mitschke J, et al. (2011) An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803. Proc Natl Acad Sci USA 108(5):2124–2129. 10. Zinchenko VV, Piven IV, Melnik VA, Shestakov SV (1999) Vectors for the complementation analysis of cyanobacterial mutants. Russ J Genet 35:228–232. 11. Hein S, Scholz I, Voß B, Hess WR (2013) Adaptation and modification of three CRISPR loci in two closely related cyanobacteria. RNA Biol 10(5):852–864. 12. Georg J, et al. (2009) Evidence for a major role of antisense RNAs in cyanobacterial gene regulation. Mol Syst Biol 5:305. 13. Steglich C, et al. (2008) The challenge of regulation in a minimal photoautotroph: non-coding RNAs in Prochlorococcus. PLoS Genet 4(8):e1000173. 14. Mohamed A, Jansson C (1989) Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803. Plant Mol Biol 13(6):693–700. 17 15. Reyes JC, Florencio FJ (1995) A novel mechanism of glutamine synthetase inactivation by ammonium in the cyanobacterium Synechocystis sp. PCC 6803. Involvement of an inactivating protein. FEBS Lett 367(1):45–48. 16. Navarro F, Martín-Figueroa E, Florencio FJ (2000) Electron transport controls transcription of the thioredoxin gene (trxA) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 43(1):23–32. 17. Galmozzi CV, Fernández-Avila MJ, Reyes JC, Florencio FJ, Muro-Pastor MI (2007) The ammoniuminactivated cyanobacterial glutamine synthetase I is reactivated in vivo by a mechanism involving proteolytic removal of its inactivating factors. Mol Microbiol 65(1):166–179. 18. Urban JH, Vogel J (2007) Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Res 35(3):1018–1037. 19. Corcoran CP, et al. (2012) Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA. Mol Microbiol 84(3):428–445. 20. García-Domínguez M, Reyes JC, Florencio FJ (2000) NtcA represses transcription of gifA and gifB, genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. PCC 6803. Mol Microbiol 35(5):1192–1201. 21. Wright PR, et al. (2014) CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res 42(Web Server issue):W119–123. 22. Lorenz R, et al. (2011) ViennaRNA Package 2.0. Algorithms Mol Biol 6:26. 23. Wright PR, et al. (2013) Comparative genomics boosts target prediction for bacterial small RNAs. Proc Natl Acad Sci USA 110(37):E3487–3496. 24. Muro-Pastor MI, Reyes JC, Florencio FJ (2005) Ammonium assimilation in cyanobacteria. Photosyn Res 83(2):135–150. 25. Schwarz R, Forchhammer K (2005) Acclimation of unicellular cyanobacteria to macronutrient deficiency: emergence of a complex network of cellular responses. Microbiology (Reading, Engl) 151(Pt 8):2503–2514. 26. Ohashi Y, et al. (2011) Regulation of nitrate assimilation in cyanobacteria. J Exp Bot 62(4):1411– 1424. 27. Espinosa J, et al. (2014) PipX, the coactivator of NtcA, is a global regulator in cyanobacteria. Proc Natl Acad Sci USA 111(23):E2423–2430. 28. Quast C, et al. (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41(Database issue):D590–596. 29. Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res 25(6):1203– 1210. 30. Sittka A, Pfeiffer V, Tedin K, Vogel J (2007) The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol Microbiol 63(1):193–217. 18