NsiR3, a ni ogen s ess-inducible small RNA, egula es
p oline oxidase exp ession in he cyanobac e ium Nos oc
sp. PCC 7120
Isid o
´
Al a ez-Esc ibano
1
, Manuel B enes-
´
Al a ez
1
, El i a Olmedo-Ve d
1
, Jens Geo g
2
,
Wol gang R. Hess
2
, Agus ı´n Vioque
1
and Alicia M. Mu o-Pas o
1
1 Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones Cien ı´ icas and Uni e sidad de Se illa, Se illa, Spain
2 Gene ics and Expe imen al Bioin o ma ics, Facul y o Biology, Uni e si y o F eibu g, F eibu g, Ge many
Keywo ds
he e ocys ; N cA; pos - ansc ip ional
egula ion; Pu A; egula o y RNA
Co espondence
A. Vioque, Ins i u o de Bioquı´mica Vege al y
Fo osı´n esis, Consejo Supe io de
In es igaciones Cien ı´ icas and Uni e sidad
de Se illa, A da. Am´
e ico Vespucio 49,
Se illa 41092, Spain
Tel: +34-954489519
E-mail: [email p o ec ed]
(Recei ed 14 May 2020, e ised 17 July
2020, accep ed 10 Augus 2020)
doi:10.1111/ ebs.15516
NsiR3 (ni ogen s ess-inducible RNA 3) is a small noncoding RNA s ongly
conse ed in he e ocys - o ming cyanobac e ia. In Nos oc sp. PCC 7120, an-
sc ip ion o NsiR3 is induced by ni ogen s a a ion and depends on he global
ni ogen egula o N cA. A conse ed N cA-binding si e is cen e ed a ound
posi ion −42.5 wi h espec o he ansc ip ion s a si e o NsiR3 homologs,
and N cA binds in i o o a DNA agmen con aining his sequence. In he
absence o combined ni ogen, NsiR3 exp ession is induced in all cells along
he Nos oc ilamen bu much mo e s ongly in he e ocys s, di e en ia ed cells
de o ed o ni ogen ixa ion. Co-exp ession analysis o ansc ip omic da a
ob ained om mic oa ays hyb idized wi h RNA ob ained om Nos oc wild-
ype o mu an s ains g own in he p esence o ammonium o in he absence
o combined ni ogen e ealed ha he exp ession p o ile o gene pu A (p oline
oxidase) co ela es nega i ely wi h ha o NsiR3. Using a he e ologous sys em
in Esche ichia coli, we show ha NsiR3 binds o he 50-UTR o pu A mRNA,
esul ing in educed exp ession o a epo e gene. O e exp ession o NsiR3 in
Nos oc esul ed in s ong educ ion o pu A mRNA accumula ion, u he sup-
po ing he nega i e egula ion o pu A by NsiR3. The highe exp ession o
NsiR3 in he e ocys s e sus ege a i e cells o he N
2
- ixing ilamen could con-
ibu e o he p e iously desc ibed absence o pu A mRNA and o he ca a-
bolic pa hway o p oduce glu ama e om a ginine ia p oline speci ically in
he e ocys s. Pos - ansc ip ional egula ion by NsiR3 ep esen s an indi ec
N cA-ope a ed egula o y mechanism o pu A exp ession.
Da abase
Mic oa ay da a a e a ailable in GEO da abase unde accession numbe s GSE120377 and
GSE150191.
In oduc ion
In some ilamen ous cyanobac e ia, he acclima ion o
ni ogen de iciency in ol es di e en ia ion o he e o-
cys s, a cell ype specialized o he ixa ion o
a mosphe ic ni ogen [1]. G ow h a he expense o N
2
in ol es comple e me abolic emodeling so ha wo
di e en cell ypes wi h di e en me abolic capabili ies
Abb e ia ions
2-OG, 2-oxoglu a a e; dRNASeq, di e en ial RNA sequencing; FC, old change; GEO, Gene Exp ession Omnibus; GOGAT, glu amine
oxoglu a a e amino ans e ase; GS, glu amine syn he ase; GSA, glu ama e γ-semialdehyde; LB, Lu ia B o h; OAA, oxaloace a e; OAC,
o ni hine–ammonium cycle; P5C, Δ
1
-py oline-5-ca boxyla e; Sm, s ep omycin; Sp, spec inomycin; sRNA, small RNA; β-Asp-A g, β-
aspa yla ginine; STRR, sho andemly epea ed epe i i e; TCA, ica boxylic acid cycle.
1614 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
( ege a i e cells and he e ocys s) coope a e o achie e
g ow h o he ilamen as a whole [2]. The di e en ia-
ion o he e ocys s in ol es complex ansc ip ional
changes bo h in ege a i e cells and in he e ocys s no
only o achie e mo phological di e en ia ion o he e-
ocys s bu also o unde go me abolic adap a ions
equi ed o g ow h a he expense o N
2
[3,4].
Small RNAs (sRNAs) cons i u e a ele an class o
pos - ansc ip ional egula o s in ol ed in he adap a-
ion o bac e ial me abolism o di e en en i onmen al
si ua ions [5]. sRNAs can coo dina e changes in gene
exp ession in esponse o en i onmen al s esses. Simila
o he obse a ions made in o he g oups o bac e ia,
cyanobac e ia exhibi abundan ansc ip ion o non-
coding RNAs, including an isense RNAs and sRNAs,
and ansc ip ion o many o hem is egula ed in
esponse o ni ogen a ailabili y [6–9]. Se e al ni ogen-
egula ed noncoding RNAs ha e been iden i ied and
cha ac e ized in he ilamen ous, he e ocys - o ming
cyanobac e ium Nos oc sp. PCC 7120 [3,10–14]. Fu -
he mo e, he e ocys -speci ic ansc ip ion o noncoding
RNAs has been desc ibed [3,11,15,16], and some o
hose ansc ip s a ec he p ocess o he e ocys di e -
en ia ion [17] o he egula ion o enzyma ic ac i i ies
whose le els mus be adjus ed speci ically in he e ocys s
e sus ege a i e cells [13].
NsiR3 is an sRNA whose exp ession is induced
upon ni ogen dep i a ion and depends on N cA, he
global ni ogen egula o in cyanobac e ia [9,11].I
was o iginally desc ibed as conse ed in 27 he e ocys -
o ming cyanobac e ia bu absen in nonhe e ocys ous
s ains [11], sugges ing a possible ole o NsiR3 ela ed
o he e ocys unc ion. Howe e , no egula o y a ge
has so a been iden i ied o NsiR3.
Compu a ional p edic ion o mRNAs egula ed by a
gi en sRNA can be misleading because he in e ac ions
be ween sRNAs and hei a ge s ake place h ough
sho , o en discon inuous complemen a y sequences
[18]. Expe imen al iden i ica ion o possible a ge s
based on simple analysis o di e ences in exp ession
changes upon al e a ion o he amoun o he sRNA is
in many cases impeded by seconda y e ec s. Fu he -
mo e, such app oaches ely on he assump ion ha he
pos - ansc ip ional egula ion exe ed by he sRNA
esul s in clea changes in he amoun o he a ge
mRNA, which is no necessa ily ue [19]. Fo his ea-
son, he combina ion o compu a ional and mo e
sophis ica ed expe imen al app oaches is o en mo e
p oduc i e ( e iewed in Re . [20]).
In his wo k, we show ha upon ni ogen dep i a-
ion, exp ession o NsiR3 is mo e s ongly induced in
he e ocys s han in ege a i e cells. By using a no el
app oach based on co ela ion o exp ession analysis
we iden i y pu A, ha encodes p oline oxidase, as a
a ge o NsiR3 because o he s ong nega i e co ela-
ion wi h NsiR3. Finally, we demons a e he in e ac-
ion be ween NsiR3 and he 50-UTR o he pu A
mRNA, and he nega i e egula ion exe ed by NsiR3
on he exp ession o pu A in Nos oc sp. PCC 7120.
Resul s
NsiR3 is conse ed in he e ocys - o ming
cyanobac e ia, and i s ansc ip ion is induced
upon ni ogen s ess
The ni ogen- egula ed ansc ip ion o NsiR3 was dis-
co e ed in a p e ious dRNASeq expe imen [9]. Phylo-
gene ic conse a ion o NsiR3 homologs ac oss
cyanobac e ial genomes was la e desc ibed [11], and
we ha e now iden i ied NsiR3 homologs in addi ional
cyanobac e ia, con i ming ha NsiR3 appea s
es ic ed o he e ocys - o ming s ains (Fig. 1).
In Nos oc sp. PCC 7120, NsiR3 is ansc ibed down-
s eam o all4558 and in he opposi e o ien a ion
(Fig. 2A). The p edic ed seconda y s uc u e con ains
wo possible s em–loops ac ing as ansc ip ional e -
mina o s (Fig. 2B); he second one (T2) is also ound
in closely ela ed cyanobac e ial s ains and is o med
by STRR (sho andemly epea ed epe i i e) impe -
ec 7-n epea s ha a e p esen in genomes o ila-
men ous cyanobac e ia usually in in e genic egions
bu in some case wi hin coding sequences. Thei unc-
ion, i any, is unknown [21,22] (Fig. 2C). Exp ession
o NsiR3 is induced upon ni ogen s ess (Fig. 2D,E).
Induc ion upon emo al o combined ni ogen is
quick, and he amoun s o NsiR3 emain high up o
24 h. T ans e om ammonium-con aining medium o
ni a e-con aining medium also induces NsiR3 exp es-
sion bu o lowe le els and he induc ion is ansien
(Fig. 2D,E), e lec ing he ac ha ni ogen s ess in
hese condi ions is quickly elie ed by de ep ession o
he ni ope on which encodes ni a e educ ase and
ni i e educ ase, he enzymes in ol ed in assimila ion
o ni a e [23,24]. Consis en wi h he p edic ed sec-
onda y s uc u e, wo RNAs, o 45 and 115 nucleo-
ides, we e de ec ed by no he n hyb idiza ion wi h he
NsiR3 p obe. The 45-nucleo ide RNA (NsiR3S) is o
he expec ed size om he expe imen ally de e mined
ansc ip ional s a si e (TSS) o NsiR3 [9] o he i s
p edic ed e mina o , T1. The majo de ec ed band is
115 nucleo ides long (NsiR3L) and ma ches he calcu-
la ed leng h om he TSS o he second p edic ed e -
mina o , T2, u he downs eam (Fig. 2B). NsiR3L is
he mos abundan o he wo species o NsiR3; he e-
o e, i was used in all he expe imen s desc ibed he e.
1615The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
I.
´
Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase
Fig. 1. Alignmen o NsiR3 RNA. The sequences o NsiR3S om di e en cyanobac e ia we e aligned wi h CLUSTAL OMEGA. Nucleo ide
posi ions conse ed in he 70 s ains whe e NsiR3 was ound a e indica ed by as e isks. Each sequence is indica ed by he o ganism name
ollowed by he GenBank accession numbe (www.ncbi.nlm.nih.go /genbank). The NsiR3 sequence om Nos oc sp. PCC 7120 is
highligh ed in bold.
1616 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
sRNA egula ion o p oline oxidase I.
´
Al a ez-Esc ibano e al.
N cA di ec ly egula es NsiR3 exp ession
T ansc ip ion o NsiR3 in Nos oc sp. PCC 7120
depends on N cA [9,11]. Alignmen o he p omo e
egion om all 70 iden i ied nsiR3 sequences shows
ha a pu a i e conse ed N cA-binding sequence
appea s cen e ed a ound posi ion −42.5 in 67 o hem
(Fig. 3), which is compa ible wi h di ec ac i a ion o
NsiR3 ansc ip ion by N cA.
To e i y he in e ac ion be ween N cA and he p o-
mo e egion o nsiR3, we pe o med an elec-
opho e ic mobili y shi assay wi h pu i ied N cA
Fig. 2. The ni ogen s ess-inducible (NsiR3). (A) Schema ic ep esen a ion o he egion encoding NsiR3 in Nos oc sp. PCC 7120. The wo
lanking genes a e indica ed. The ben a ow ep esen s he ansc ip ional s a a posi ion 5452083 , and he s em–loops ep esen he
wo ansc ip ional e mina o s o NsiR3. The posi ion o he N cA-binding si e is also indica ed. (B) Seconda y s uc u e model o NsiR3
om Nos oc sp. PCC 7120 based on he consensus ob ained wi h RNAali old om he alignmen s in Fig. 1and C his image. The
hep anucleo ide epea s (STRR) a e amed. (C) The sequences o NsiR3 encoded in hose s ains ha could ha e a second ansc ip ional
e mina o (T2) u he downs eam T1 we e aligned using CLUSTAL OMEGA. The p edic ed seed egion is highligh ed in yellow. Accession
numbe s a e indica ed in Fig. 1. (D) Ni ogen- esponsi e exp ession o NsiR3 in Nos oc sp. PCC 7120. Exp ession was analyzed by no he n
blo in cells g own in he p esence o ammonium and ans e ed o medium con aining no sou ce o combined ni ogen (N
2
) o con aining
ni a e (NO
3) o he numbe o hou s indica ed. The uppe panel shows hyb idiza ion o he NsiR3 p obe. The lowe panel shows
hyb idiza ion o a p obe o 5S RNA used as loading and ans e con ol. Sizes (n ) a e indica ed on he le . The expe imen was epea ed
ou imes wi h simila esul s. The no he n blo con aining he highes numbe o ime poin s is shown. (E) Quan i ica ion o NsiR3L in he
blo shown in (D) upon ni ogen emo al (black, N
2
) o upon ni ogen emo al ollowed by ni a e addi ion ( ed, NO
3).
1617The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
I.
´
Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase
Fig. 3. Alignmen o he p omo e sequences o nsiR3. The sequences ups eam nsiR3 we e aligned wi h CLUSTAL OMEGA. Nucleo ide
posi ions ully conse ed a e indica ed by as e isks. The consensus sequence is ep esen ed wi h WebLogo [49]. The likely −10 elemen s
a e boxed, and wo expe imen ally de e mined TSS om Nos oc sp. PCC 7120 [9] and Nodula ia spumigena CCY9414 [50] a e ci cled in
ed. Accession numbe s a e indica ed in Fig. 1.
1618 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
sRNA egula ion o p oline oxidase I.
´
Al a ez-Esc ibano e al.

p o ein and a DNA agmen ex ending om posi ions
−118 o +31 wi h espec o he TSS o nsiR3 ha
includes he possible N cA-binding sequence. In he
p esence o N cA, a e a ded complex was obse ed
wi h he agmen con aining he wild- ype p omo e
sequence (Fig. 4A). Howe e , he ac ion o agmen
bound by N cA was s ongly (nine old) educed (Fig. 4
A,B) when he assay was pe o med wi h a DNA ag-
men con aining a e sion o he nsiR3 p omo e wi h
he N cA-binding si e mu a ed (GTG changed o
CAC). These esul s demons a e he binding o N cA
o a speci ic sequence o he nsiR3 p omo e .
NsiR3 accumula es di e en ially in he e ocys s
bu is no essen ial o diazo ophic g ow h
I was p e iously shown ha NsiR3 ansc ip ion is
dependen on N cA bu independen o He R [9,11],
he mas e egula o o he e ocys di e en ia ion.
The e o e, we expec ha NsiR3 ansc ip ion would
be induced in all cells o he ilamen upon ni ogen
s ess. To cha ac e ize he pa e n o exp ession o
NsiR3, we p epa ed a plasmid con aining a ansc ip-
ional usion o he NsiR3 p omo e o he g p gene
(pSAM341, see Table S4). The plasmid was in oduced
in Nos oc sp. PCC 7120 by conjuga ion, and exp es-
sion o GFP was analyzed by con ocal luo escence
mic oscopy (Fig. 5). Filamen s g owing in ammonium-
con aining medium had e y low g een luo escence,
while ilamen s g owing in medium lacking combined
ni ogen showed luo escence in all cells, bu peaks o
g een luo escence we e associa ed wi h cells ha we e
di e en ia ing as he e ocys s, as indica ed by hei
lowe ed au o luo escence. In e es ingly, induc ion o
he nsiR3 p omo e occu s ea ly in he e ocys de elop-
men because signi ican g een luo escence was
al eady obse ed in imma u e he e ocys s, s ill ha ing
subs an ial ed luo escence (no shown).
The conse a ion o NsiR3 in he e ocys - o ming
cyanobac e ia and i s N cA-dependen exp ession sug-
ges s ha NsiR3 is in ol ed in some aspec o he eg-
ula ion o he esponse o ni ogen s ess in hese
complex cyanobac e ia ha includes di e en ia ion o
specialized cells. To explo e he possible unc ion o
NsiR3, we cons uc ed a Nos oc s ain lacking NsiR3
(ΔnsiR3). This s ain g ew simila ly o wild- ype in
media con aining ammonium and ni a e, o lacking
combined ni ogen (Fig. 6); he e o e, he di e en ia-
ion o unc ional he e ocys s is no comp omised in
he absence o NsiR3.
NsiR3 is a egula o o pu A (al 0540)
Bac e ial sRNAs impac hei a ge mRNAs o en by
ep essing he ini ia ion o ansla ion ia binding o
hei 50-UTRs [5]. Hence, he mRNAs do no associa e
wi h ibosomes and may become mo e ulne able o
iboendonucleases, leading o educed mRNA le el. To
iden i y po en ial a ge s o NsiR3 whose exp ession
would be nega i ely egula ed by NsiR3 in Nos oc sp.
PCC 7120, we decided o sea ch o genes whose exp es-
sion p o ile showed a nega i e co ela ion wi h he
exp ession o NsiR3. Fo his pu pose, we ha e used a
no el app oach based on co ela ion analysis o da a
ob ained om hyb idiza ion o high-densi y mic oa -
ays [3]. In o de o iden i y hose genes wi h nega i e
co ela ion wi h NsiR3, we analyzed da a ob ained om
hyb idiza ion o RNA samples ex ac ed om he
ΔnsiR3 mu an g own in ammonium-con aining med-
ium ( wo eplica es) o om he ΔnsiR3 mu an a e
8 h o incuba ion in medium lacking combined ni ogen
( wo eplica es). In addi ion, in o de o inc ease he
Fig. 4. N cA binds o he nsiR3 p omo e . (A) Elec opho e ic
mobili y shi assays showing binding o pu i ied His- agged N cA
p o ein o a DNA agmen con aining he wild- ype p omo e o
NsiR3 om Nos oc sp. PCC 7120 (le ) o a mu a ed e sion
al e ed in he posi ions indica ed in ed ( igh ). Th ee imes mo e
p obe was used in he assay wi h he mu an agmen han wi h
he wild- ype agmen . A ep esen a i e expe imen is shown. (B)
Quan i ica ion o he au o adiog am p esen ed in panel (A). The
ac ion o p obe bound by N cA was calcula ed o he wild- ype
(black) o he mu a ed ( ed) e sion.
1619The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
I.
´
Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase
sensi i i y o he co ela ion analysis, p e iously a ail-
able da a om hyb idiza ion o he same mic oa ay
pla o m wi h o he 20 samples (bo h om he wild- ype
s ain and om a he R mu an s ain ei he in he p es-
ence o ammonium o a e di e en pe iods o ni ogen
dep i a ion) [3] we e included in he analysis. The
exp ession da a om he 24 samples (Table S1) we e
analyzed as desc ibed [3], and a co ela ion able was
ob ained o NsiR3 (Table S2). The p obe wi h he
s onges nega i e co ela ion wi h NsiR3 (−0.943) was
ha o gene al 0540 (pu A), encoding p oline oxidase.
Mo eo e , ano he p obe co esponding o he 50-UTR
o al 0540 had a s ong nega i e co ela ion as well
(−0.884). Figu e 7A shows he exp ession p o ile o
NsiR3 and pu A in he 24 samples hyb idized o he
a ays. We also con i med by no he n blo he opposi e
Fig. 5. Exp ession pa e n o he nsiR3
p omo e in Nos oc ilamen s. Con ocal
luo escence images ( ed channel, g een
channel, bo h channels me ged, and b igh -
ield image) o Nos oc ilamen s ca ying
he g p gene unde he con ol o he nsiR3
p omo e (plasmid pSAM341) and g owing
on op o medium con aining ammonium,
NHþ
4(A), o lacking any sou ce o combined
ni ogen, N
2
(B). Quan i ica ion o he
signals o he g een channel (GFP) is
shown o he segmen s indica ed by a blue
line in he co esponding b igh - ield images.
All images we e acqui ed wi h he same
sensi i i y se ings so ha in ensi ies can
be compa ed. Scale ba s, 20 µm.
Fig. 6. The ΔnsiR3 s ain can di e en ia e he e ocys s. Cells we e g own in he p esence o ni a e, washed wi h BG11
0
, and esuspended
a 0.1 μg chlo ophyllmL
−1
. Fi e old se ial dilu ions o liquid cul u es o wild- ype o ou independen ΔnsiR3 isola es we e p epa ed. Ten
mic oli e o he cell suspension and o h ee i e old se ial dilu ions we e pla ed on BG11
0
pla es con aining ammonium (NHþ
4), ni a e
(NO
3), o no sou ce o combined ni ogen (N
2
). Pic u es we e aken a e 10 days o incuba ion a 30 °C.
1620 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
sRNA egula ion o p oline oxidase I.
´
Al a ez-Esc ibano e al.
exp ession dynamics o pu A wi h NsiR3 ha was
deduced om he co-exp ession analysis (Fig. 7B).
The esul s desc ibed abo e s ongly sugges ha he
exp ession o pu A migh be egula ed by NsiR3. In
ac , we could p edic wi h In aRNA [25] an in e ac-
ion be ween NsiR3 and he 50-UTR o pu A mRNA
(Fig. 8A). NsiR3 would bind o posi ions −5 o−17
wi h espec o he ini ia ion codon o pu A, occluding
he ibosome-binding si e.
To e i y he in e ac ion be ween NsiR3 and he 50-
UTR o pu A, we used a he e ologous epo e sys em
[26] in which he 50-UTR plus he i s 60 n o he
pu A coding sequence was ansla ionally used o he
gene encoding supe olde GFP (s g p). This cons uc
was co-exp essed in E. coli ei he wi h NsiR3 o wi h a
con ol, un ela ed RNA. The ini ia ion codon o pu A
was changed om GTG o ATG o op imal exp es-
sion in E. coli. The luo escence o cells ca ying s g p
usions signi ican ly dec eased when NsiR3 was co-ex-
p essed, indica ing a nega i e e ec o NsiR3 on
exp ession o s GFP (Fig. 8B).
To e i y he in e ac ions be ween NsiR3 and he
mRNA o pu A, we c ea ed a mu a ed e sion o NsiR3
al e ed in posi ion 14 (G o C, NsiR3 Mu 14) (Fig. 8A).
The mu a ion in NsiR3 elimina ed he in e ac ion
be ween NsiR3 and he mRNA o pu A (Fig. 8B). We
designed a compensa o y mu a ion in he 50-UTR o
pu A (Comp-14*17) ha would es o e he in e ac ion
wi h NsiR3 (Mu 14) (Fig. 8A). When NsiR3 (Mu 14)
was combined wi h he mu a ed e sion o he 50-UTR
o pu A, he educ ion o luo escence was es o ed
(Fig. 8B). All oge he , hese esul s e i y he in e ac-
ion o NsiR3 wi h he 50-UTR o pu A a he posi ions
p edic ed by In aRNA. In consequence, an inhibi o y
e ec o NsiR3 on pu A in he in i o con ex o he
he e ologous E. coli sys em is suppo ed.
NsiR3 ep esses he exp ession o pu A in
Nos oc sp. PCC 7120
To analyze he egula o y e ec o NsiR3 on pu A in
Nos oc sp. PCC 7120, we gene a ed a Nos oc s ain
Fig. 7. Nega i e co ela ion o pu A and
nsiR3 exp ession. (A) Exp ession le el o
nsiR3 ( ed) and pu A (al 0540, black), in he
24 RNA samples hyb idized o he
mic oa ays used o he co-exp ession
analysis. (B) pu A exp ession a e emo al
o combined ni ogen. RNA was ex ac ed
om Nos oc cul u es a di e en imes
a e ni ogen emo al and subjec ed o
no he n blo . The il e was hyb idized wi h
p obes o pu A ( op), nsiR3 (middle), and
npB (bo om), ha was used as loading
con ol. The expe imen was epea ed h ee
imes wi h simila esul s. The no he n blo
con aining he highes numbe o ime
poin s is shown. (C) Quan i ica ion o he
blo shown in (B). The amoun o pu A
mRNA (black) and NsiR3 ( ed) is exp essed
as pe cen age o he maximum. Fo pu A,
he ull-leng h ansc ip (a ow in B) was
used in quan i ica ion. Sizes o ibosomal
RNAs a e indica ed in (B).
1621The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
I.
´
Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase
wi h con olled exp ession o NsiR3 by complemen ing
s ain ΔnsiR3 wi h a plasmid con aining nsiR3 unde
con ol o he coppe -inducible pe E p omo e
(ΔnsiR3+P
pe E
::nsiR3). Accumula ion o he mRNA o
pu A was analyzed by no he n blo in he wild- ype,
ΔnsiR3, and ΔnsiR3+P
pe E
::nsiR3 s ains a di e en
imes a e ni ogen emo al and coppe addi ion
(Fig. 9A). A s onge educ ion in he amoun o he
ull-leng h pu A mRNA was obse ed in he
ΔnsiR3+P
pe E
::nsiR3 s ain han in he wild- ype, in
ag eemen wi h he obse a ion ha ΔnsiR3+P
pe E
::
nsiR3 accumula ed abou 40 imes mo e NsiR3 han
he wild- ype (Fig. 9A,B). The dele ion o nsiR3 in he
ΔnsiR3 s ain esul ed in le els o he pu A mRNA
ha we e no signi ican ly educed a e 24 h in he
absence o combined ni ogen. These esul s indica e a
s ong nega i e co ela ion be ween he amoun o
NsiR3 and he amoun o he pu A mRNA. As a con-
ol, we hyb idized he same il e wi h a p obe o
gene ag E encoding he bi unc ional enzyme a ginine
dihyd olase/o ni hine cyclodeaminase ha ac s
ups eam p oline oxidase in he ca abolic pa hway
om a ginine o p oline [27]. The dynamic o changes
in he amoun o ag E mRNA upon ni ogen emo al
was di e en o ha o pu A. In he wild- ype s ain,
whe eas he amoun o pu A mRNA was educed o
abou 40% a e 24 h in he absence o combined
ni ogen, no signi ican change was obse ed in he
case o ag E. In addi ion, he le els o ag E mRNA
did no co ela ed o changes in he amoun o NsiR3
(Fig. 9), as p edic ed o a mRNA ha is no a a ge
o NsiR3.
Discussion
In his wo k, we cha ac e ize NsiR3, a highly con-
se ed sRNA in he e ocys ous cyanobac e ia. NsiR3
was iden i ied in he genomes o se en y he e ocys -
o ming cyanobac e ia bu was no ound in unicellu-
la o ilamen ous s ains ha do no de elop he e o-
cys s. This dis ibu ion o he nsiR3 gene, in con as
o ha o o he N cA- egula ed sRNAs such as
NsiR4, also ound in unicellula s ains [10], sugges s a
ele an unc ion ela ed o he speci ic me abolic ai s
o he e ocys - o ming s ains. nsiR3 is no ansc ibed
in he p esence o ammonium, bu i s ansc ip ion is
s ongly induced upon ans e o ni ogen- ee med-
ium, sugges ing a possible unc ion in he esponse o
ni ogen s ess. Fu he mo e, nsiR3 ansc ip ion is
also induced upon ans e om ammonium- o
ni a e-con aining medium. Howe e , in his case he
induc ion is lowe and ansien . This can be explained
because he ni ogen s ess is quickly elie ed upon
induc ion o he ni a e assimila ion pa hway in he
p esence o ni a e [23]. We show ha he global egu-
la o o ni ogen assimila ion, N cA, binds di ec ly o
he nsiR3 p omo e , explaining he p e ious obse a-
ion ha N cA is equi ed o exp ession o NsiR3
[9,11]. By means o a usion o he g p gene, we show
ha exp ession o NsiR3, al hough induced in all cells
o he ilamen unde ni ogen dep i a ion, is much
s onge in he e ocys s han in ege a i e cells. This
obse a ion migh be explained by he highe concen-
a ion o N cA p esen in he he e ocys e sus in eg-
e a i e cells [28,29]. Al e na i ely, he nsiR3 p omo e
Fig. 8. Ve i ica ion o NsiR3 in e ac ion wi h he 50-UTR o pu A
using an in i o epo e sys em. (A) P edic ed in e ac ion be ween
NsiR3 and he 50-UTR o pu A mRNA acco ding o INTARNA [25].
Nucleo ides a e numbe ed wi h espec o he s a o he coding
sequence (ini ia ion codon is unde lined). The ibosome-binding si e
in he pu A mRNA is highligh ed in yellow. A mu a ion in oduced
in NsiR3 a posi ion 14 (G o C, Mu 14) and he co esponding
compensa o y mu a ion in pu A 50-UTR posi ion −17 (C o G,
Comp-14*17) a e indica ed in ed and blue, espec i ely. (B)
Fluo escence measu emen s o E. coli DH5αcul u es bea ing
combina ions o plasmids exp essing di e en e sions o NsiR3
and pu A::s g p usions. Plasmid pJV300 (encoding a con ol RNA)
was used as con ol. The da a a e p esen ed as he
mean s anda d de ia ion o cul u es om eigh independen
colonies a e sub ac ion o luo escence in cells bea ing pXG-0
(***P<0.0001, S uden - es ).
1622 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
sRNA egula ion o p oline oxidase I.
´
Al a ez-Esc ibano e al.
Pseudomonas s u ze i A1501. P oc Na l Acad Sci USA
113, E4348–E4356.
34 Rippka R, De uelles J, Wa e bu y JB, He dman M &
S anie RY (1979) Gene ic assignmen s, s ain his o ies
and p ope ies o pu e cul u es o cyanobac e ia.
Mic obiology 111,1–61.
35 Samb ook J & Russell DW (2001) Molecula Cloning:
A Labo a o y Manual. Cold Sp ing Ha bo
Labo a o y, Cold Sp ing Ha bo , NY.
36 Mu o-Pas o AM, Vallada es A, Flo es E & He e o A
(1999) The he C gene is a di ec a ge o he N cA
ansc ip ional egula o in cyanobac e ial he e ocys
de elopmen . J Bac e iol 181, 6664–6669.
37 U ban JH & Vogel J (2007) T ansla ional con ol and
a ge ecogni ion by Esche ichia coli small RNAs
in i o.Nucleic Acids Res 35, 1018–1037.
38 W igh PR, Rich e AS, Papen o K, Mann M, Vogel
J, Hess WR, Backo en R & Geo g J (2013)
Compa a i e genomics boos s a ge p edic ion o
bac e ial small RNAs. P oc Na l Acad Sci USA 110,
E3487–E3496.
39 Elhai J & Wolk CP (1988) Conjugal ans e o DNA
o cyanobac e ia. Me hods Enzymol 167, 747–754.
40 Me ino-Pue o V, Ma iscal V, Mullineaux CW, He e o
A & Flo es E (2010) F a p o eins in luencing ilamen
in eg i y, diazo ophy and localiza ion o sep al p o ein
SepJ in he he e ocys - o ming cyanobac e ium
Anabaena sp. Mol Mic obiol 75, 1159–1170.
41 Cai Y & Wolk CP (1990) Use o a condi ionally le hal
gene in Anabaena sp. s ain PCC 7120 o selec o
double ecombinan s and o en ap inse ion sequences.
J Bac e iol 172, 3138–3145.
42 Buikema WJ & Haselko n R (2001) Exp ession o he
Anabaena he R gene om a coppe - egula ed p omo e
leads o he e ocys di e en ia ion unde ep essing
condi ions. P oc Na l Acad Sci USA 98, 2729–2734.
43 Mohamed A & Jansson C (1989) In luence o ligh on
accumula ion o pho osyn hesis-speci ic ansc ip s in
he cyanobac e ium Synechocys is 6803. Plan Mol Biol
13, 693–700.
44 Vioque A (1992) Analysis o he gene encoding he
RNA subuni o ibonuclease P om cyanobac e ia.
Nucleic Acids Res 20, 6331–6337.
45 Al schul SF, Gish W, Mille W, Mye s EW & Lipman
DJ (1990) Basic local alignmen sea ch ool. J Mol Biol
215, 403–410.
46 Madei a F, Pa k YM, Lee J, Buso N, Gu T,
Madhusoodanan N, Basu ka P, Ti ey ARN, Po e
SC, Finn RD e al. (2019) The EMBL-EBI sea ch and
sequence analysis ools APIs in 2019. Nucleic Acids Res
47, W636–W641.
47 Lo enz R, Be nha SH, H¨
one zu Siede dissen C,
Ta e H, Flamm C, S adle PF & Ho acke IL (2011)
ViennaRNA package 2.0. Algo i hms Mol Biol 6, 26.
48 Ri chie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W
& Smy h GK (2015) limma powe s di e en ial
exp ession analyses o RNA-sequencing and
mic oa ay s udies. Nucleic Acids Res 43, e47.
49 C ooks GE, Hon G, Chandonia J-M & B enne SE
(2004) WebLogo: a sequence logo gene a o . Genome
Res 14, 1188–1190.
50 Voss B, Bolhuis H, Fewe DP, Kop M, M¨
oke F, Haas
F, El-Shehawy R, Hayes P, Be gman B, Si onen K
e al. (2013) Insigh s in o he physiology and ecology o
he b ackish-wa e -adap ed Cyanobac e ium Nodula ia
spumigena CCY9414 based on a genome- ansc ip ome
analysis. PLoS One 8, e60224.
Suppo ing in o ma ion
Addi ional suppo ing in o ma ion may be ound
online in he Suppo ing In o ma ion sec ion a he end
o he a icle.
Table S1. Di e en ially_exp essed_genes_all_samples.
Table S2. Pea son_co ela ion_o _NsiR3_and_all_-
genes.
Table S3. S ains.
Table S4. Plasmids.
Table S5. Oligonucleo ides.
Table S6. Sequences.
1629The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies
I.
´
Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase