In e cellula Di usion o a Fluo escen Suc ose Analog ia he Sep al
Junc ions in a Filamen ous Cyanobac e ium
Dennis J. Nü nbe g,
a
Vicen e Ma iscal,
b
Jan Bo nikoel,
c
Me cedes Nie es-Mo ión,
b
No be K auß,
a
An onia He e o,
b
I is Maldene ,
c
En ique Flo es,
b
Con ad W. Mullineaux
a
School o Biological and Chemical Sciences, Queen Ma y Uni e si y o London, London, Uni ed Kingdom
a
; Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo
Supe io de In es igaciones Cien í icas and Uni e sidad de Se illa, Se ille, Spain
b
; Depa men o Mic obiology/O ganismic In e ac ions, Uni e si y o Tübingen, Tübingen,
Ge many
c
D.J.N. and V.M. con ibu ed equally o his wo k.
ABSTRACT Many ilamen ous cyanobac e ia p oduce specialized ni ogen- ixing cells called he e ocys s, which a e loca ed a
semi egula in e als along he ilamen wi h abou 10 o 20 pho osyn he ic ege a i e cells in be ween. Ni ogen ixa ion in
hese complex mul icellula bac e ia depends on me aboli e exchange be ween he wo cell ypes, wi h he he e ocys s supplying
combined-ni ogen compounds bu dependen on he ege a i e cells o pho osyn he ically p oduced ca bon compounds.
He e, we used a luo escen ace o p obe in e cellula me aboli e exchange in he ilamen ous he e ocys - o ming cyanobac e-
ium Anabaena sp. s ain PCC 7120. We show ha esculin, a luo escen suc ose analog, is inco po a ed by a suc ose impo sys-
em in o he cy oplasm o Anabaena cells. The cy oplasmic esculin is apidly and e e sibly exchanged ac oss ege a i e-
ege a i e and ege a i e-he e ocys cell junc ions. Ou measu emen s e eal he kine ics o esculin exchange and also
show ha in e cellula me abolic communica ion is los in a signi ican ac ion o olde he e ocys s. SepJ, F aC, and F aD
a e p o eins loca ed a he in e cellula sep a and a e sugges ed o o m s uc u es analogous o gap junc ions. We show
ha a ⌬sepJ ⌬ aC ⌬ aD iple mu an shows an al e ed sep um s uc u e wi h hinne sep a bu a dense pep idoglycan
laye . In e cellula di usion o esculin and luo escein de i a i es is impai ed in his mu an , which also shows a g ea ly
educed equency o nanopo es in he in e cellula sep al c oss walls. These indings sugges ha F aC, F aD, and SepJ a e
impo an o he o ma ion o junc ional s uc u es ha cons i u e he majo pa hway o eeding he e ocys s wi h
suc ose.
IMPORTANCE Anabaena and i s ela i es a e ilamen ous cyanobac e ia ha exhibi a sophis ica ed o m o p oka yo ic mul i-
cellula i y, wi h he o ma ion o di e en ia ed cell ypes, including no mal pho osyn he ic cells and specialized ni ogen- ixing
cells called he e ocys s. The ques ion o how he e ocys s communica e and exchange me aboli es wi h o he cells in he ilamen
is key o unde s anding his o m o bac e ial mul icellula i y. He e we p o ide he i s in o ma ion on he in e cellula ex-
change o a physiologically impo an molecule, suc ose. We show ha a luo escen suc ose analog can be impo ed in o he
Anabaena cy oplasm by a suc ose impo sys em. Once in he cy oplasm, i is apidly and e e sibly exchanged among all o he
cells in he ilamen by di usion ac oss he sep al junc ions. Pho osyn he ically p oduced suc ose likely ollows he same ou e
om cy oplasm o cy oplasm. We iden i y some o he sep al p o eins in ol ed in suc ose exchange, and ou esul s indica e ha
hese p o eins o m s uc u es unc ionally analogous o me azoan gap junc ions.
Recei ed 7 Oc obe 2014 Accep ed 11 Feb ua y 2015 Published 17 Ma ch 2015
Ci a ion Nü nbe g DJ, Ma iscal V, Bo nikoel J, Nie es-Mo ión M, K auß N, He e o A, Maldene I, Flo es E, Mullineaux CW. 2015. In e cellula di usion o a luo escen suc ose
analog ia he sep al junc ions in a ilamen ous cyanobac e ium. mBio 6(2):e02109-14. doi:10.1128/mBio.02109-14.
In i ed Edi o Elisabe h Gan , Uni e si y o Ma yland, College Pa k Edi o Douglas G. Capone, Uni e si y o Sou he n Cali o nia
Copy igh © 2015 Nü nbe g e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-Noncomme cial-Sha eAlike 3.0 Unpo ed
license, which pe mi s un es ic ed noncomme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Add ess co espondence o Con ad W. Mullineaux, [email p o ec ed], En ique Flo es, [email p o ec ed], o I is Maldene , [email p o ec ed].
The ilamen ous cyanobac e ium Anabaena sp. s ain PCC 7120
(he e Anabaena) is a mul icellula p oka yo e. Simul aneous
oxygenic pho osyn hesis and ni ogen ixa ion a e achie ed by
loca ing he oxygen-sensi i e ni ogenase in specialized di e en-
ia ed cells called he e ocys s. He e ocys di e en ia ion in ol es
complex me abolic and mo phological changes (1, 2). Du ing
combined-ni ogen s a a ion, he e ocys s o m a in e als o 10
o 20 cells, es ablishing a spacing pa e n along he ilamen (3)
ha may be con olled by a di usible p oduc o he pa S gene
ac ing on he mas e egula o He R (4–6). He e ocys s main ain a
mic ooxic cy oplasm by building ex a en elope laye s as di u-
sion ba ie s o O
2
(7) and by disman ling pho osys em II and
ac i a ing espi a ion (1, 2). He e ocys s and ege a i e cells a e
mu ually dependen : while ege a i e cells supply ca bon skele-
ons, he e ocys s supply combined-ni ogen compounds (1, 2).
Likely ehicles o combined-ni ogen supply om he e ocys s o
ege a i e cells a e glu amine (8, 9) and
␤
-aspa yl-a ginine, a
b eakdown p oduc o he s o age compound cyanophycin (10,
11). The mos likely ca ie o ixed ca bon is suc ose, since di-
azo ophic g ow h equi es suc ose p oduc ion in he ege a i e
RESEARCH ARTICLE c ossma k
Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14 ®mbio.asm.o g 1
cells and b eakdown by in e ases in he he e ocys s (12–15).
Howe e , amino acids such as glu ama e and alanine may also be
ans e ed o he e ocys s (16, 17).
In Anabaena, wo ou es o in e cellula me aboli e exchange
ha e been p oposed (18–20): ei he ia a con inuous pe iplasm
(21, 22) o by di usion om cy oplasm o cy oplasm ia cell-cell
connec ions (23) in ol ing he sep al p o eins SepJ (also known as
F aG) (24, 25), F aC, and F aD (26, 27). These p o eins a e likely
componen s o s uc u es ha ha e been e med mic oplasmod-
esma a (28, 29) o sep osomes (30). Analyses o dele ion mu an s
show he impo ance o hese p o eins o ilamen in eg i y, di-
azo ophic g ow h, and in e cellula communica ion, consis en
wi h he idea ha SepJ, F aC, and F aD o m s uc u es ha allow
he in e cellula mo emen o small cy oplasmic molecules (23–
27). By analogy wi h me azoan gap junc ions ( e iewed in e e -
ence 31), hese s uc u es ha e been e med sep al junc ions (32).
The mu ein sacculus is gene ally hicke in cyanobac e ia han
in he mos widely s udied G am-nega i e bac e ia, such as Esch-
e ichia coli, bu hinne han in G am-posi i e bac e ia (33–35).
Thus, he cyanobac e ial mu ein sacculus is likely composed o
se e al pep idoglycan laye s. In Anabaena, he hickness o he
mu ein sacculus may co espond o wo in e linked pep idogly-
can laye s (30). Mu ein sacculi ha e been isola ed om se e al
he e ocys - o ming cyanobac e ia, and sacculi co esponding o
se e al cell uni s ha e been ob ained (36–38). This implies ha he
pep idoglycan laye s o adjacen cells a e used in a subs an ial
ac ion o he in e cellula sep a o a ilamen . In ma e ial isola ed
om Nos oc punc i o me, he sep al pep idoglycan can be seen by
elec on mic oscopy as discs con aining holes abou 20 nm in
diame e ha ha e been e med nanopo es and a e ele an o
in e cellula communica ion (38). Nanopo es in he in e cellula
pep idoglycan would be equi ed o accommoda e p o ein s uc-
u es linking he cy oplasms o adjacen cells. The low-densi y
a eas obse ed by elec on mic oscopy in he in e cellula pep i-
doglycan o Anabaena (30), Anabaena a iabilis (39), and Mas-
igocladus laminosus (40) likely co espond o he sep al pep i-
doglycan nanopo es. Addi ionally, an elec on omog aphic s udy
o he sep um be ween cells in Anabaena shows ha hese s uc-
u es a e also p esen be ween ege a i e cells and he e ocys s
(41). Cell wall amidases AmiC2 (NpF1846) and AmiC1 (Al 0092),
which ha e been ecen ly cha ac e ized in N. punc i o me and
Anabaena, espec i ely (37, 42), a e equi ed o make he sep al
pep idoglycan nanopo es. Mu an s lacking hese p o eins a e im-
pai edincelldi e en ia ionandexchangeo he luo escen ace
molecule calcein, sugges ing ha modi ica ion o he sep al pep-
idoglycan by hese enzymes is essen ial o in e cellula commu-
nica ion (38, 43).
In e cellula communica ion in ilamen ous cyanobac e ia has
been s udied by using luo escence eco e y a e pho obleaching
(FRAP). FRAP equi es a sui able luo escen ace o be in o-
duced in o he app op ia e cell compa men . Pe iplasmic com-
munica ion was s udied by exp essing g een luo escen p o ein
(GFP) (22, 44) o he smalle luo escen p o ein iLOV (45) ex-
po ed in o he pe iplasm ia he win-a ginine ansloca ion sys-
em. GFP is no exchanged om cy oplasm o cy oplasm, bu
cy oplasmic exchange could be examined by loading he smalle
luo escen ace molecules calcein and 5-ca boxy luo escein
(5-CF) in o he cy oplasm o Anabaena (23, 46) and o he ila-
men ous cyanobac e ia (23, 38, 40) by using an es e i ied cell-
pe mea ing p ecu so ha is p ocessed by cy oplasmic es e ases o
elease a hyd ophilic luo escen p oduc . FRAP e eals apid
ans e o calcein and 5-CF be ween he cy oplasms o adjacen
cells, impai ed in mu an s lacking SepJ, F aC, o F aD (23, 26, 46).
Plan suc ose up ake anspo e s (SUTs) (47) ha e been
s udied by moni o ing he up ake o he luo escen couma in
␤
-glucoside esculin (48, 49). Type I SUTs impo esculin a a a e
simila o ha o suc ose (47, 50), showing ha esculin is ecog-
nized and anspo ed simila ly o suc ose. He e we explo e he
up ake and in e cellula exchange o esculin in Anabaena. Fluo-
escence mic oscopy and FRAP show ha esculin is impo ed in o
he cy oplasm and can hen be exchanged apidly and e e sibly
among ege a i e cells and be ween ege a i e cells and he e o-
cys s. A iple mu an lacking SepJ, F aC, and F aD is impai ed in
in e cellula esculin exchange. Compa ed o he wild ype, i
shows an al e ed pep idoglycan s uc u e and a much lowe e-
quencyo nanopo esin he pep idoglycan a he in e cellula c oss
walls.
RESULTS
An Anabaena ⌬sepJ ⌬ aC ⌬ aD iple mu an shows an al-
e ed pep idoglycan s uc u e and ewe nanopo es in he sep al
c oss walls. Anabaena ⌬sepJ and ⌬ aC ⌬ aD mu an s ha e been
p e iously cons uc ed (26, 27, 46). To es whe he he simul a-
neous inac i a ion o aC, aD, and sepJ has any addi ional e ec
compa ed o he inac i a ion o aC and aD o o sepJ, a iple
mu an was c ea ed by ans e o a sepJ-inac i a ing cons uc o
⌬ aC ⌬ aD mu an s ain CSVT22 (27). S ain CSVM141 has
he p edic ed dele ions in he h ee genes and lacks wild- ype cop-
ies o hese genes (see Fig. S1 in he supplemen al ma e ial). Sim-
ila ly o s ains CSVT22 (⌬ aC ⌬ aD) and CSVM34 (⌬sepJ),
s ain CSVM141 g ew well in medium wi h combined ni ogen
(ni a e o ammonium). In he p esence o ni a e and cul u e
ea men in acco dance wi h he p o ocol desc ibed in Ma e ials
and Me hods, CSVM141 o med ilamen s gene ally sho e han
hose o he wild ype bu longe han hose ound in he ⌬sepJ and
⌬ aC ⌬ aD mu an s (see Fig. S2 in he supplemen al ma e ial).
T ans e o BG11
0
medium, which lacks combined-ni ogen
compounds, led o apid agmen a ion o CSVM141 ilamen s
(see Fig. S2B), esul ing in e y sho ilamen s a e 48 h (see
Fig. S2C). CSVM141 he e o e canno o m he e ocys s o
g ow diazo ophically.
To assess whe he he absence o he h ee sep al p o eins could
in luence sep al cha ac e is ics, we s udied he in e cellula sep a
in CSVM141 and wild- ype ilamen s g own wi h combined ni-
ogen. T ansmission elec on mic oscopy (TEM) showed ha
he cell junc ions in he iple mu an we e signi ican ly hinne
han in he wild ype (Fig. 1A). The dis ance (mean ⫾s anda d
de ia ion [SD]; n, numbe o junc ions measu ed) be ween he
cy oplasmic memb anes o adjacen cells was 27.7 ⫾1.12 nm (n⫽
16) o CSVM141 and 37.3 ⫾1.93 (n⫽23) o he wild ype
(S uden ’s es , P⫽3.3 ⫻10
⫺23
). In CSVM141, a pa icula ly
elec on-dense laye could be de ec ed be ween he adjacen cells,
indica i e o an inc eased densi y o sep al pep idoglycan.
To s udy pep idoglycan in mo e de ail, labeling wi h luo es-
cen ancomycin (Van-FL), which ma ks pep idoglycan ha is in
he p ocess o g ow h o emodeling (38), was pe o med. Van-FL
s ongly labeled he in e cellula sep a in he wild ype and he
⌬ aC ⌬ aD mu an bu much less s ongly in he ⌬sepJ and iple
mu an s (Fig. 1B). In he wild ype and he ⌬ aC ⌬ aD mu an ,
he e was a b oad sp ead in he le el o Van-FL labeling (see Fig. S3
Nü nbe g e al.
2®mbio.asm.o g Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14
in he supplemen al ma e ial). The ⌬sepJ mu an and especially
CSVM141 showed e y ew highly labeled sep a, implying lowe
pep idoglycan u no e o emodeling a es in he in e cellula
sep a in he absence o SepJ.
We hen in es iga ed whe he he a ays o sep al pep idogly-
can nanopo es ha ha e been iden i ied in N. punc i o me (38)
and Anabaena (43) a e s ill p esen in he mu an s lacking he
sep al p o eins. Isola ed sacculi we e isualized by TEM, and a
o al o 5 o 10 sep a o each mu an we e inspec ed (examples
shown in Fig. 2; see Fig. S4 in he supplemen al ma e ial). The
mean numbe o nanopo es was conside ably lowe in he h ee
mu an s han in he wild ype (Fig. 2, his og am). In e es ingly,
he sep al pep idoglycan nanopo es had signi ican ly g ea e di-
ame e in he h ee mu an s han in he wild ype, and his was
pa icula ly e iden in he s ains lacking F aC and F aD (see
Fig. S5 in he supplemen al ma e ial). Thus, he sep al p o eins
(SepJ, F aC, and F aD) a e in ol ed in sep al pep idoglycan nano-
po e o ma ion, bu i should be no ed ha some esidual nano-
po es a e s ill e ained e en in he iple mu an (Fig. 2).
The Anabaena ⌬sepJ ⌬ aC ⌬ aD iple mu an is impai ed
in in e cellula molecula exchange o ace dyes. The luo es-
cein de i a i es calcein and 5-CF (Table 1) can be loaded in o he
Anabaena cy oplasm and used o measu e he kine ics o in e cel-
lula molecula exchange in con ocal FRAP measu emen s (23,
27). These a i icial dyes mo e om cy oplasm o cy oplasm ia
he sep al junc ions, and any in ol emen o he pe iplasm is un-
likely (23). The ⌬sepJ mu an shows a dec eased a e o calcein
exchange (23, 27, 46), and ⌬ aC and ⌬ aD mu an s and he
⌬ aC ⌬ aD double mu an show dec eased a es o exchange o
bo h 5-CF and calcein (27). Howe e , hese mu an s show a esid-
ual exchange ac i i y o abou 20% o he wild- ype ac i i y. To
de e mine whe he his esidual exchange is a ec ed by a combi-
na ion o hese mu a ions, we s udied he in e cellula exchange o
calcein and 5-CF in he iple mu an by using he a e cons an o
luo escence eco e y o he bleached cell (R) as a simple measu e
o molecula exchange ac i i y (27). Ra es o calcein exchange
be ween ege a i e cells o he iple mu an we e simila o hose
obse ed in he ⌬sepJ and ⌬ aC ⌬ aD mu an s, and 5-CF ex-
change a es we e simila o hose o he ⌬ aC ⌬ aD double
mu an (Table 2). These esul s imply he exis ence o an addi-
ional pa hway o mechanism, independen o he known sep al
p o eins, o he in e cellula exchange o calcein and 5-CF ha
con ibu es abou 20% o he wild- ype ac i i y. An ob ious pos-
sibili y is ha he e a e addi ional, as ye uncha ac e ized,
channel- o ming p o eins esponsible o his esidual exchange
ac i i y and associa ed wi h he small numbe o emaining sep al
nanopo es in he iple mu an (Fig. 2).
Up ake o esculin by Anabaena. Calcein and 5-CF a e luo es-
cein de i a i es wi h no esemblance o any me aboli es known o
be physiologically impo an in Anabaena. The e o e, o p o ide
mo e physiologically ele an in o ma ion on in e cellula molec-
ula exchange, we se ou o ind a usable luo escen analog o
suc ose, which appea s o be a key ehicle o ixed ca bon ans e
om ege a i e cells o he e ocys s (12–15). We easoned ha
such a molecule migh be ac i ely impo ed in o he cy oplasm by
na i e suc ose up ake sys ems, hus emo ing he need o use cell-
pe mean es e de i a i es as wi h calcein and 5-CF (23, 27). Se -
e al luo escen suc ose analogs ha e been used o moni o su-
c ose up ake in plan s, including u in, que ce in, and esculin
(50). We ound ha esculin was signi ican ly inco po a ed and
e ained in Anabaena cells (Fig. 3). Compa ison o he dis ibu-
ions o esculin and chlo ophyll luo escence om he hylakoid
memb anes shows ha esculin luo escence o igina es om he
cy oplasm (Fig. 3A). Esculin luo escence could no be de ec ed in
he pe iplasm, whe e luo opho es make a luo escen halo ou -
side he hylakoid memb anes (22, 44, 45). Figu e S6 compa es
he dis ibu ions o esculin luo escence wi h chlo ophyll and
pe iplasmic GFP. Since he pe iplasm is a hin compa men , we
canno exclude he p esence o some esculin in he pe iplasm, bu
i is clea ha a leas mos o he esculin mus be in he cy oplasm.
Simila ly o calcein (23), esculin luo escence is somewha de-
ple ed in he hylakoid memb ane egion (Fig. 3A; see Fig. S6 in
he supplemen al ma e ial), con i ming ha esculin is loca ed in
he cy oplasm a he han in he hylakoid lumen. Nea ly all ila-
men s show esculin luo escence, bu signi ican a ia ion in lu-
o escence in ensi y (Fig. 3A) indica es a ia ion in esculin up ake
compe ence.
Esculin luo escence is pH dependen (51), and luo escence
emission spec a a pH 4.5 o 10 show ha he luo escence yield
inc eases wi h he pH (see Fig. S7A in he supplemen al ma e ial).
Thecy oplasmicpH o Anabaena ege a i e cells is abou 7.0 when
hey a e g own in BG11 medium (52). Assuming ha he pH in
o he cell compa men s alls wi hin he ange o 6.5 o 8.0, esculin
luo escence yield would be simila o wi hin a ac o o ~0.8 o 1.3
(see Fig. S7A). We ound ha esculin luo escence is una ec ed by
O
2
concen a ion (see Fig. S7B), and he e o e, compa ison o
luo escence in ege a i e cells and he e ocys s should no be
p oblema ic.
Anabaena exp esses suc ose anspo ac i i y (53). To es
whe he esculin is aken up by a speci ic suc ose anspo e , we
FIG 1 Ul as uc u e and emodeling o he sep al pep idoglycan in wild- ype
Anabaena and mu an s ains lacking sep al p o eins. (A) T ansmission elec-
on mic og aphs o he sep um be ween wo ege a i e cells o wild- ype
Anabaena ( op) and be ween wo ege a i e cells o s ain CSVM141 (⌬sepJ
⌬ aC ⌬ aD) (bo om). Scale ba s, 200 nm. (B) Fluo escence mic og aphs
showing labeling wi h Van-FL in wild- ype Anabaena and mu an s CSVM34
(⌬sepJ), CSVT22 (⌬ aC ⌬ aD), and CSVM141 (⌬sepJ ⌬ aC ⌬ aD).
Suc ose Supply o Cyanobac e ial He e ocys s
Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14 ®mbio.asm.o g 3
de eloped a luo ome ic assay o esculin up ake (Fig. 4). Esculin
was aken up linea ly o a leas 70 min. Addi ion o suc ose
g ea ly educed he a e o esculin up ake, sugges ing ha bo h
molecules compe e o he same up ake mechanism (Fig. 4).
Esculin as a luo escen p obe o in e cellula communica-
ion. FRAP expe imen s o p obe in e cellula communica ion
use a con ocal lase scanning mic oscope o bleach luo escence in
one o mo e cells. The subsequen edis ibu ion o luo escence
be ween cells e eals he kine ics o in e cellula exchange o he
luo opho e (23). Expe imen s wi h esculin we e less s aigh o -
wa d han hose wi h luo escein de i a i es. The e is weak bu
de ec able backg ound au o luo escence om he cells a he
wa eleng hs used o esculin de ec ion (Fig. 3B), and esculin lu-
o escence is easily bleached while eco ding image se ies in he
con ocal mic oscope. By using low lase powe and a wide con o-
calpinhole o e icien ligh collec iona he expense o esolu ion
FIG 2 Sep al nanopo es in Anabaena. Shown a e ep esen a i e ansmission elec on mic og aphs o sacculi om wild- ype Anabaena and mu an s CSVM34
(⌬sepJ), CSVT22 (⌬ aC ⌬ aD), and CSVM141 (⌬sepJ ⌬ aC ⌬ aD) (scale ba s, 500 nm). Fo u he examples, see Fig. S4 in he supplemen al ma e ial. The
his og am shows he mean numbe o nanopo es pe sep um ⫾SD o he di e en s ains. S uden ’s es s showed ha he di e ences be ween he wild ype
and all o he mu an s we e signi ican (CSVT22, P⫽5.9 ⫻10
⫺3
; CSVM34, P⫽7⫻10
⫺3
; CSVM141, P⫽3.7 ⫻10
⫺3
).
TABLE 1 P edic ed physicochemical p ope ies o esculin, suc ose, and he hyd olyzed in acellula o ms o wo luo escein de i a i es used o
p obe in e cellula communica ion in Anabaena
a
Pa ame e Suc ose Esculin Calcein 5-CF
Molecula mass (Da) 342.3 340.3 622.5 376.3
Pola su ace a ea (Å
2
)189.5 145.9 231.7 113.3
Sol en -accessible molecula su ace a ea (Å
2
)456.5 414.6 794.8 448.4
Van de Waals olume (Å
3
)289.0 278.0 510.4 299.1
Minimal p ojec ion a ea (Å
2
)57.1 45.8 67.0 59.2
Leng h pe pendicula o minimal p ojec ion a ea(Å) 11.5 14.8 19.7 13.1
Maximal p ojec ion a ea (Å
2
)78.0 90.1 139.1 91.0
Leng h pe pendicula o maximal p ojec ion a ea (Å) 9.2 7.0 10.4 9.7
Cha ge(s) o di e en species a pH 7.0 (% abundance[s]) 0 (100) 0 (93), ⫺1 (7) ⫺3 (48), ⫺2 (25), ⫺4 (25), ⫺5 (2) ⫺1 (98), ⫺2 (2)
a
See e e ences 23, 26, 27, and 46. P ope ies a e p edic ed o pH 7.0, co esponding o he pH in he cy oplasm o ege a i e cells (52).
Nü nbe g e al.
4®mbio.asm.o g Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14
in he zdi ec ion, we could eco d epea images wi h negligible
pho obleaching. An impo an con ol is o es o he possibili y
o spon aneous eco e y om pho obleaching by bleaching ou
he en i e ilamen , so ha he e is no possibili y o luo escence
eco e y due o edis ibu ion o he luo opho e. Such a con ol
indica es a spon aneous eco e y o abou 7.5% o he ini ial lu-
o escence (Table 3), which complica es da a analysis. Finally, es-
culin FRAP measu emen s always showed incomple e luo es-
cence eco e y, simila ly o 5-CF (27). This incomple e eco e y
indica es ha a p opo ion o esculin canno be exchanged be-
ween cells, accoun ing o ~50 o 75% o he esculin luo escence
(Table 3). Esculin binds o some p o eins (54), and he e could be
speci ic suc ose-binding ac o s in he cy oplasm. P o ein-bound
esculin will p obably be una ailable o in e cellula exchange by
any ou e. The sep al junc ions a e impe meable o GFP and
he e o e mus ha e a po e size ha excludes many (and possibly
all) p o eins (4, 22). The immobile ac ion p ecluded accu a e
quan i a ion o he “exchange coe icien ” E(23) among ege a-
i e cells. E ela es he a e o dye mo emen be ween wo adjacen
cells o he di e ence in dye concen a ion be ween he cells, and
i squan i a ion equi es i ing o he simula ed ime de elopmen
o dye dis ibu ion in he ilamen o he expe imen al da a (23).
He e, we used a simple way o quan i y kine ics o esculin ans e
be ween ege a i e cells by measu ing he eco e y a e cons an R
as p e iously desc ibed (27; see Ma e ials and Me hods).
In e cellula di usion o esculin in Anabaena ilamen s.
We used FRAP o p obe he in e cellula ans e o esculin in
Anabaena ilamen s by bleaching esculin luo escence in a single
cell and moni o ing i s luo escence eco e y o e ime (Fig. 5). In
di e en ia ed ilamen s, we bleached esculin luo escence in eg-
TABLE 2 Ra es o calcein and 5-CF exchange be ween wild- ype and
mu an ege a i e Anabaena cells
a
Anabaena s ain
Mean R(s
⫺1
)⫾SD (n)
Calcein 5-CF
PCC 7120 (wild ype) 0.097 ⫾0.023 (18) 0.080 ⫾0.013 (37)
CSVM34 (⌬sepJ) 0.023 ⫾0.007 (12) 0.054 ⫾0.007 (54)
CSVT22 (⌬ aC ⌬ aD) 0.015 ⫾0.003 (21) 0.017 ⫾0.003 (42)
CSVM141 (⌬sepJ ⌬ aC ⌬ aD) 0.028 ⫾0.006 (21) 0.022 ⫾0.002 (84)
a
Calcein and 5-CF we e loaded in o cells o BG11-g own ilamen s o he s ains
indica ed, he ace was bleached in one cell, and he luo escence eco e y a e
cons an , R, was de e mined as desc ibed in Ma e ials and Me hods. Da a a e mean
alues ⫾SDs (12 o 84 ilamen s we e analyzed o each s ain and ace ). S uden ’s
es s indica e ha di e ences in calcein ans e be ween s ains PCC 7120 and
CSVM34 and be ween PCC 7120 and CSVT22 a e signi ican (P⬍10
⫺4
) and he
di e ence be ween CSVM141 and CSVT22 migh also be signi ican (P⫽0.04).
Di e ences in 5-CF ans e be ween s ains PCC7120 and CSVM34, PCC7120 and
CSVT22, CSVM34 and CSVT22, and CSVM141 and CSVT34 a e all signi ican (P⬍
10
⫺5
).
FIG 3 Up ake o esculin by wild- ype Anabaena ilamen s. (A) Esculin-labeled cells (g own in BG11). (B) Con ol wi h unlabeled cells. Scale ba s, 10
␮
m. Esculin
luo escence (443 o 490 nm) is cyan (le ), chlo ophyll luo escence (670 o 720 nm) is magen a (cen e ), and o e laid images a e shown on he igh .
FIG 4 Time cou se o esculin up ake in Anabaena cells and e ec o suc ose
compe i ion. Cells we e g own in BG11 o BG11
0
medium wi h o wi hou
combined ni ogen. Up ake o esculin (100
␮
M) was measu ed wi h o wi h-
ou suc ose a 10 mM. E o ba s ep esen SDs (n⫽3 o 5). Ra es o up ake
di e ed signi ican ly o BG11 wi h o wi hou suc ose (P⫽0.00003), BG11
0
wi h o wi hou suc ose (P⫽0.002), and BG11 e sus BG11
0
wi hou suc ose
(P⫽0.05). Absolu e esculin up ake alues assume ha he in acellula luo-
escence yield is simila o ha in he bu e .
Suc ose Supply o Cyanobac e ial He e ocys s
Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14 ®mbio.asm.o g 5

e a i e cells and in he e ocys s, which could be dis inguished by
hei enla ged size and educed chlo ophyll luo escence. Bo h cell
ypes show eco e y unde all o he condi ions es ed, indica ing
ha esculin can be ans e ed be ween ege a i e cells (Fig. 5A
and B) and om ege a i e cells o he e ocys s (Fig. 5C and D).
Fluo escence eco e y g ea ly exceeds he spon aneous eco e y
o esculin luo escence (Table 3), showing ha he e is in e cellu-
la molecula exchange. Esculin ans e shows he cha ac e is ics
o di usion a he han ac i e anspo , since luo escen esculin
always lows down he concen a ion g adien and he ans e
uns o equilib ium (Fig. 5). Re e sibili y o esculin ans e om
ege a i e cells o he e ocys s was es ed by bleaching ege a i e
cells neighbo ing a he e ocys . I a single ege a i e cell nex o a
he e ocys is bleached, luo escence eco e y mainly e lec s mo-
lecula exchange wi h he nex ege a i e cell a he han wi h he
he e ocys because o he as e exchange among ege a i e cells
(Table 3). The e o e, we chose a sho ilamen o Anabaena wi h
a e minal he e ocys and bleached esculin luo escence in all o
he ege a i e cells. The subsequen decay o he e ocys luo es-
cence (Fig. 5E and F) shows ha esculin can be ans e ed om
he e ocys s o ege a i e cells. Esculin ans e occu s a compa a-
ble a es in bo h di ec ions (Fig. 5D and F), indica ing in e cellula
di usion o esculin wi hou signi ican ac i e anspo . Esculin
luo escence is no obse ed conspicuously o consis en ly a he
pe iphe y o he cells (Fig. 3A; see Fig. S6 in he supplemen al
ma e ial), sugges ing ha exchange occu s p edominan ly om
cy oplasm o cy oplasm, esembling he p e iously cha ac e ized
in e cellula di usion o calcein and 5-CF (23, 27).
Kine ics o esculin exchange and in luence o sep al p o eins
SepJ, F aC, and F aD. In wild- ype Anabaena ilamen s, esculin
exchange is signi ican ly as e among ege a i e cells han be-
ween ege a i e cells and he e ocys s (Table 3), al hough he di -
e ence is less p onounced han wi h calcein (23). Esculin ex-
change among ege a i e cells did no become signi ican ly as e
ollowing combined-ni ogen emo al (Table 3), in con as o
calcein (23). Esculin in lux in o he e ocys s is no signi ican ly
as e in a mu an ha lacks he cyanophycin syn he ase CphA1
and is he e o e unable o o m cyanophycin plugs a he he e o-
cys cell poles (55) (Table 3). This indica es ha esculin ans e is
no e a ded by any cyanophycin plugs ha may be p esen . Con-
sis en wi h his inding, we could no de ec any in e ac ion be-
ween esculin and cyanophycin in i o (see Fig. S8 in he supple-
men al ma e ial).
SepJ, F aC, and F aD a e impo an o he in e cellula ex-
change o luo escen ace s (23, 27, 46). We in es iga ed he
in luence o hese p o eins on he in e cellula ans e o esculin
by using ⌬sepJ,⌬ aC ⌬ aD, and ⌬sepJ ⌬ aC ⌬ aD mu an s as
desc ibed abo e. These mu an s show ilamen agmen a ion un-
de some condi ions (24, 26) (see Fig. S2 in he supplemen al
ma e ial); howe e , we always chose ilamen s a leas 5 cells long
o FRAP measu emen s. In BG11-g own cul u es, all o he mu-
an s showed lowe luo escence eco e y a es han he wild ype
(Table 3), consis en wi h he in ol emen o hese sep al p o eins
in heexchange o esculin among ege a i e cells. Howe e , slowe
esidual esculin exchange was obse ed e en in he iple mu an
(Table 3), as is also he case wi h calcein and 5-CF (Table 2). The
⌬sepJ mu an does no g ow diazo ophically and is a es ed ea ly
in he e ocys di e en ia ion (46) and so canno be used o es o
he in ol emen o SepJ in esculin exchange be ween ege a i e
cells and he e ocys s. Howe e , while he ⌬ aC ⌬ aD mu an is
also incapable o sus ained diazo ophic g ow h, i su i es o
mo e han 48 h ollowing a ni ogen s ep down, p oducing he -
e ocys s du ing his ime (27). This allows a es o he in ol e-
men o F aC and F aD in he e ocys – ege a i e-cell exchange.
FRAP shows ha he ans e o esculin in o ⌬ aC ⌬ aD he e o-
cys s is slowe han in he wild ype by a ac o o ⬎3 (Table 3).
Loss o me abolic communica ion in olde he e ocys s. In
wild- ype Anabaena g own diazo ophically o 48 h, some he -
e ocys s show no esculin luo escence, despi e he p esence o es-
culin in he neighbo ing ege a i e cells (Fig. 6A and B). This
sugges s ha some he e ocys s a e de icien in esculin exchange
wi h hei ege a i e neighbo s. We quan i ied esculin equilib a-
ion be ween he e ocys s and ege a i e cells by di iding he escu-
lin luo escence in ensi y in each he e ocys by he mean luo es-
cence in ensi y in i s immedia e ege a i e neighbo s (I
H
/I
V
a io).
The I
H
/I
V
a io should be ~1 o “communica ing” he e ocys s,
whe e esculin equilib a es ac oss he cell junc ions, bu ~0 o
he e ocys s ha a e de icien in esculin exchange and also incapa-
ble o di ec esculin up ake. The I
H
/I
V
a io shows a bimodal dis-
ibu ion wi h peaks close o 0 and 1 (Fig. 6C), consis en wi h wo
such popula ions o he e ocys s. Fluo escence in he noncommu-
nica ing he e ocys s was no signi ican ly abo e he backg ound
seen in unlabeled cells. Communica ing and noncommunica ing
he e ocys s a e some imes p esen in he same Anabaena ilamen
(Fig. 6B). The equency o communica ing he e ocys s is almos
100% 24 h a e a ni ogen s ep down bu hen d ops o ~70% a
48 h and subsequen ly emains a ha le el (Fig. 6D). The e o e,
young he e ocys s a e nea ly all capable o esculin exchange, bu
du ing con inued diazo ophic g ow h, a popula ion o ~30% o
noncommunica ing he e ocys s builds up. Communica ing and
noncommunica ing he e ocys s a e equally likely o be loca ed a
he e minus o he ilamen : we ound ha a 48 h a e a ni ogen
TABLE 3 Kine ics o esculin exchange om FRAP measu emen s on
Anabaena ilamen s
a
Measu emen (no. o eplica es)
Mean R
(s
⫺1
)⫾SD
Mean
F
i
⫾SD
Vege a i e cells in p esence o ni a e
Wild- ype Anabaena (29)* 0.137 ⫾0.050 0.518 ⫾0.143
CSVM34 (⌬sepJ) (25)† 0.099 ⫾0.042 0.571 ⫾0.145
CSVT22 (⌬ aC ⌬ aD) (30)‡ 0.069 ⫾0.029 0.605 ⫾0.072
CSVM141 (⌬sepJ ⌬ aC ⌬ aD) (21)§ 0.069 ⫾0.030 0.671 ⫾0.068
Vege a i e cells o wild- ype Anabaena 48 h
a e ni ogen dep i a ion (38)¶
0.142 ⫾0.046 0.507 ⫾0.110
He e ocys s 48 h a e ni ogen dep i a ion
Wild- ype Anabaena (33)** 0.060 ⫾0.054 0.757 ⫾0.069
CSVT22 (⌬ aC ⌬ aD) (18)†† 0.017 ⫾0.024 0.736 ⫾0.097
CSS7 (cphA1::C.S3) (24) 0.074 ⫾0.069 0.640 ⫾0.160
Spon aneous luo escence eco e y o
wild- ype Anabaena (21)
0.925 ⫾0.049
a
The mean exponen ial eco e y a e cons an s (R) and immobile ac ions (F
i
) o
ilamen s g own wi h o wi hou combined ni ogen we e measu ed. R alues we e
s anda dized by di iding by 2 o cells wi h wo connec ing junc ions (i.e., all o he cells
excep hose a he e minus o he ilamen ). F
i
is de ined by (I
I
⫺I
E
)/(I
I
⫺I
0
), whe e
I
I
is he ini ial luo escence in ensi y be o e bleaching, I
0
is he luo escence in ensi y
immedia ely a e bleaching, and I
E
is he inal luo escence in ensi y. The ex en o
spon aneous luo escence eco e y is gi en by 1 ⫺F
i
. S uden ’s es s show ha Ris
signi ican ly di e en be ween * and † (P⫽0.004), * and ‡ (P⬍0.00001), * and § (P⬍
0.00001), † and ‡ (P⫽0.004), † and § (P⫽0.008), ** and †† (P⫽0.002), * and **
(P⬍0.00001), ‡ and †† (P⬍0.00001), and ¶ and ** (P⬍0.00001). R alues o cells
showing 1 ⫺F
i
⫽⬍0.075 (equi alen o he spon aneous luo escence eco e y in he
absence o di usion) we e conside ed o be 0.
Nü nbe g e al.
6®mbio.asm.o g Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14
FIG 5 Examples o FRAP expe imen s moni o ing in e cellula exchange o esculin in wild- ype Anabaena ilamen s. A and B, esculin exchange among
ege a i e cells; C and D, esculin ans e om ege a i e cells o a e minal he e ocys ; E and F, esculin ans e om a e minal he e ocys o ege a i e cells. (A,
C, and E) Fluo escence images om FRAP ime se ies showing esculin luo escence p io o bleaching (p e), immedia ely a e bleaching ( ⫽0), and a he la e
ime poin s indica ed. Scale ba s, 5
␮
m. (B and D) Fluo escence eco e y cu es o bleached cells. Fluo escence is exp essed in consis en a bi a y uni s (a.u.).
(E and F) Esculin mo emen om a e minal he e ocys (a owhead) o he neighbo ing ege a i e cells, obse ed by moni o ing he e ocys luo escence a e
bleaching o all o he ege a i e cells. Filled ci cles, he e ocys luo escence in successi e images p io o bleaching. Open ci cles, he e ocys luo escence e sus
ime a e bleaching luo escence in he ege a i e cells. He e ocys luo escence a ⫽0 is al eady lowe ed by di usion o esculin ou o he he e ocys du ing
bleaching, p obably combined wi h some di ec bleaching o he he e ocys esculin due o sca e ed ligh .
Suc ose Supply o Cyanobac e ial He e ocys s
Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14 ®mbio.asm.o g 7
s ep down, 52% o noncommunica ing he e ocys s we e e minal
(n⫽42) and also ha 52% o communica ing he e ocys s we e
e minal (n⫽149). This sugges s ha loss o communica ion is
no a consequence o ilamen b eakage. A cphA1 mu an de icien
in cyanophycin syn he ase (52) showed a simila equency o
noncommunica ing he e ocys s (see Fig. S9 in he supplemen al
ma e ial), indica ing ha cyanophycin plugs a e also no he cause
o he e ocys noncommunica ion. He e ocys s a e incapable o
u he cell di ision and ha e a limi ed li e ime be o e hey senesce
and die (56). To es he possibili y ha he noncommunica ing
he e ocys s a e senescen , we es o ed combined ni ogen o a
diazo ophic cul u e. This p e en s he gene a ion o new he -
e ocys s, he e o e leading o an aging he e ocys popula ion
and he g adual disappea ance o he e ocys s om he cul u e
(57). He e ocys equency dec eases ollowing combined-
ni ogen addi ion, wi h a simul aneous dec ease in he p opo -
ion o communica ing he e ocys s (Fig. 6E). This sugges s ha
he noncommunica ing he e ocys s a e hose in he ea ly s ages o
senescence,p io o any ob ious mo phological changes (Fig.6A).
DISCUSSION
Esculin is a luo escen ace ha closely esembles suc ose (Ta-
ble 1). We ound ha esculin is impo ed in o he cy oplasm o
Anabaena cells by a suc ose up ake sys em (Fig. 4), as i is in plan s
(48). The e o e, imaging o esculin luo escence in Anabaena
should gi e a good guide o he beha io o suc ose. Esculin in he
Anabaena cy oplasm includes a mobile pool ha apidly and e-
e sibly exchanges among ege a i e cells and be ween ege a i e
cells and he e ocys s (Fig. 5). Esculin luo escence could no be
de ec ed in he pe iplasm (Fig. 3 and 6; see Fig. S6 in he supple-
men al ma e ial), and exchange is educed in mu an s lacking sep-
al junc ion p o eins, sugges ing ha he majo ou e o in e cel-
lula ans e o esculin is di usion om cy oplasm o cy oplasm
ac oss cell junc ions. Communica ing he e ocys s showed, on a -
e age, le els o esculin luo escence simila o hose o hei ege-
a i e neighbo s (Fig. 6C), sugges ing ull equilib a ion o esculin
ac oss he cell junc ion. Re e sible he e ocys – ege a i e-cell ex-
change is also indica ed by he simila kine ics o esculin mo e-
men in bo h di ec ions (Fig. 5).
Ou indings on he ul as uc u e and molecula exchange
cha ac e is icso a mu an lacking sep alp o eins shed ligh on he
mechanism o in e cellula esculin exchange. S uc u es ha ha e
been e med mic oplasmodesma a o sep osomes ha e long been
known o be p esen in he in e cellula sep a o he ilamen s o
he e ocys - o ming cyanobac e ia (29, 58–60). Cell wall pe o a-
ions ha ha e been e med nanopo es, ecen ly epo ed o be
p esen in sep al pep idoglycan discs (38), may be he places whe e
hese s uc u es a e se he mu ein sacculus (30, 32, 39, 40, 43).
Th ee known p o eins ha a e equi ed o making long ilamen s
in Anabaena, SepJ, F aC, and F aD, a e loca ed a he in e cellula
sep a whe e hey appea o play a ole in cell-cell adhesion and
acili a ing communica ion be ween adjacen cells (23, 24, 26).
Such unc ions would equi e a physical in e ac ion be ween he
p o eins con ibu ed by each o he adjacen cells, implying ha
hese p o eins a e se he sep al pep idoglycan. We ound a sig-
ni ican ly dec eased numbe o nanopo es in mu an s lacking
combina ions o SepJ, F aC, and F aD compa ed o he wild ype.
FIG 6 Esculin labeling o Anabaena he e ocys s. (A and B) Fluo escence mic og aphs showing esculin-labeled wild- ype Anabaena ilamen s 48 h a e a
combined-ni ogen s ep down. Communica ing he e ocys s a e indica ed by whi e a ows, and noncommunica ing he e ocys s (de ined by an I
H
/I
V
a io o
⬍0.2) a e indica ed by yellow a ows. I
H
, esculin luo escence in ensi y in he he e ocys ; I
V
, mean esculin luo escence in ensi y in immedia ely neighbo ing
ege a i e cells. Chlo ophyll luo escence is magen a, esculin luo escence is cyan, and he b igh - ield image is in g ayscale. Panels A (bo om) and B a e me ged
images. Scale ba s, 5
␮
m in panel A and 10
␮
m in panel B. (C) F equency dis ibu ion o I
H
/I
V
a ios o he e ocys s in ilamen s esculin labeled as in panels A and
B. (D and E) F equencies o communica ing (black) and noncommunica ing (g ay) he e ocys s e sus ime ollowing a combined-ni ogen s ep down (D) and
a e he addi ion o combined ni ogen o a diazo ophic cul u e (E). De ached he e ocys s we e no included in he s a is ics.
Nü nbe g e al.
8®mbio.asm.o g Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14
The mu an s lack he ypical nanopo e a ay seen in he wild ype
and ins ead show jus a ew andomly placed nanopo es. A
s aigh o wa d hypo hesis is ha SepJ, F aC, and F aD a e com-
ponen s o he sep al junc ions and ha sep al junc ion complexes
con aining hese p o eins in luence he ac i i y o he AmiC ype
amidases o o m a sep al nanopo e a ay. Howe e , he p esence
o a ew esidual nanopo es in he ⌬ aC ⌬ aD ⌬sepJ iple mu-
an indica es ei he ha he e a e pep idoglycan hyd olases ha
can o m some po es in he absence o any sep al junc ion p o eins
o ha o he sep al junc ion p o eins, ye o be iden i ied, a e
p esen in he in e cellula sep a. In e es ingly, he esidual nano-
po es ha e a signi ican ly g ea e mean diame e han hose seen
in he wild ype (see Fig. S5 in he supplemen al ma e ial). This is
consis en wi h he idea ha he esidual sep al channels in he
⌬sepJ ⌬ aC ⌬ aD iple mu an ha e a molecula composi ion
di e en om ha o he majo i y o he channels seen in he wild
ype, which a e likely o include SepJ, F aC, and F aD among hei
componen s.
In he ⌬sepJ ⌬ aC ⌬ aD iple mu an , he cy oplasmic mem-
b anes o he adjacen cells a e e y close o each o he , and a
highly elec on-dense laye (p esumably pep idoglycan) is ob-
se ed be ween hem. In e cellula junc ions ha a e hinne han
in he wild ype ha e also been obse ed in he ⌬sepJ single mu an
s ain CSVM34 (30). As wi h me azoan gap junc ions (31), sep al
junc ion complexes migh de e mine a ixed dis ance be ween he
cy oplasmic memb anes o he adjacen cells, and he absence o
hose complexes migh p oduce he collapse o he sep um be-
ween hose cells. Mu an s lacking SepJ show lowe labeling wi h a
luo escen ancomycin de i a i e (Fig. 1; see Fig. S3 in he sup-
plemen al ma e ial), which is indica i e o a lowe a e o sep al
pep idoglycan emodeling (38) in he absence o his p o ein.
Mu an s lacking SepJ, F aC, and F aD a e all impai ed in in-
e cellula esculin exchange (Table 3), consis en wi h he idea
ha he sep al junc ions a e c ucial o suc ose exchange be ween
cells. Loss o F aC and F aD slows esculin exchange be ween eg-
e a i e cells and he e ocys s by abou 70% and slows esculin ex-
change among ege a i e cells by abou 50% (Table 3). F aC and
F aD a e in ol ed in he o ganiza ion o SepJ a he sep a (26),
which may explain why esculin exchange is as s ongly a ec ed in
he ⌬ aC ⌬ aD mu an s ain as in he ⌬sepJ ⌬ aC ⌬ aD iple
mu an (Table 3). SepJ, F aC, and F aD (and possibly o he p o-
eins) may o m a amily o in e cellula channels wi h di e en
selec i i ies o molecula size and cha ge. Residual in e cellula
exchange o esculin and luo escein de i a i es in he iple mu-
an may be due o addi ional, uncha ac e ized channels be e
capable o ansmi ing esculin han la ge and mo e nega i ely
cha ged molecules (Table 1). This is consis en wi h he esidual
sep al nanopo es in he iple mu an (Fig. 2).
A ou e o exchange o ca bon and ni ogen compounds be-
ween he e ocys s and ege a i e cells could in ol e di usion
h ough a con inuous pe iplasm (22), ollowed by ac i e impo
in o he cy oplasm (57). Howe e , he apid exchange o esculin a
he e ocys – ege a i e-cell sep al junc ions sugges s ha di usion
om cy oplasm o cy oplasm ac oss hese junc ions is he main
ou e o suc ose ans e . Me azoan gap junc ions p o ide a p ec-
eden o ap o ein amily(in hiscase,connexins) o minga ange
o di usion channels wi h di e en pe meabili y cha ac e is ics
(31). Some Anabaena sep al junc ion s uc u es could be op i-
mized o he exchange o suc ose, consis en wi h he idea ha
suc ose is he p incipal me aboli e used o supply he e ocys s wi h
pho osyn ha e (12–15).
Du ing p olonged diazo ophic g ow h, ~30% o he e ocys s
do no show esculin luo escence (Fig. 6). Al hough he e ocys s
migh be capable o di ec suc ose up ake (57), he noncommu-
nica ing he e ocys s mus be incapable o esculin up ake by his
ou e, and om he neighbo ing ege a i e cells, since hei escu-
lin luo escence is negligible (Fig. 6). Changes in he noncommu-
nica ing popula ion ollowing emo al and eaddi ion o com-
bined ni ogen (Fig. 6) sugges ha his popula ion consis s o
he e ocys s in he ea ly s ages o senescence. He e ocys s can con-
sume ~50% o he ilamen ’s pho osyn ha e (1), so i mus be
impo an o supply only ully unc ional he e ocys s wi h su-
c ose. I may be c ucial o su i al o close communica ion
wi h a dying he e ocys be o e i lyses, o a oid apid leakage o
me aboli es in o he medium. Loss o communica ion could
esul om closu e o hal -channels on ei he side o he
he e ocys – ege a i e-cell sep al junc ion, pe haps compa able o
he egula ion o me azoan gap junc ion ac i i y (61). The pa i-
ioning o suc ose be ween communica ing cells in he ilamen
may be con olled by suc ose me abolism in indi idual cells, a
simple bu e ec i e way o ensu e ha he pho osyn ha e supply
mee s hepho osyn ha edemando indi idual ege a i ecellsand
he e ocys s.
MATERIALS AND METHODS
S ains, g ow h condi ions, and PCR analysis. The sepJ, aC, and aD
genes a e open eading ames al 2338,al 2392, and al 2393 o he
Anabaena genome, espec i ely (62). Mu an s ains CSS7 (cphA1::C.S3),
CSVT22 (⌬ aC ⌬ aD), and CSVM34 (⌬sepJ) ha e been p e iously de-
sc ibed (27, 46, 55). S ain CSVM141 was cons uc ed by ans e ing an
inac i a ing sepJ cons uc o mu an CSVT22 as p e iously desc ibed o
he cons uc ion o CSVM34 om s ain PCC 7120 (46). CSVM141 ca -
ies unma ked dele ions in aC, aD, and sepJ. DNA was isola ed om
Anabaena as desc ibed in e e ence 63. PCR was pe o med by s anda d
p ocedu es (64), and he PCR p oduc s we e esol ed by elec opho esis
in 0.8% aga ose gels. Fo he oligodeoxynucleo ide p ime s used, see Ta-
ble S1 in he supplemen al ma e ial.
Anabaena s ains we e g own in liquid BG11 medium (65) wi h an i-
bio ics app op ia e o mu an s ain CSS7 in conical lasks wi h shaking
(80 o 100 pm) a 30°C in whi e ligh a ~15 o 75
␮
E·m
⫺2
·s
⫺1
.
He e ocys o ma ion was induced by g ow h in BG11
0
medium (65),
which lacks combined ni ogen. Fo ans e be ween media, cells we e
ha es ed by cen i uga ion (3,000 ⫻g) and washed h ee imes in he
app op ia e medium. To de e mine ilamen leng h, lasks con aining
BG11 medium we e inocula ed wi h ma e ial om pla es and incuba ed
o 3 o 4 days a 30°C (80 o 90 pm, 25
␮
E·m
⫺2
·s
⫺1
). The cul u es we e
hen ha es ed, adjus ed o 2
␮
g o chlo ophyll a(Chl) · ml
⫺1
and incu-
ba ed in esh BG11 medium o 24 h. Filamen s we e coun ed a ha ime
o ha es ed, washed, and esuspended in BG11
0
medium and u he
incuba ed unde he cul u e condi ions desc ibed abo e. Samples we e
aken wi h g ea ca e o p e en dis up ion o he ilamen s and placed on
solidi ied medium (BG11 o BG11
0
). Samples we e isualized and pho o-
g aphed by s anda d ligh mic oscopy.
Elec on mic oscopy. Ul a hin sec ion p epa a ion was done as de-
sc ibed in e e ence 40. B ie ly, ilamen s o he s ains indica ed ha we e
g own in BG11 medium we e ha es ed by gen le cen i uga ion and ixed
wi h 4% (w / ol) glu a aldehyde and 2% (w / ol) KMnO
4
, dehyd a ed
wi h inc easing concen a ions o e hanol, and embedded in a aldi e, and
ul a hin sec ions we e p epa ed and pos s ained wi h u anyl ace a e and
lead ci a e (66). The samples we e examined wi h a JEOL JEM-1230
elec on mic oscope a 80 kV. The sacculi we e isola ed and analyzed as
p e iously desc ibed (38, 67). In b ie , a e b eakage o he cells by soni-
Suc ose Supply o Cyanobac e ial He e ocys s
Ma ch/Ap il 2015 Volume 6 Issue 2 e02109-14 ®mbio.asm.o g 9