Mini e iew
P edic ing subs a e exchange in ma ine
dia om-he e ocys ous cyanobac e ia symbioses
Me cedes Nie es-Mo ión,
1
En ique Flo es
2
*and
Rachel A. Fos e
1
*
1
Depa men o Ecology, En i onmen and Plan
Sciences, S ockholm Uni e si y, S ockholm, 106 91,
Sweden.
2
Ins i u o de Bioquímica Vege al y Fo osín esis, CSIC
and Uni e sidad de Se illa, Amé ico Vespucio 49,
Se ille, E-41092, Spain.
Summa y
In he open ocean, some phy oplank on es ablish
symbiosis wi h cyanobac e ia. Some pa ne ships
in ol e dia oms as hos s and he e ocys ous cyano-
bac e ia as symbion s. He e ocys s a e specialized
cells o ni ogen fixa ion, and a unc ion o he sym-
bio ic cyanobac e ia is o p o ide he hos wi h ni o-
gen. Howe e , bo h pa ne s a e pho osyn he ic and
capable o ca bon fixa ion, and he possible me abo-
li es exchanged and mechanisms o ans e a e
poo ly unde s ood. The symbion cellula loca ion
a ies om in e nal o pa ial o ully ex e nal, and
his is eflec ed in he symbion genome size and
con en . In o de o iden i y he memb ane ans-
po e s po en ially in ol ed in me aboli e exchange,
we compa e he d a genomes o h ee di e en ly
loca ed symbion s wi h known anspo e s mainly
om model ee-li ing he e ocys ous cyanobac e ia.
The ypes and numbe s o anspo e s a e di ec ly
ela ed o he symbion cellula loca ion: es ic ed in
he endosymbion s and wide in he ex e nal symbi-
on . Th ee p oposed models o me aboli e exchange
a e sugges ed which ake in o accoun he ype o
anspo e s in he symbion s and he influence o
hei cellula loca ion on he a ailable nu ien pools.
These models p o ide a basis o se e al hypo heses
ha gi en he impo ance o hese symbioses in
global N and C budge s, wa an u u e es ing.
In oduc ion
In as expanses o open ocean en i onmen s concen a-
ions o dissol ed ino ganic ni ogen a e below analy ical
de ec ion. He e, mic oo ganisms capable o educing di-
ni ogen (N
2
), which comp ises 78% o he a mosphe e, a e
a an ad an age and ypically domina e. The p ocess o N
2
fixa ion, o he educ ion o N
2
o ammonium, is pe o med
by a small and di e se g oup o bac e ia and a chaea
(Young, 1992). Some N
2
fixing (diazo ophic) popula ions in
he open ocean a e symbio ic, wi h a ew gene a o mic o-
algae, specifically dia oms as hos s and he e ocys ous cya-
nobac e ia as symbion s (Fos e and O’Mullan, 2008)
(Fig. 1A–C). Collec i ely hese symbioses a e e e ed o as
dia om diazo ophic associa ions (DDAs). DDAs a e globally
dis ibu ed and a e conside ed majo con ibu o s o bo h N
and ca bon (C) cycles due o seasonal blooms wi h high N
2
and C fixa ion a es and apid sinking (Mague e al., 1974;
Ven ick, 1974; Ca pen e e al., 1999; Sub amaniam e al.,
2008; Ka l e al., 2016). Despi e hei ecogni ion as globally
significan , he e s ill emain la ge gaps in ou unde s anding
o DDAs, especially how he pa ne s in e ac and acqui e
(and sha e) he elemen s necessa y o me abolism and
g ow h. He e, we analysed he a ailable DDA symbion
genomes o iden i y memb ane anspo e s po en ially
in ol ed in in e ac ions wi h he hos pa ne s. This allowed
us o ecognize a ge p o eins o u he in es iga ion by
gene ic app oaches, o ins ance by he he e ologous
exp ession and analysis o genes om he symbion s.
The dia om diazo ophic associa ions
The he e ocys ous cyanobac e ial symbion s
The he e ocys ous cyanobac e ial symbion s o DDAs
ha e been cha ac e ized by se e al gene ic ma ke s,
including 16S RNA, he ni H gene, which encodes he
ni ogenase educ ase componen (Fe p o ein) o he
ni ogenase complex o N
2
fixa ion, and he he R gene,
Recei ed 17 Janua y, 2020; e ised 2 Ap il, 2020; accep ed 3 Ap il,
2020. *Fo co espondence. E-mail efl[email p o ec ed]; Tel.
+34954489523; E-mail ac[email p o ec ed]; Tel. 4608161207.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s
use and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modifica ions o
adap a ions a e made.
En i onmen al Mic obiology (2020) 22(6), 2027–2052 doi:10.1111/1462-2920.15013
which encodes a key egula o o cell di e en ia ion in
he e ocys ous cyanobac e ia (Janson e al., 1999; Fos e
and Zeh , 2006). Based on hese phylogenies he symbi-
on s a e ela ed o o he he e ocys ous cyanobac e ia
in he o de Nos ocales (e.g. Anabaena sp., Nos oc
sp. and Calo h ix sp.). No ably, he symbion s a e mo e
ela ed be ween hem han o any o he known he e ocys-
ous cyanobac e ium (Capu o e al., 2019). In spi e o
ha , symbion he R and ni H sequences a e ela i ely
di e gen (84% and 91% iden i y espec i ely) sugges ing
a high specifici y in he pa ne ships (Janson e al., 1999;
Fos e and Zeh , 2006). In o he wo ds, one pa icula
symbion s ain associa es wi h one pa icula hos
genus, and he d i e o he specifici y is cu en ly
unknown (Janson e al., 1999; Fos e and Zeh , 2006).
Mo phologically, he symbion s a y in e ms o filamen
leng h and ape , and all possess e minal he e ocys s.
The symbion s a y in hei cellula loca ion: in e nal,
pa ial and ex e nal. ‘In e nal’symbion s ha e pene a ed
he dia om’s cy oplasm; ‘pa ial’(o ‘pe iplasmic’) e e s o
symbion s ha eside be ween he dia om’s cy oplasmic
memb ane and us ule (ou e silicified cell wall o dia-
oms); and ‘ex e nal’a e symbion s ha a e a ached o
he su ace o he dia om (Villa eal, 1989, 1990; Capu o
e al., 2019). Impo an ly, he ex e nal symbion (Calo h ix
hizosoleniae, CalSC01) can be g own independen ly o
i s hos in he labo a o y (Fos e e al., 2010) (see
Fig. 1D). D a genomes a e a ailable o he he e ocys-
ous symbion s: Richelia in acellula is Rin HH01 (in e -
nal, associa ed wi h Hemiaulus hauckii), 3.24 Mbp; R.
in acellula is Rin HM01 (in e nal, associa ed wi h H.
memb anaceus), 2.21 Mbp; R.in acellula is Rin RC01
(pa ial, associa ed wi h Rhizosolenia cle ei), 5.4 Mbp;
and Calo h ix hizosoleniae CalSC01 (ex e nal, associ-
a ed wi h Chae oce os comp esus), 5.97 Mbp (Hil on
e al., 2013; Hil on, 2014). This shows ha genome size
is di ec ly ela ed o he cellula loca ion o he symbion s.
Gene al ea u es (size, GC con en and pe cen coding)
o he symbion genomes a e simila o o he Nos ocales,
including ee-li ing and acul a i e and obliga e symbio ic
s ains o mul icellula plan s (e.g. Anabaena sp. PCC
7120, Nos oc punc i o me PCC 73102 and Nos oc
azollae 0708 espec i ely; PRJNA244, PRJNA216 and
PRJNA30807 espec i ely) (Meeks e al., 2001; Hil on
e al., 2013). The R.in acellula is Rin HM01 d a
genome lacks se e al sequences expec ed o a ull
genome due o low sequencing co e age (Hil on e al.,
2013), and he e o e i was no compa ed u he .
The symbio ic dia oms
Dia oms a e single-celled euka yo ic plank on widely dis-
ibu ed in aqua ic en i onmen s ha con ibu e signifi-
can ly (20%) o global p ima y p oduc ion (Field e al.,
1998). Mos dia oms domina e coas al en i onmen s,
whe e dissol ed nu ien s a e high, and like o he mic o-
algae, dia oms can u ilize ni a e and ammonium
(Guilla d and Kilham, 1977; A mb us , 2009). In addi ion,
dia oms possess a comple e u ea cycle (Allen e al.,
2011). On he o he hand, in oligo ophic ma ine
Fig. 1. Epifluo escen images aken o
wild popula ions (A–C) o he a ious
Dia om Diazo oph Associa ions
(DDAs) and he ex e nal symbion
Calo h ix SC01 a e isola ion (D). Cells
imaged in he field used a blue exci a-
ion fil e (450–490 nm) o dis inguish
he he e ocys ous cyanobac e ial sym-
bion s (yellow-o ange) om he dia om
chlo oplas s ( ed). A. One Rhizosolenia
cle ei-R. in acellula is (Rin RC01)
symbiosis. No e he longe filamen o
Richelia when associa ed wi h
Rhizosolenia.B.Achaino h ee
Hemiaulus hauckii dia oms associa ed
wi h Richelia in acellula is (Rin HH01).
C. A chain o >10 Chae oce os com-
p essus dia oms wi h Calo h ix hizo-
soleniae (CalSC01) a ached o he
ou side. No e ha he spines a e no
isible o he dia om and he ape o
he Calo h ix symbion di e s om ha
o Richelia.D.TheCalSC01isola e
se e al mon hs a e isola ion (imaged
wi h blue exci a ion). No e he longe
filamen s when g owing eely o i s
hos dia om. Scale ba s a e app oxi-
ma ely 10 μm. [Colo figu e can be
iewed a wileyonlinelib a y.com]
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2028 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
en i onmen s, some dia oms o m a symbiosis wi h cya-
nobac e ia (Villa eal, 1992; Fos e and O’Mullan, 2008).
In gene al, we know a less abou he symbio ic hos dia-
oms compa ed wi h hei espec i e symbion s. The hos
dia oms di e d ama ically in cell size (e.g. Hemiaulus
hauckii,12–35 μm; H.memb anaceus,30–70 μm;
Rhizosolenia cle ei,7–250 μm; Chae oce os comp e-
ssus,7–40 μm). The Hemiaulus spp. and Chae oce os
spp. hos s a e capable o o ming long chains (>50 cells),
while R.cle ei end o be soli a y. These obse a ions
aise ques ions on how/i symbion me abolism (e.g. N
2
fixa ion) is influenced by hos cell size, and whe he /how
subs a es a e also exchanged be ween symbio ic cells.
The gene ic iden i y o he hos s was only ecen ly cha ac-
e ized o a ew gene ic ma ke s and esul ed in cong u-
en phylogenies wi h he espec i e symbion phylogenies
sugges ing co-e olu ion (Capu o e al., 2019).
The symbiosis
An impo an , in e es ing, and o en challenging cha ac e -
is ic o any symbiosis is defining he unc ion o each pa -
ne . In he DDAs he symbion unc ion is ob ious, since
only he symbion can educe N
2
, and he p o ision o
fixed N o he dia om hos s has been shown on he cellula
le el (Fos e e al., 2011), bu he mechanism o N ans e
is uns udied. In e es ial symbioses in ol ing he e ocys-
ous cyanobac e ia, he symbio ic popula ions o en eside
in da kened ca i ies and ely (he e o ophically) on hei
hos plan s o educed C subs a es (Söde bäck and
Be gman, 1993). In DDAs, bo h pa ne s a e pho osyn-
he ic, and hence capable o C fixa ion, bu he possible
exchange and ans e o C subs a es is unknown. None-
heless, in e es ingly, a ecen cellula model o DDAs
es ima es ha 25% o C fixed by he hos is ans e ed o
he symbion , since he C equi emen by he symbion o
N
2
fixa ion is highe han he fixa ion o C p edic ed om
i s own pho osyn hesis (Inomu a e al., 2020).
Since all DDAs ha e e aded long- e m isola ion
(Villa eal, 1989, 1990), i is di ficul o s udy expe imen ally
how he pa ne s in e ac , sha e and po en ially compe e
o subs a es. He e, we ha e iden ified a ious candida e
anspo e s in he symbio ic R.in acellula is/C. hizo-
soleniae (he ea e Richelia/Calo h ix) d a genomes by
compa ison mainly o hose o model he e ocys ous cya-
nobac e ia. We ha e ocused on anspo e s o C, N and
some o he elemen s (e.g. i on, phospho us and sul u )
ha a e impo an o he basis o he pa ne ship.
No e on me hodology
To iden i y pa icula gene p oduc s in he DDA symbi-
on s, we ha e pe o med BLASTp analyses (Al schul
e al., 1997). We gene ally used p o eins o known
unc ion om Anabaena sp. PCC 7120 (he ea e Ana-
baena) as que ies, al hough in some pa icula cases
well-cha ac e ized p o eins om o he sou ces, mainly fil-
amen ous cyanobac e ia (Nos oc punc i o me ATCC
29133, T ichodesmium e y h aeum IMS101) we e used.
The symbion s a e also p edic ed o ha e some mem-
b ane p o eins no ound in o he cyanobac e ia, and
such memb ane p o eins we e compa ed wi h ans-
po e s om o he biological sou ces.
To define o hologues, we ha e ollowed conse a i e
c i e ia. Thus, gene ally, we checked ha he symbion ’s
p o ein was o app oxima ely he same leng h as he
que y p o ein, which, combined wi h significan simila i y,
ga e e y low Expec alues (e.g. <10
−50
o <10
−150
[indica ed as <e-50 o <e-150] o p o eins o 150–500
amino acid esidues app oxima ely). On he o he hand,
compa isons wi h Expec alues o , e.g. >10
−20
(indi-
ca ed as >e-20) we e conside ed o deno e simila bu
no necessa ily o hologous p o eins. This is especially
common in memb ane p o eins ha can belong o he
same amily o anspo e s bu ecognize di e en sub-
s a es. The alignmen s, using Clus al O (Madei a e al.,
2019), o some examples o p o eins ha gi e significan
simila i y o be conside ed o hologues a e p esen ed in
Figs. S1–S5.
Ou e memb ane ansloca o s
Cyanobac e ia a e dide m bac e ia, i.e. hey con ain an
ou e memb ane (OM) ou side o he cy oplasmic mem-
b ane (Hahn and Schlei , 2014). The OM cha ac e is i-
cally con ains nume ous p o eins ha ake a β-ba el
con o ma ion, and he OM ou e leafle con ains lipopoly-
saccha ide (LPS) as a cha ac e is ic componen .
Al hough cyanobac e ial LPS is no iden ical o ha o he
bes -s udied G am-nega i e bac e ia (Hahn and Schlei ,
2014), he genome o he he e ocys ous symbion s o
DDAs encode a numbe o OM inse ion p o eins (BamA
amily p o eins) as well as p o eins in ol ed in LPS syn-
hesis and anspo ha collec i ely sugges he p es-
ence o a ma u e OM in he symbion s (Table S1).
Hence, ma e ials ans e ed be ween he dia om and he
symbion in DDAs mus a e se he OM. Subs a e ans-
loca ion ac oss he OM gene ally akes place h ough
po ins, which a e ime ic β-ba el p o eins (Yamashi a
and Buchanan, 2010).
The e a e cu en ly abou 90 ecognized amilies o
β-ba el po ins in he T anspo e Classifica ion Da abase
[TCDB (Saie J e al., 2016); h p://www. cdb.o g/], and
cyanobac e ia possess cha ac e is ic po ins ha cons i-
u e one o hose amilies (TCDB #1.B.23). A cha ac e is-
ic ea u e o he cyanobac e ial po ins is he p esence o
an N- e minal domain wi h simila i y o ‘S-laye homology’
domains ha may connec he OM o he pep idoglycan
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2029
laye . These cyanobac e ial po ins show simila i y o he
Op B- ype po ins ha media e ans e o saccha ides in
bac e iasuchasPseudomonas ae uginosa ( an den Be g,
2012). Indeed, one o hese po ins om he acul a i e
symbion N.punc i o me (Npun_R5320) has been shown
o acili a e up ake o glucose and uc ose in o he cyano-
bac e ium (Ekman e al., 2013). None heless, i appea s
ha hese po ins ulfil classical po in unc ion gene ally pe -
mi ing he ans e o small molecules and ions h ough he
OM (Hahn and Schlei , 2014). The Anabaena genome
encodes se en Op B- ype po ins, o which All4499 and
Al 4550 appea o be pa icula ly abundan (Mosla ac
e al., 2007b; Nicolaisen e al., 2009).
Using All4499 and Npun_R5320 as que ies, we ound
ou homologues in CalSC01 and wo in each o he
Rin RC01 and Rin HH01 genomes (Table 1). Thus, he
ex e nal, acul a i e symbion CalSC01 is mo e simila o
Anabaena in numbe o po ins han he pa ial and in e -
nal symbion s (Rin RC01 and Rin HH01, espec i ely). In
he la e wo, one o he po ins (Rin RC01_7172,
Rin HH01_240) may be addi ionally subjec ed o egula-
ion by zinc (Zn), since he DNA sequence ups eam o
he encoding gene con ains a possible binding si e o
he Zu ansc ip ion ac o , which is in ol ed in Zn
homeos asis (Sein-Echaluce e al., 2015). The o he OM
po in (Rin RC01_1265, Rin HH01_6530), which shows
he highes simila i y o All4499 and Npun_R5320
(Table 1), may ep esen , he e o e, he gene al po in o
he endosymbion s.
The Anabaena genome encodes an unusually high
numbe o TonB-dependen OM anspo e s (Hahn and
Schlei , 2014), which media e up ake h ough he OM o
i on (Fe) complexes o i amin B
12
. This up ake is ene -
gized by he in e ac ion o a pe iplasmic domain o he
TonB-dependen OM anspo e wi h he inne memb ane
p o ein TonB (Yamashi a and Buchanan, 2010). Nei he
Rin RC01 no Rin HH01 appea o bea any TonB-
dependen anspo e , whe eas CalSC01 shows wo
TonB-dependen Fe complex anspo e s (see Table 4
below) and one TonB-dependen i amin B
12
anspo e
(Table 1). This poin s o a di e en s a egy o ace ele-
men up ake in he symbion s ains (see below) ha is
likely influenced by he symbion loca ion: he nu ien
pool o he ex e nal symbion (CalSC01) is ha o he
Table 1. Ou e memb ane (OM) p o eins encoded in he DDA symbion genomes.
Que y
CalSC01
(ex e nal)
Rin RC01
(pa ial)
Rin HH01
(in e nal) P o ein/ unc ion
All4499 2790721588
Ga0265390_12245
(1.5e-78)
Rin RC_1265 (0.0) Rin HH _6530
(0.0)
OM po in Op B
2790720937
Ga0265390_11703
(2.3e-81)
Rin RC_7172
Zn egula ed (?)
(1e-60)
Rin HH _240
Zn egula ed (?)
(1e-112)
2790720522
Ga0265390_113824
(6.5e-85)
2790722855
Ga0265390_13252
(6.5e-95)
Npun_R5320 2790721588
Ga0265390_12245
(1e-129)
Rin RC_1265
(1e-126)
Rin HH_6530
(1e-128)
OM po in Op B
2790720937
Ga0265390_11703
(1e-133)
Rin RC_7172
Zn egula ed (?)
(2e-54)
Rin HH_240
Zn egula ed (?)
(1e-107)
2790720522
Ga0265390_113824
(9e-134)
2790722855
Ga0265390_13252
(5e-127)
Al 4028-Al 4029 2790719984
Ga0265390_110111
(2e-122; 2e-119)
nd nd Vi amin B
12
anspo e
Al 2887 (HgdD) 2790721775
Ga0265390_124212
(0.0)
Rin RC_2765
(0.0)
Rin HH_21590*
(0.0)
TolC-like OM expo e
ORFs om he symbion s (CalSC01, Rin RC01, Rin HH01) iden ified in BLASTp analysis (Expec alues indica ed in pa en hesis) using he indi-
ca ed p o ein om Anabaena (All, Al ) o Nos oc punc i o me (Npun) as a que y. An as e isk indica es a possibly incomple e sequence; nd, no
de ec ed; (?), Zn- egula ion is possible bu no ye demons a ed.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2030 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
su ound, while he endosymbion s lack di ec con ac wi h
he ou side wa e column and a e limi ed (i no elian ) o
he hos cy oplasm (Rin HH01) o egion be ween us ule
and he hos cy oplasmic memb ane (Rin RC01).
TolC- ype expo e s a e ime ic p o eins ha make
an OM channel in he o m o a β-ba el and a pe iplas-
mic channel o med by α-helixes (Yamashi a and
Buchanan, 2010). TolC expo e s a e commonly able o
unc ion oge he wi h se e al di e en plasma mem-
b ane expo e s hus media ing he expo o di e en
subs ances om he cell. The Anabaena genome
encodes only one TolC-like p o ein, HgdD (Al 2887),
which can pa icipa e in he expo o di e en sub-
s ances including he e ocys -specific glycolipids
(Mosla ac e al., 2007a) and oxic compounds such as
e hidium b omide (Hahn e al., 2012). Like Anabaena,
each o he h ee symbion s has only one TolC-like p o-
ein (Table 1), which likely is in ol ed in mul iple expo
unc ions.
Cy oplasmic memb ane anspo e s: ABC and MFS
anspo e s
Cy oplasmic memb ane anspo e s a e cu en ly classi-
fied in o nume ous phylogene ically dis inc supe amilies
and amilies (see TCDB [Saie J e al., 2016]; h p://
www. cdb.o g/). Two such supe amilies wi h nume ous
p o ein membe s commonly e e ed o in his a icle a e
he ATP-Binding Casse e anspo e supe amily (ABC;
TCDB #3.A.1) and he Majo Facili a o Supe amily
(MFS; TCDB #2.A.1). The ABC anspo e supe amily is
one o he la ges amilies among anspo sys ems wi h
a wide dis ibu ion in all h ee domains o li e. ABC ans-
po e s can be di ided in o expo e s, ound in bo h
euka yo es and p oka yo es, and impo e s, which, wi h a
ew euka yo ic excep ions, a e la gely ound in p oka yo es
media ing he up ake o nu ien s (Wilkens, 2015). ABC
impo e s gene ally con ain one pe iplasmic solu e-binding
p o ein (SBP), which binds he ligand in he pe iplasm o
deli e y o he app op ia e memb ane anspo e complex;
wo in eg al memb ane p o eins ( ansmemb ane domains
[TMDs]) ha o m he solu e- ansloca ion pa hway; and
wo nucleo ide-binding p o eins o domains (NBD) ha
hyd olyze ATP in he cy oplasm (Cui and Da idson,
2011). MFS p o eins a e he la ges amily o seconda y
anspo e s and allow he anspo o a la ge a ie y o
ions and solu es ac oss memb anes (Reddy e al., 2012).
They comp ise acili a o s, sympo e s and an ipo e s,
which mo e subs a es ac oss memb anes ia acili a ed
di usion, co- anspo o exchange espec i ely (Yan,
2015). MFS p o eins a e in eg al memb ane p o eins ha
gene ally possess 12 o 14 ansmemb ane segmen s
(TMSs) (Reddy e al., 2012).
C-compound anspo e s
Cyanobac e ia a e mainly pho oau o ophic, fixing ca bon
dioxide (CO
2
), bu many s ains can also assimila e some
o ganic compounds including suga s, mainly glucose, uc-
ose and suc ose (Rippka e al., 1979). Dia oms a e also
p edominan ly pho osyn he ic and many a e acul a i e
he e o ophs (Hellebus and Lewin, 1977). In bo h dia oms
and cyanobac e ia, CO
2
fixa ion is ca alysed by ibulose-
1,5-bisphospha e ca boxylase/oxygenase (RubisCO).
Since bo h pa ne s o DDAs possess RubisCO, a key
ques ion o he symbion s (especially o he in e nal
and pa ial symbion s: Rin HH01 and Rin RC01) is how
hey ob ain and/o compe e o C, especially o cope
wi h he high a e o N
2
fixa ion.
Ino ganic ca bon
Simila o many o he au o ophs, bo h cyanobac e ia and
dia oms ha e de eloped C Concen a ion Mechanisms
(CCMs) o inc ease he concen a ion o CO
2
a ound
RubisCO (Ra en e al., 2008). The CCMs o dia oms a e
conside ed gene ically di e se and include high in e spe-
cies a ia ions (Young e al., 2016; Shen e al., 2017).
In gene al, cyanobac e ial and dia om CCMs show
unc ional and composi ional simila i ies, and bo h pos-
sess subcellula compa men s o housing RubisCO,
i.e. ca boxysomes in cyanobac e ia and py enoids in dia-
oms, bu hei CCM genes a e no homologous (Young
and Hopkinson, 2017). The CCMs o dia oms and cyano-
bac e ia a e composed o bica bona e (HCO
3
−
) ans-
po e s and ca bonic anhyd ases (CAs) ha media e he
in e con e sion o CO
2
and HCO
3
−
, and cyanobac e ia
possess, in addi ion, CO
2
anspo e s (Kaplan and
Reinhold, 1999; Badge e al., 2006; Came on e al.,
2014). CAs a y in numbe , sub ype (α,ß,γ, o he s) and
localiza ion in model dia oms such as Phaeodac ylum
ico nu um and Thalassiosi a pseudonana (Hopkinson
e al., 2016).
The DDA symbion s con ain he ccmKLMNO ope on
(wi h wo copies o ccmK in each o Rin HH01, Rin RC01
and CalSC01) o β-ca boxysomes and he gene o ca -
boxysomal CA, which a e essen ial o ca boxysome
unc ionali y. The up ake sys ems o CO
2
and HCO
3
−
species (ino ganic C, Ci) in cyanobac e ia include ABC
and MFS anspo e s o HCO
3
−
and a specialized ype
o NAD(P)H dehyd ogenase (NDH-1; pho osyn he ic
complex I) ha unc ions in apping CO
2
(P ice, 2011;
Schulle e al., 2020). In dia oms bo h CO
2
and HCO
3
−
en e he cell om he en i onmen . HCO
3
−
is ac i ely
anspo ed by memb ane-embedded anspo e s, while
CO
2
elies on a di usi e flux media ed by he ‘chlo oplas
pump’, he ac i e pumping o HCO
3
−
o he chlo oplas
(Hopkinson e al., 2011).
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2031

The Anabaena genome encodes wo CO
2
up ake sys-
ems o he NDH-1 ype, he NDH-1
3
and NDH-1
4
p o ein
complexes, and h ee HCO
3
−
impo e s, including one
ABC anspo e (high a fini y) and wo sodium-dependen
anspo e s (Sb A, high a fini y; BicA, low a fini y)
(He e o and Flo es, 2019), whe eas he symbion ’sCi
up ake sys ems appea o be influenced by he dia om
hos . The chlo oplas pump mechanism and subsequen
dis ibu ion o Ci is pa icula ly in e es ing in ela ion o
symbion cellula loca ion and hei espec i e genome
con en o Ci anspo . Thus, none o he endosymbion s
has genes encoding high-a fini y HCO
3
−
anspo e s,
whe eas he ex e nal symbion CalSC01 has a gene
encoding an MFS high-a fini y HCO
3
−
anspo e , Sb A
(Table 2; Fig. 2A). The absence o he high-a fini y
HCO
3
−
anspo e s in he in e nal symbion s may eflec
he influence o he ‘chlo oplas pump’and CA mecha-
nisms o i s hos , which d i e high HCO
3
−
concen a ions
wi hin he icini y o Rin RC01 and Rin HH01 symbion s.
Low HCO
3
−
concen a ions a e common in he su ace
ocean and could d i e he e en ion o he high-a fini y
HCO
3
−
anspo e (Sb A) in he ex e nal symbion ,
CalSC01. Thus, an open and impo an ques ion in each
symbiosis is he up ake kine ics o Ci, and how he sym-
bion cellula loca ion influences he ac i i y and pa ne
in e ac ion.
In e es ingly, he h ee symbion s bea genes encoding
p o eins simila o he SulP- amily HCO
3
−
anspo e
BicA. CalSC01 and Rin RC01 each shows wo genes
encoding BicA-like p o eins, whe eas Rin HH01 shows
he possible bicA gene agmen ed in ou consecu i e
sequences, pe haps eflec ing incomple e sequencing
co e age o , al e na i ely, ha i is a gene in he p ocess
o degene a ion (Table 2, see also Table 5 below).
Because SulP anspo e s o HCO
3
−
and sul a e a e
e y simila o each o he , expe imen al es s a e needed
o define he ac ual subs a e o hose p o eins. Addi ion-
ally, each o he h ee symbion s encodes he compo-
nen s o an NDH-1 ype (NDH-1
3
)o CO
2
up ake sys em.
Whe eas CalSC01 is expec ed o fixCO
2
(a leas when
g owing independen ly o he dia om), he p esence o
RubisCO, ca boxysome genes, an NDH-1 complex and,
likely, a BicA anspo e in he in e nal and pa ial symbi-
on s suppo he idea ha hey can also fixCO
2
, which
is consis en wi h high a es o C fixa ion measu ed in
field popula ions (Ca pen e e al., 1999; Sub amaniam
e al., 2008).
O ganic ca bon
Despi e he ac ha he main ophic mode o cyano-
bac e ia is pho oau o ophy, some s ains, including some
he e ocys ous ypes ha engage in symbioses wi h e -
es ial plan s, ha e he capaci y o suga up ake
suppo ing he e o ophic g ow h. Thus, GlcP (MFS ans-
po e o glucose) and F (ABC anspo e o uc ose)
in symbio ically compe en N.punc i o me (Ekman e al.,
2013) and F in A. a iabilis (Unge e e al., 2008) a e
well-known examples o anspo e s o some kind o
he e o ophic g ow h (Wolk and Sha e , 1976; Summe s
e al., 1995). Addi ionally, mixo ophic g ow h wi h di e -
en C sou ces (Mala inszky e al., 2017) and he iden ifi-
ca ion o componen s o ABC glucoside anspo e s
in ol ed in suga -s imula ed g ow h in Anabaena ha e
been ecen ly epo ed (Nie es-Mo ión and Flo es, 2018).
Su p isingly, no homologues o GlcP o F p o eins a e
encoded in any o he dia om symbion s. Howe e , he
h ee symbion s may encode he ABC anspo e Gls
(Table 2; Fig. 2A), which in Anabaena unc ions in he
up ake o glucosides (Nie es-Mo ión and Flo es, 2018).
CalSC01 bea s wo homologous p o eins o each GlsQ
and GlsP (TMDs), which migh sugges he unc ion o
wo ABC-glucoside anspo e s (Fig. 2A) as i has been
epo ed in Anabaena (Nie es-Mo ión and Flo es, 2018).
In con as , due o he low simila i y o he pu a i e SBP
o ei he Richelia s ain o he Anabaena SBP GlsR
(Table 2), expe imen al confi ma ion o hei subs a es is
equi ed. The Anabaena Al 3705 MFS sympo e is a
possible glycoside anspo e , al hough i could no
expe imen ally anspo glucosides in Anabaena
(Nie es-Mo ión e al., 2017). Whe eas Rin HH01 and
Rin RC01 bea a homologous p o ein, CalSC01 con ains
wo possible homologues o Al 3705 (Table 2; Fig. 2A).
The p esence o hese anspo e s aises he possibili y
ha he symbion s can assimila e a suga such as
suc ose, which in Anabaena has a p incipal ole in he
ans e o educed C om ege a i e cells o he e ocys s
(López-Igual e al., 2010; Nü nbe g e al., 2015). Consis-
en ly, he h ee symbion s encode in e ases, which a e
enzymes ha i e e sibly spli suc ose in o glucose and
uc ose (Table 2).
In cyanobac e ia, suc ose is syn hesized by he com-
bined ac ion o suc ose-phospha e syn hase (Sps) and
suc ose-phospha e phospha ase (Spp), which cons i u e
an i e e sible pa hway o suc ose biosyn hesis, o by he
e e sible enzyme suc ose syn hase (Sus) (Sale no and
Cu a i, 2003). Whe eas Rin RC01 and CalSC01 bea
genes encoding Sps and Spp, Rin HH01 lacks hese
enzymes o he al e na i e suc ose syn hase. Hence,
Rin HH01 has he capaci y o hyd olyze bu no syn he-
size suc ose. The e o e, a mixo ophic g ow h, fixing CO
2
and assimila ing suga s p o ided by he hos , is possible
o he symbion s, and his could be especially ele an in
he case o he in e nal symbion Rin HH01. While
CalSC01 and Rin RC01 encode in e ases In A and
In B, he only in e ase p esen in Rin HH01 is mos sim-
ila o Anabaena In B, which is he e ocys -specific
(López-Igual e al., 2010). I would, he e o e, be o g ea
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2032 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
Table 2. Ca bon up ake anspo e s and ela ed p o eins encoded in he DDA symbion genomes.
Que y
CalSC01
(ex e nal)
Rin RC01
(pa ial)
Rin HH01
(in e nal) P o ein/ unc ion
Al 2877 (CmpA) Al 2878 (CmpB)
Al 2879 (CmpC) Al 2880 (CmpD)
nd nd nd ABC Bica bona e anspo e :
CmpA (SBP)
CmpB (TMD)
CmpC (NBD)
CmpD (NBD)
All2134 2790721,442
Ga0265390_12075
(3e-178)
nd nd High-a fini y bica bona e:Na
+
sympo e , Sb A
BicA om Synechococcus sp. PCC
7002
2790719465
Ga0265390_10577
(0.0)
Rin RC_3892
(0.0)
Rin HH_3960#
(4e-12)
Rin HH_3970#
(1e-103)
Rin HH_3980#
(3e-21)
Rin HH_3990#
(5e-32)
Low-a fini y, SulP- amily bica bona e:
Na
+
sympo e , BicA
( wo simila p o eins in Anabaena:
All1304, Al 1635)2790720482
Ga0265390_11353
(0.0)
Rin RC_4851
(0.0)
Al 4156 (NdhF) 2790722617
Ga0265390_130615
(0.0)
Downs eam genes:
2790722616
2790722615
Rin RC_4170
(0.0)
Downs eam
genes:
Rin RC_4169
Rin RC_4168
Rin HH_18810
(0.0)
Downs eam
genes:
Rin HH_18800
Rin HH_18780
Th ee-gene clus e s ha encode an
NDH-1 ype (NDH-1
3
)o CO
2
up ake
complex
Al 2532 (GlsQ) 2790723713
Ga0265390_139111
(TMD, 3e-61)
2790721923
Ga0265390_12555
(TMD, 6.9e-56)
Rin RC_0814
(TMD, 9e-62)
Rin HH_290
(TMD, 8e-52)
Rin HH_15070
(TMD, 9e-47)
Glucoside ABC anspo e Gls:
GlsQ (TMD)
GlsP (TMD)
GlsR (SBP)
GlsC (NBD)
GlsD (NBD)
All0261 (GlsP) 2790722162
Ga0265390_127217
(TMD, 2e-55)
2,790721031
Ga0265390_117612
(TMD, 3.95 e-61)
All1916 (GlsR) 2790721653
Ga0265390_12342
(SBP, 2e-52)
Rin RC_6152
(SBP, 4e-11)
Rin HH_17430
(SBP, 9e-21)
Al 4781 (GlsC) 2790719311
Ga0265390_10456
(NBD, 8e-118)
Rin RC_6050
(NBD, 1e-92)
Rin HH_19540
(NBD, 1e-166)
All1823 (GlsD) 2790721447
Ga0265390_12083
(NBD, 2e-117)
Al 3705 2790723960
Ga0265390_14405
(1.3e-71)
2790720797
Ga0265390_11606
(3.18e-169)
Rin RC_2529
(0.0)
Rin HH_3250**
(1e-39)
Rin HH_3260**
(1e-174)
Glycoside-Pen oside-Hexu onide:
Ca ion MFS sympo e
Al 1521 2790722378
Ga0265390_12903
(0.0)
Rin RC_4014
(0.0)
nd In e ase In A
Al 0819 2790722061
Ga0265390_12672
(0.0)
Rin RC_3156
(0.0)
Rin HH_3860
(0.0)
In e ase In B (he e ocys -specificin
Anabaena)
All3028 (SBP) 2790720991
Ga0265390_11733
(SBP, 0.0)
Rin RC_2147
(SPB, 1e-134)
Rin HH_12690
(SBP, 0.0)
TRAP ca boxyla e anspo e .
Consis s o SBP, small TMD (Dc Q),
and la ge TMD (Dc M)
Al 3026 (small TMD) 2790722400
Ga0265390_12928
(TMD small, 8e-59)
Rin RC_3100
(TMD small,
8e-93)
Rin HH_15260
(TMD small,
1e-83)
Al 3027 (la ge TMD) 2790722399
Ga0265390_12927
(TMD la ge, 0.0)
Rin RC_3101
(TMD la ge, 0.0)
Rin HH_15250
(TMD la ge, 0.0)
(Con inues)
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2033
in e es o loca e and de e mine he unc ionali y o In B
in he endosymbion s ains Rin RC01 and Rin HH01.
T ipa i e ATP-independen pe iplasmic (TRAP) ans-
po e s a e gene ally ca boxyla e anspo e s (Mulligan
e al., 2011). Anabaena con ains h ee genes encoding a
TRAP anspo e ha media es he up ake o py u a e
and o he monoca boxyla e 2-oxoacids (Pe nil e al.,
2010). Genes encoding homologues o he Anabaena
TRAP anspo e p o eins can be de ec ed in he h ee
symbion s (Table 2; Fig. 2A). A ecen field in es iga ion
epo ed he co-exp ession o he Rin RC01 gene o he
TRAP solu e ecep o and EAMA-like anspo e s in he
dia om hos Rhizosolenia (Ha ke e al., 2018). EAMA
anspo e s belong o he D ug/Me aboli e T anspo e
supe amily (TCDB #2.A.7), a e associa ed wi h iose-
phospha e ansloca o s loca ed on he plas id memb ane,
and unc ion in he model dia om P. ico nu um o expo
ca bohyd a es de i ed om pho osyn hesis (Moog e al.,
2015). Hence, i was sugges ed ha he dia om hos was
supplying suga subs a es o he symbion (Ha ke e al.,
2018). Howe e , conside ing ha he Rin RC01 esides
ou side he hos dia om cy oplasm in he pe iplasmic
space, an addi ional anspo mechanism ac oss he hos
cell memb ane is equi ed. Finally, i indeed educed C
subs a es a e anspo ed om he hos , o he candida es
o simple o ganic molecule anspo a e a p edic ed ace-
a e pe mease p esen in CalSC01 and Rin RC01, and a
p edic ed p oline anspo e in CalSC01 and Rin HH01
(Table 2). Iden i ying he subs a e(s) o hese anspo e s
would be o g ea in e es o unde s and he physiology o
he DDAs, especially in he case o he in e nal symbion
(Rin HH01), in which subs a e(s) can be aken up di ec ly
om he hos dia om’s cy oplasm.
N-compound anspo e s
Cyanobac e ia and dia oms ha e he abili y o assimila e
ino ganic and simple o ganic N compounds, and some
cyanobac e ia fix a mosphe ic N
2
. Howe e , he
en i onmen s whe e he DDAs a e epo ed a e no o i-
ously N deple e, since concen a ions o ino ganic and
o ganic N a e below analy ical de ec ion. Hence, he hos
dia oms a e dependen on he symbion s o N.
P ima y N sou ces
Ni ogenase, he enzyme esponsible o N
2
fixa ion, is
inac i a ed in he p esence o O
2
, and he e o e N
2
-fixing
cyanobac e ia ha e o sepa a e spa ially o empo ally N
2
fixa ion and oxygenic pho osyn hesis (Flo es e al.,
2015). Unde condi ions o combined-N dep i a ion,
cyanobac e ia o he o de Nos ocales (including,
e.g. Anabaena,Calo h ix,Nos oc and Richelia) p oduce
di e en ia ed cells called he e ocys s whe e N
2
fixa ion
akes place. A common obse a ion in e es ial-based
symbioses wi h he e ocys ous cyanobac e ia is ha he -
e ocys equency (no mally abou 7%-10%) inc eases
(e.g. o 17%–60%) when he symbion s a e li ing in
symbioses a he han in a ee-li ing s a e (Meeks,
2009). The Richelia/Calo h ix symbion s, howe e , a e
unique symbion s in ha hey possess e minal he e o-
cys s, and he e o e canno inc ease he numbe o he -
e ocys s, bu a he main ain a high a io o he e ocys s
o ege a i e cells i he numbe o ege a i e cells is
small. In Rin HH01 and CalSC01 filamen s he la e is
ue, since hey ypically ha e 1–3 ege a i e cells, how-
e e , filamen s end o be longe in he case o Rin RC01
(Fig. 1; see also Villa eal, 1992). Longe filamen s in
Rin RC01 and no ably in ee-li ing CalSC01 imply mo e
ege a i e cells pe o ming C fixa ion and hence a highe
C supply om hei own pho osyn hesis. I is cu en ly
unknown how filamen leng h is egula ed in hese
o ganisms.
He e ocys o ma ion in ol es mo phological and me a-
bolic changes ha allow he exp ession and unc ion o
ni ogenase (Flo es e al., 2019b). One o hese mo pho-
logical changes consis s in he deposi ion o wo en e-
lope laye s ou side o he OM: he glycolipid laye (HGL)
Table 2. Con inued
Que y
CalSC01
(ex e nal)
Rin RC01
(pa ial)
Rin HH01
(in e nal) P o ein/ unc ion
Ac P o Rh.capsula us
a
2790722390
Ga0265390_129110
(e-45)
Rin RC_5151
(e-49)
nd Ca ion/ace a e pe mease
Pu P o B.sub ilis
b
2790719613
Ga0265390_10674
(4e-61)
nd Rin HH_18700
(e-67)
Nu i ional high-a fini y sodium/p oline
pe mease Pu P
a.Rh., Rhodobac e .
b.Bacillus.
ORFs om he symbion s (CalSC01, Rin RC01, Rin HH01) iden ified in BLASTp analysis (Expec alues indica ed in pa en hesis) using he indi-
ca ed p o ein om Anabaena (unless indica ed o he wise) as a que y. Double as e isks designa e genes ha a e spli ; Hash indica es genes ha
a e agmen ed; nd, no de ec ed.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2034 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
ha educes he pe mea ion o O
2
, and he polysaccha-
ide laye (HEP) ha p o ides p o ec ion (Nicolaisen
e al., 2009). The HGL is composed o a y alcohols gly-
cosidically linked o suga esidues. Whe eas HGLs
made o C6 suga ha e been ound in eshwa e ee-
li ing cyanobac e ia and some s ains o ben hic
Calo h ix (Baue sachs e al., 2009), Richelia (Rin HH01,
Rin RC01) con ains no el HGLs wi h a C5 suga , ibose,
a he han a C6 suga (Schou en e al., 2013; Bale e al.,
2015). Recen ly, a no el HGL was cha ac e ized o a
new Calo h ix sp. CCY1611 isola ed om he No h
A lan ic (Bale e al., 2018); i is unknown i CCY1611 is
simila and/o gene ically ela ed o CalSC01, and he
HGLs o CalSC01 a e uncha ac e ized. The C5 suga
ound in Rin RC01 and Rin HH01 migh be explained by
an adap a ion o he endosymbion o he high O
2
con-
cen a ion wi hin he dia om hos (Walsby, 1985). This
adap a ion illus a es he impo ance o N
2
fixa ion in he
DDAs, and de ails o he p oduc ion o he he e ocys
en elope in he symbion s will me i specific esea ch in
he u u e.
In addi ion o N
2
, he e ocys ous cyanobac e ia can
assimila e om he en i onmen a ious sou ces o N
including ammonium, ni a e, ni i e, u ea and some
amino acids (He e o and Flo es, 2019). The in acellula
con e sion o hese compounds o ammonium is equi ed
o N inco po a ion in o ca bon skele ons o p oduce
o ganic N compounds. Ammonium is a p e e ed N
sou ce o many o ganisms and i s up ake in en i on-
men s wi h low ex e nal concen a ions in ol es he Am
Fig. 2. Upse diag am showing he in e sec ion o each se o anspo e s in he model cyanobac e ium Anabaena sp. PCC 7120 and he DDA sym-
bion s Calo h ix CalSC01, Richelia Rin RC01, and Richelia Rin HH01 o (A) ca bon, (B) ni ogen, (C) i on and (D) phospho us. The C, N, Fe and P
anspo e con en in CalSC01 is simila o Anabaena and highe in numbe han anspo e s p esen in he endosymbion s Rin RC01 and
Rin HH01. A. No e he p esence o Sb A, Gls_2 and MFS_2 in he ex e nal symbion and hei absence in he endosymbion s. TRAP ca boxyla e
anspo e , NDH-1
3
and he Gls_1 a e p esen in he h ee symbion s. CalSC01 bea s wo homologous p o eins each o GlsQ and GlsP (Table 2)
sugges ing he p esence o wo glucoside ABC anspo e s (Gls_1 and Gls_2) as in Anabaena. We e e o he second anspo e as ha con aining
Ga0265390_12555 and _117612. Two possible MFS anspo e s o glycosides a e iden ified in CalSC01: MFS_1 (Ga0265390_14405) and MFS_2
(Ga0265390_11606) (see Table 2). B. No e he ammonium anspo e (Am ) and ni a e/ni i e MFS anspo e a e only p esen in he ex e nal sym-
bion . The h ee symbion s bea a possible amino acid pe mease (APC supe amily), which is no p esen in Anabaena. C. The h ee symbion s bea
he e ic i on ABC anspo e and he e ous i on pe mease E eU, as in Anabaena, whe eas unce ain y o SchE in he symbion s is designa ed
wi h an as e isk (see he ex ). D. No e he p esence o he phosphona e ABC anspo e and a second phospha e ABC anspo e (Phospha e
ABC anspo e 2) in he ex e nal symbion CalSC01. We e e o he second phospha e ABC anspo e 2 as ha con aining Ga0265390_11512,
_12441, _12442, _12443, _11047 (see Table 5).
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2035
S e ano ic e al., 2011). Once Fe(III) is bound o he side-
opho e in he medium, he complex is aken up by he
OM p o ein SchT (Al 0397), a TonB-dependen anspo e
(Nicolaisen e al., 2008), and hen ansloca ed in o he
cy oplasm by he ABC anspo e FhuBCD (S e ano ic
e al., 2011; Rudol e al., 2016). Addi ionally, Anabaena
exp esses o he TonB-dependen anspo e s in ol ed in
Fe and coppe (Cu) up ake including Iu A2 (Al 2581; Rud-
ol e al., 2016) and IacT (All4026; Nicolaisen e al., 2010).
Only he ex e nal symbion CalSC01 con ains homologues
o side opho e biosyn hesis genes (see Table S3),
al hough i s genome encodes a p o ein wi h only low simi-
la i y o he SchE expo e (Table 4; Fig. 2C). Because
bac e ia o en exp ess ecep o s o side opho es ha hey
do no p oduce o o Fe con ained in hei hos s (B aun
and Killman, 1998; Ra ledge and Do e , 2000), CalSC01
homologues o schizokenin ecep o SchT and TonB-
dependen anspo e s Iu A2 and IacT (Table 4; Fig. 2C)
migh be in ol ed in he up ake o side opho es sec e ed
by CalSC01 i sel as well as by o he bac e ia. CalSC01
bea s also he ABC- anspo e FhuBCD equi ed o
Fe
3+
-side opho e acquisi ion (Table 4; Fig. 2C). Because
side opho es a e sec e ed in esponse o low dissol ed Fe
concen a ion (Wilhelm and T ick, 1994), hese ans-
po e s may allow CalSC01 o main ain Fe homeos asis in
a low Fe concen a ion en i onmen . Mo eo e , CalSC01
is expec ed o acqui e Fe h ough o he pa hways, as
suppo ed by he p esence o ano he Fe
+3
ABC ans-
po e (homologue o E.coli Fu ABC), a Fe
3+
o e ic-
dici a e ABC anspo e (homologue o E.coli FecBCDE),
ahigh-a fini y Fe
2+
/Pb
2+
pe mease, and (possibly) a
FeoB-like e ous i on anspo e (Table 4; Fig. 2C).
Finally, al hough euka yo es canno p oduce side opho es,
some, including dia oms, can access he bound Fe om
side opho es (Amin e al., 2009; Kazamia e al., 2018);
pe haps a simila s a egy occu s in he Chae oce os-
Calo h ix symbiosis and he hos dia om uses i s symbi-
on ’s side opho e syn hesis o i s own Fe acquisi ion.
Rin HH01 and Rin RC01 symbion s lack any TonB-
dependen anspo sys em and he ABC anspo e
FhuBCD equi ed o e ic-side opho e acquisi ion,
bu hei genomes encode homologues o a e ic i on
ABC anspo e (Fu ABC homologue), he high-a fini y
Fe
2+
/Pb
2+
pe mease and only in Rin RC01, possibly he
FeoB-like anspo e (Table 4; Fig. 2C). Residing inside
he dia om, he concen a ion o Fe migh be su ficien
and appa en ly, bo h Rin HH01 and Rin RC01 symbion s
e ol ed o inco po a e ino ganic o o ganic o ms o
Fe(III) and Fe(II) by pa hway(s) di e en o he side-
opho e up ake sys ems, as also epo ed o Syn-
echocys is sp. PCC 6803 and T.e y h aeum IMS101
(Roe and Ba beau, 2014; Jiang e al., 2015). The p es-
ence o p o eins simila o schizokinen expo e SchE in
he h ee DDM symbion s (Table 4) should be aken wi h
cau ion, since hose p o eins a e indeed mos simila o
bac e ial AmpG (Expec alues <e-100), which is
in ol ed in he up ake o cell wall deg ada ion p oduc s.
In summa y, whe eas endosymbion s Rin RC01 and
Rin HH01 appea o be non-side opho e-u ilizing cyano-
bac e ia ha ing o he pa hways o Fe homeos asis,
CalSC01, li ing ex e nally a ached, appea s o be able
o use side opho es and ha e mo e s a egies o Fe
up ake.
Sul a e ABC anspo e and sul a e pe mease
Sul a e is a mac onu ien , which is equi ed o pho osyn-
he ic o ganisms. Sul u is p esen in p o eins, lipids, elec-
on anspo componen s and many cellula me aboli es.
The limi ed in acellula s o age o S implies he up ake,
mainly in he o m o sul a e anion, om he en i onmen
h ough specific anspo e s. The main p oka yo ic ans-
po e in ol ed in sul a e up ake is in he SulT g oup o
ABC anspo e s (TCDB #3.A.1.6). This anspo e ,
Sbp/CysPTWA, is esponsible o sul a e and hiosul a e
up ake in p oka yo es (Saie J e al., 1999). I is com-
posed o wo TMD p o eins, CysT and CysW, he NBD
CysA p o ein (homodime - o ming CysA) and he sul a e-
binding (Sbp) and hiosul a e-binding (CysP) pe iplasmic
SBPs (H yniewicz e al., 1990; Si ko e al., 1990).
Table 5. Con inued
Que y
CalSC01
(ex e nal)
Rin RC01
(pa ial)
Rin HH01
(in e nal) P o ein/ unc ion
(NBD, 7e-169)
_113152 (TMD) 2790720875
Ga0265390_11654
(TMD, 4e-153)
_113151 (TMD) 2790720874
Ga0265390_11653
(TMD, 1e-165)
ORFs om he symbion s (CalSC01, Rin RC01, Rin HH01) iden ified in BLASTp analysis (Expec alues indica ed in pa en hesis) using he indi-
ca ed p o ein om Anabaena (unless indica ed o he wise) as a que y. Expec alues in Anabaena ha co espond o compa ison o he E.coli
p o eins a e p o ided in pa en hesis. An as e isk indica es a possibly incomple e sequence; nd, no de ec ed.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2042 M. Nie es-Mo ión, E. Flo es and R. A. Fos e

Anabaena and he h ee symbion s bea he genes ha
encode he componen s o his anspo e (Table 5),
which suppo s he idea ha hey can ake up sul a e.
P o eins o he SulP amily a e pe meases ha can
anspo se e al di e en anions including sul a e (TCDB
#2.A.53). Anabaena and he h ee symbion s con ain se -
e al p o eins o his amily, some o which could be sul a e
anspo e s, whe eas o he s could be bica bona e ans-
po e s as hey a e mos simila o he bica bona e ans-
po e BicA om oceanic cyanobac e ia (P ice e al., 2004)
men ioned ea lie (Tables 2 and 5). The iden ifica ion o he
subs a e anspo ed by each pa icula SulP- amily p o ein
will be o much in e es and equi es u he in es iga ion.
Phospha e and phosphona e anspo e s
Simila o Fe, phospho us (P) is o en a limi ing nu ien in
ma ine sys ems since i can be ound a low concen a-
ions o e en in o ms no biologically a ailable
(Schindle , 1977; Ka l e al., 2001). The phospho us com-
pounds and s a egies o acquisi ion unde P limi a ion
ha e been cha ac e ized in some model sys ems, and
some p og ess has ecen ly been made on ma ine phy o-
plank on ( e iewed in Lin e al., 2016). The p e e ed o m
o P o phy oplank on is ino ganic phospha e (Pi). How-
e e , unde he ch onically low Pi condi ions ypical o he
oligo ophic ocean whe e DDAs eside, some phy oplank-
on u ilize dissol ed o ganic phospho us (DOP) sub-
s a es (Cui e al., 2015) and/o educe hei cellula
P quo as by using non-P lipids in hei lipid memb anes
(Van Mooy e al., 2009). DOP u iliza ion has been less
s udied in dia oms, and mos wo k o da e has used alka-
line phospha ase ac i i y assays as indica i e o DOP u i-
liza ion o , ecen ly, quan i a i e p ofiling o ansc ip s
and p o eins unde P deficien condi ions (Pe y, 1976;
Dyh man e al., 2006, 2012). In he DDA hos s, i is no
known how o which P subs a es a e u ilized.
Cyanobac e ia ha e e ol ed mechanisms and egula-
o y adap a ions o acqui e Pi since i is a c ucial equi e-
men o fixN
2
(Ba din e al., 1996) and main ain g ow h.
Two majo anspo e s o Pi acquisi ion in bac e ia a e
he PhoT and PiT sys ems. The PhoT anspo e s
(Phospha e Up ake T anspo e Family; TCDB #3.A.1.7)
a e high-a fini y ABC anspo e s ha a e no mally
exp essed in cells g owing unde low-Pi concen a ions
(Rao and To iani, 1990). The E.coli PhoT sys em Ps
comp ises a pe iplasmic SBP (Ps S), wo TMD p o eins
(Ps A and Ps C) and he NBD p o ein (Ps B). On he
o he hand, p o eins in he PiT (Ino ganic Phospha e
T anspo e ) amily (TCDB #2.A.20) no mally consis o
10–12 TMSs and media e anspo o Pi—complexed
wi h a me al di alen ca ion—in a sympo mechanism
wi h H
+
o Na
+
ions (Jackson e al., 2008). In E.coli, Pi A
and Pi B a e low-a fini y anspo sys ems ha unc ion
when he ex e nal Pi concen a ion is highe han 20 μM,
which ep esses he Ps sys em (Rao and To iani,
1990). Anabaena con ains wo PhoT (All4575 o All4572
and All0911 o All0907) and wo PiT (Al 2336 and
Al 3096) anspo e s. Each o he h ee symbion s con-
ains he componen s o a leas one PhoT ABC ans-
po e ha is mos simila o he Anabaena All4575 o
All4572 anspo e (Table 5; Fig. 2D). Whe eas
Rin RC01 and Rin HH01 bea homologues o he Ana-
baena PiT anspo e All2336, CalSC01 lacks his Pi
anspo e , as do se e al eshwa e s ains and mos
ma ine picocyanobac e ia (Su e al., 2007; Scanlan e al.,
2009) (Table 5; Fig. 2D). Ne e heless, only he CalSC01
symbion con ains an ex a gene clus e and wo inde-
penden genes ha oge he may encode ano he PhoT
amily ABC anspo e (Table 5; Fig. 2D), esembling he
duplica e o mul iple Ps sys ems ound in some eshwa-
e and e es ial cyanobac e ial s ains (Pi e al., 2010;
Hudek e al., 2016).
Some cyanobac e ia can also u ilize phosphona es, as
fi s demons a ed o he ma ine N
2
-fixing cyanobac e-
ium T.e y h aeum IMS101 (Dyh man e al., 2006). Pho-
sphona es a e o ganic molecules con aining a co alen
bond be ween a oms o P and C, which a e de i ed om
he deg ada ion o glycolipids, glycop o eins, an ibio ics
o phosphonolipids (Kolowi h e al., 2001). Bac e ia,
some euka yo es, and plan s a e known o syn hesize
phosphona es (Ho igushi, 1984; Kugle e al., 1990),
howe e , only p oka yo es and some ungi a e capable o
acqui ing phosphona es as a sou ce o P, N o C
(Konono a and Nesmeyano a, 2002). In E.coli, he ABC
anspo e PhnCDE media es he up ake o pho-
sphona es. Impo an ly, assimila ion o phosphona es
also equi es he hyd olysis o he C─P bond, which in E.
coli and many o he bac e ia is ca ied ou by he
enzymes o he C-P lyase pa hway (Ho e-Jensen e al.,
2014). T ichodesmium e y h aeum con ains he genes
encoding he phosphona e ABC anspo e (Table 5;
Fig. 2D) and he phnG o phnM genes encoding he C-P
lyase pa hway (Dyh man e al., 2006). We, he e o e,
used he T.e y h aeum genes o look o phosphona e
u iliza ion genes in he DDA symbion s [al hough Ana-
baena also seems o con ain he phosphona e u iliza ion
pa hway (Ho e-Jensen e al., 2014), i has no been
expe imen ally cha ac e ized]. Among he DDA symbi-
on s, only CalSC01 bea s he phosphona e anspo e
(Table 5; Fig. 2D) and some homologues o genes
encoding he C-P lyase pa hway (al hough he pa hway
gene clus e appea s o be incomple ely sequenced).
In summa y, he endosymbion s Rin HH01 and
Rin RC01 con ain high-a fini y and low-a fini y phospha e
anspo e s, whe eas he ex e nal symbion CalSC01
con ains high-a fini y phospha e anspo e s and a pho-
sphona e u iliza ion pa hway. Concen a ions o Pi a e
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2043
o en low in he egions in which he DDAs h i e
(i.e. 10-yea a e age, 25-m dep h in No h Pacific sub-
opical gy e: 70 39 nmol L
−1
; Bjö kman e al., 2018);
hence he absence o he low-a fini y PiT anspo e in
he ex e nal symbion (CalSC01) is no su p ising.
Mic onu ien anspo e s in Rin HH01
Gi en he cy oplasmic loca ion o Rin HH01 wi hin he dia-
om, we checked he p esence o possible anspo e s o
mic onu ien s in his endosymbion . Rin HH01 con ains
possible anspo e s o so me als, including a Na
+
:H
+
exchange (Rin HH_10180; TCDB #2.A.36), a K
+
channel
(Rin HH_22210; TCBD #1.A.1), he K AB anspo e o
K
+
(Rin HH_14160, _14170; TCBD #2.A.38.4) and a pos-
sible anspo e o Mg
2+
(Rin HH_14380; TCDB #1.
A.26). Rin HH01 also con ains possible ABC anspo e s
o Mn
2+
and/o Zn
2+
(Rin HH_220, _230, _21250 and
Rin HH_5870, _5880, _5890; bo h belonging o TCDB #3.
A.1.15), Ni
2+
and/o Co
2+
(Rin HH_19750, _19760,
_19770; TCDB #3.A.1.23) and molybda e
(Rin HH_20340, _20350; TCDB #3.A.1.8). Finally, possi-
ble ola e-biop e in anspo e s (Rin HH_9790, _1390;
TCDB#2.A.71) a e also p esen . The p esence o hese
anspo e s in Rin HH01 iden ifies some essen ial mic o-
nu ien s ha he endosymbion can ake up ac i ely om
he dia om’s cy oplasm.
P oposed C and N fixa ion and me aboli e exchange
(C, N, Fe, S and P) model in he dia om-
cyanobac e ium symbioses
Based on he main knowledge o he symbiosis be ween
dia oms and diazo ophic cyanobac e ia and he new
insigh s in oduced in his a icle ega ding di e en ans-
po mechanisms in h ee symbion s, we p opose he ol-
lowing models. In each model, he symbion cellula
loca ion and how his migh a ou o limi he symbion
acquisi ion and anspo o a pa icula subs a e is
conside ed.
Model o Rin HH01
Al hough Rin HH01 esides inside i s hos , i is expec ed
ha he symbion also pe o ms CO
2
fixa ion because o
he p esence o RubisCO, ca boxysome genes and an
NDH-1 complex (Fig. 3A). (The p esence o a bica bon-
a e anspo e , BicA, in Rin HH01 is unsu e). O pa icu-
la in e es is he expec ed concen a ion g adien o Ci
wi hin he hos dia om cy oplasm, in which HCO
3
−
con-
cen a ion is p edic ed o be se e al- old highe nea e o
he chlo oplas (Hopkinson e al., 2016; Young and Hop-
kinson, 2017). Rin HH01 is commonly obse ed su -
ounded by he hos chlo oplas (Capu o e al., 2019).
In e es ingly, a compe i ion o C up ake, based on an
ex a C equi emen o N
2
fixa ion in Richelia, could exis
be ween he pa ne s. This compe i ion migh ely on he
up ake o C compounds om he hos media ed by
Richelia anspo e s, including he up ake o glucosides
by ABC (Gls) o MFS anspo e s and he up ake o ca -
boxyla es such as 2-OG by he TRAP anspo e . Mo e-
o e , since Rin HH01 appea s o ha e he capabili y o
hyd olyzing bu no syn hesizing suc ose, his disaccha-
ide would p o ide educed C o he symbion . Indeed, an
in e ase (In B) is p esen in Rin HH01 ha po en ially
unc ions in he clea age in o glucose and uc ose o
suc ose aken up om he hos (Fig. 3A).
Rin HH01 con ains he comple e sui e o genes o ca y
ou N
2
fixa ion bu lacks any Am ammonium anspo e
and he ni a e/ni i e and u ea u iliza ion pa hways, which
de aul s he exchange o N- ela ed compounds o he dia-
om. The e o e, he educed N compounds mus be
exchanged by o he anspo e s, such as amino acid
ABC anspo e s N-I and N-II ( aking up glu ama e and
o he amino acids), an oligopep ide anspo e (possible
glu a hione anspo e ), he polyamine ABC anspo e ,
a p oline pe mease, he possible ammonium/amino acid
pe mease Rin HH_4450, and he DME pe mease (wi h
an incomple e sequence) (Tables 2 and 3; Fig. 3A). Due
o he lack o GOGAT in Rin HH01, we p opose he
up ake o glu ama e om he dia om wi h he pa icipa ion
o amino acid anspo e (s) such as N-I and N-II. Glu a-
ma e could be exchanged wi h glu amine in a p ocess
esembling he Gln o Glu exchange be ween he e o-
cys s and ege a i e cells in ee-li ing he e ocys ous
cyanobac e ia. Addi ionally, he p esence o cyanophycin
me abolism genes bu absence o a ginine ca abolism
genes in Rin HH01 s ongly suppo s he ans e o a gi-
nine om he symbion o he hos . Hence, o N supply
o he dia om, glu amine and a ginine a e candida e ehi-
cles. Rega ding Fe, S and P acquisi ion, Rin HH01 p e-
sen s a low numbe o anspo e s wi h espec o
Anabaena and CalSC01 (Fig. 2). Rin HH01 migh acqui e
Fe h ough he Fe
3+
ABC anspo e Fu ABC and he
Fe
2+
pe mease E eU. Sul a e anspo is iden ical in all
h ee symbion s and Anabaena and cha ac e ized by he
Sbp/CysPTWA ABC anspo e and a SulP pe mease.
Rin HH01 is limi ed o Pi, using a high-a fini y Ps (PhoT)
ABC anspo e and he low-a fini y PiT anspo e .
Model o Rin RC01
Al hough Rin RC01 esides be ween he cy oplasmic
memb ane and he us ule o he dia om, i has C and N
compound up ake capabili ies simila o hose in
Rin HH01. Fo example, genes encoding Am ammonium
anspo e s o ni a e/ni i e and u ea assimila ion pa h-
ways a e also missing. On he o he hand, genes
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2044 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
Fig. 3. P oposed models o me aboli e
exchange and anspo mechanisms in he
DDAs. The filamen o he symbion s is com-
posed o a single e minal he e ocys and a
a iable numbe o ege a i e cells. Some
dia om hos s de elop chains. A. Model o
he Hemiaulus hauckii-Richelia in acellula is
(Rin HH01) symbiosis. F om wo o ou fila-
men s o Richelia pe dia om can eside in
he hos cy oplasm (Bus os-Díaz e al., 2019;
Capu o e al., 2019). No e he whi e a eas
showing he spaces be ween wo single dia-
oms in a chain. B. Model o he
Rhizosolenia cle ei-Richelia in acellula is
(Rin RC01) symbiosis. The symbion is
loca ed be ween he dia om plasma mem-
b ane and he us ule wi h he e minal he -
e ocys close o he dia om al e (Taylo ,
1982). No e ha he numbe o ege a i e
cells in he symbion is highe han in he wo
o he symbion s. C. Model o Chae oce os
comp essus-Calo h ix hizosoleniae
(CalSC01) symbiosis. The dia om hos con-
ains spines o which he symbion a aches
ans e sely wi h he he e ocys (No is,
1961). Symbion anspo e s and ela ed
p o eins a e shown amed. 2OG,
2-oxoglu a a e; aa(s), amino acid(s);
Amm/aa pe mease, Rin HH_4450/
Rin RC_1364/ Ga0265390_112922 p o eins;
DME, D ug/Me aboli e Expo e ; EAMA,
D ug/Me aboli e T anspo e supe amily p o-
ein; Gls, ABC glucoside anspo e ; MFS,
Majo Facili a o Supe amily; N-I and N-II,
amino acid ABC anspo e s; TRAP, T ipa -
i e ATP-independen pe iplasmic T ans-
po e . O he anspo e s a e indica ed by
hei o mal names: BicA, NDH-1
3
,
OppABCD, Po ADB and Sb A.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2045
encoding anspo e s o he up ake o glucosides, amino
acids, pep ides (possibly glu a hione) and polyamines a e
p esen , as well as genes encoding possible amino acid
expo e s including a DME p o ein and Rin RC_1364 ha
migh also anspo ammonium. Significan di e ences
wi h espec o Rin HH01 a e he p esence in Rin RC01
o he genes encoding suc ose biosyn hesis p o eins, a
second in e ase (In A) and an ace a e pe mease
ins ead o he p oline pe mease (Fig. 3B). Addi ionally,
he p esence o a BicA anspo e in Rin RC01 is mo e
likely han in Rin HH01. On he o he hand, Rin RC01
lacks cyanophycin making he exchange o a ginine less
likely han in Rin HH01. I on, S and Pi up ake capabili ies
a e also simila be ween Rin RC01 and Rin HH01. Thus,
o he han some specific me aboli es ha may be
exchanged (e.g. a ginine and p oline in Rin HH01/H.
hauckii; bica bona e and ace a e in Rin RC01/R.cle ei),
he main di e ence be ween he symbio ic associa ions
in ol ing Rin RC01 and Rin HH01 mus eside in he
physiology o and/o dependency on he hos dia om.
Whe eas Rin HH01 akes up nu ien s di ec ly om he
dia om’s cy oplasm, Rin RC01 akes up nu ien s om
he ‘pe iplasmic space’, implying ha ma e ials ha he
cyanobac e ium ob ains om he dia om a e expo ed
h ough he dia om’s cy oplasmic memb ane. The spe-
cific s udy o expo mechanisms in he dia om will be
he e o e o much in e es . The possibili y ha Rin RC01
akes up nu ien s mo e om he ex e nal medium
(ocean) han om he dia om is no suppo ed by ou find-
ings, which show a ema kably simila a ay o ans-
po e s in Rin RC01 and Rin HH01.
Model o CalSC01
CalSC01 esides ex e nal o he us ule o he dia om
and can g ow as a ee-li ing o ganism, hus i is a acul-
a i e symbion (Fos e e al., 2010). Consis en ly, he
memb ane anspo e epe oi e o his symbion is mo e
simila o ha o ypical ee-li ing he e ocys ous cyano-
bac e ia, including high-a fini y bica bona e anspo e
(Sb A) and ammonium (Am ) and ni a e/ni i e pe me-
ases (Fig. 3C). Addi ionally, in con as o he endosymbi-
on s, CalSC01 con ains a phosphona e u iliza ion
pa hway and, no ably, has he capabili y o inco po a ing
Fe wi h a comple e concou se o side opho es. Thus,
ammonium, ni i e/ni a e, phosphona e and e ic i on-
side opho e complexes a e mos likely ob ained om he
su ounding ma ine medium. On he o he hand, he
ex e nal symbion (CalSC01) has se e al anspo e s
simila o hose o he endosymbion s (Fig. 3), sugges ing
ha i may acul a i ely use esou ces ob ained om he
hos ’s phycosphe e (nu ien - ich a ea su ounding phy o-
plank on cells), hus b oadening i s g ow h op ions. Com-
pounds ob ained om he dia om hos could include
glucosides, amino acids, pep ides (possibly glu a hione),
polyamines and ca boxyla es. Rega ding N nu i ion o he
hos , CalSC01 migh beha e simila ly o he endosymbi-
on s p o iding a ginine and glu amine (as we hypo hesize
o Rin HH01) o glu amine (which is possible o
Rin RC01). Addi ionally, he p esence o Am p o eins in
CalSC01 sugges s he possibili y o an exchange o
ammonium, which would be mo e likely in CalSC01 han
in he endosymbion s. Gi en ha CalSC01 is capable o
g ow h in he labo a o y wi h epo ed e idence on solid
g ow h medium (Fos e e al., 2010), i could po en ially be
a model sys em o es he unc ionali y o se e al o he
anspo e s men ioned he e.
Concluding ema ks
In his a icle, we ha e ocused on he memb ane ans-
po e s encoded in he genomes o globally dis ibu ed
and biogeochemically significan N
2
-fixing symbion s o
dia oms wi h special emphasis on he acquisi ion o mac-
onu ien s. Ou findings show ha he endosymbion s,
ei he in e nal o pa ial (Rin HH01 and Rin RC01 espec-
i ely), con ain a simila a ay o anspo e s, sugges ing
a simila dependence on he dia om hos physiology,
whe eas he ex e nal symbion (CalSC01) has ans-
po e s simila o hose o he endosymbion s and addi-
ionally o he anspo e s use ul o li e in a dilu e ocean.
Based on cu en knowledge, he only ole o he
cyanobac e ial symbion s is o p o ide hei hos dia oms
wi h fixed N. He e ocys ous cyanobac e ia ha engage in
e es ial symbioses such as Nos oc azollae and N.
punc i o me elease N in he o m o ammonium o hei
plan pa ne s, whe eas ano he Nos oc symbion
eleases o ganic N, mainly ci ulline and glu amine
( e iewed in Meeks and Elhai, 2002). The amoun o N
2
-
de i ed ammonium eleased o e es ial hos plan s a -
ies emendously (40%–90% o fixed N) and is la gely
con olled by he GS ac i i y o he espec i e symbion s
(Meeks, 2009). Cu en ly, he amoun o N and he chem-
ical o m in which N is ans e ed in he DDAs is
unknown. GS and N
2
fixa ion ac i i ies in he a ious
DDAs a e expec ed o a y gi en he absence o GOGAT
in Rin HH01 and e en ion in he o he symbion s. I will
be o g ea in e es o de e mine which o m o N (ammo-
nium, amino acids) is ans e ed om he symbion and
he ole o he hos in po en ially p o iding glu ama e o
o he C skele ons o influence he ex en o N
2
fixa ion in
he symbion s. A possible ole o Am anspo e s has
been discussed in he ans e o ammonium in a ious
symbioses including he N.azollae –Azolla symbiosis
(Roy e al., 2020). He e, we ha e hypo hesized ans e
o glu amine and a ginine in DDAs, bu he p esence o
Am p o eins specifically in CalSC01 and o an
NKCC1- ype ca ion anspo e ha migh anspo
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2046 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
ammonium in he h ee symbion s make ammonium a
possible N ehicle in a leas some DDAs.
In his a icle, we ha e p oposed a numbe o ans-
po e s encoded in he DDA genomes as esponsible o
he ans e o specific compounds be ween symbion and
hos . The subs a es o many o hose anspo e s can be
p edic ed wi h easonable ce ain y om sequence analy-
sis, bu o some o he anspo e s, only he gene al
chemical na u e o he subs a e can be an icipa ed. In
addi ion o pe o ming expe imen al esea ch o co obo-
a e he ac i i y o hose anspo e s whose subs a es
canno be defined wi h ce ain y, u he esea ch is
needed. In pa icula , u u e wo k should also add ess he
composi ion o he dia om’s memb ane anspo e sys-
ems, which will be impo an o unde s and i s nu i ional
physiology in he ma ine en i onmen as well as i s pecu-
lia symbiosis wi h he pa ial and ex e nal symbion s,
which a e likely p o ided wi h nu ien s by he dia om hos .
ACKNOWLEDGEMENTS
We a e g a e ul o Daniel Lundin (Linnaeus Uni e si y, Swe-
den) o suppo in da a analysis and p esen a ion, Ignacio
Luque (CSIC, Se ille, Spain) o analysis o he p omo e
sequences o Zn- egula ed po ins, and Ma in Ekman
(S ockholm Uni e si y, Sweden) o discussion on cyano-
bac e ia in symbiosis. We also hank wo anonymous
e iewe s o hei help ul c i icisms o his a icle. This wo k
was suppo ed by G an No. 2018-04161 om The Swedish
Resea ch Council (Ve enskaps åde ) o RAF and EF, a g an
om he Knu and Alice Wallenbe g Founda ion o RAF, and
G an No. BFU2017-88202-P om he Spanish Go e nmen
co-financed by he Eu opean Regional De elopmen
Fund o EF.
Re e ences
Aichi, M., Yoshiha a, S., Yamashi a, M., Maeda, S.,
Nagai, K., and Oma a, T. (2006) Cha ac e iza ion o he
ni a e-ni i e anspo e o he majo acili a o supe amily
( he n P gene p oduc ) om he cyanobac e ium Nos oc
punc i o me s ain ATCC 29133. Biosci Bio echnol Bio-
chem 70: 2682–2689.
Allen, A.E., Dupon , C.L., Obo ník, M., Ho ák, A., Nunes-
Nesi, A., McC ow, J.P., e al. (2011) E olu ion and me a-
bolic significance o he u ea cycle in pho osyn he ic dia-
oms. Na u e 473: 203–207.
Al schul, S.F., Madden, T.L., Scha e , A.A., Zhang, J.,
Zhang, Z., Mille , W., e al. (1997) Gapped BLAST and
PSI-BLAST: a new gene a ion o p o ein da abase sea ch
p og ams. Nucleic Acids Res 25: 3389–3402.
Amin, S.A., G een, D.H., Ha , M.C., Küppe , F.C.,
Sunda, W.G., and Ca ano, C. (2009) Pho olysis o i on-
side opho e chela es p omo es bac e ial-algal mu ualism.
P oc Na l Acad Sci U S A 106: 17071–17076. h ps://doi.
o g/10.1073/pnas.0905512106.
A mb us , E.V. (2009) The li e o dia oms in he wo ld’s
oceans. Na u e 459: 185–192. h ps://doi.o g/10.1038/
na u e08057.
Badge , M.R., P ice, G.D., Long, B.M., and Woodge , F.J.
(2006) The en i onmen al plas ici y and ecological geno-
mics o he cyanobac e ial CO
2
concen a ing mechanism.
J Exp Bo 57: 249–265.
Bale, N.J., Hopmans, E.C., Do hou , D., S al, L.J.,
G ego, M., an Bleijswijk, J., e al. (2018) A no el he e o-
cys glycolipid in a pelagic N
2
-fixing cyanobac e ium o he
genus Calo h ix.O g Geochem 123:44–47.
Bale, N.J., Hopmans, E.C., Zell, C., Sob inho, R.L., Kim, J.-
H., Sinninghe Dams é, J.S., e al. (2015) Long chain gly-
colipids wi h pen ose head g oups as bioma ke s o
ma ine endosymbio ic he e ocys ous cyanobac e ia. O g
Geochem 81:1–7.
Ba din, S.F., Dan, S.F., Os e as, M.F., and Finan, T.M.
(1996) A phospha e anspo sys em is equi ed o sym-
bio ic ni ogen fixa ion by Rhizobium melilo i.J Bac e iol
178: 4540–4547.
Baue sachs, T., Compao e, J., Hopmans, E.C., S al, L.J.,
Schou en, S., and Sinninghe Dams é, J.S. (2009) Dis ibu-
ion o he e ocys glycolipids in cyanobac e ia. Phy o-
chemis y 70: 2934–2039.
Bjö kman, K.M., Duhamel, S., Chu ch, M.J., and Ka l, D.M.
(2018) Spa ial and empo al dynamics o ino ganic phos-
pha e and adenosine-50- isphospha e in he No h Pacific
Ocean. F on Ma Sci 5: 235. h ps://doi.o g/10.3389/
ma s.2018.00235.
B aun, V., and Killman, H. (1998) Bac e ial solu ions o he
i on-supply p oblem. T ends Biochem Sci 24: 104–109.
Bu na , M., He e o, A., and Flo es, E. (2014) Compa men-
alized cyanophycin me abolism in he diazo ophic fila-
men s o a he e ocys - o ming cyanobac e ium. P oc Na l
Acad Sci U S A 111: 3823–3828.
Bu na , M., Li, B., Kim, S.H., Michael, A.J., and Flo es, E.
(2018) Homospe midine biosyn hesis in he cyanobac e-
ium Anabaena equi es a deoxyhypusine syn hase homo-
logue and is essen ial o no mal diazo ophic g ow h. Mol
Mic obiol 109: 763–780.
Bu na , M., Picossi, S., Vallada es, A., He e o, A., and
Flo es, E. (2019) Ca abolic pa hway o a ginine in Ana-
baena in ol es a no el bi unc ional enzyme ha p oduces
p oline om a ginine. Mol Mic obiol 111: 883–897.
Bus os-Díaz, E.D., Ba ona-Gómez, F., and Cib ián-
Ja amillo, A. (2019) Cyanobac e ia in ni ogen-fixing sym-
bioses. In Cyanobac e ia: Basic Science o Applica ions,
Mish a, A.K., Tiwa i, D.N., and Rai, A.N. (eds): Academic
P ess, pp. 29–42. h ps://doi.o g/10.1016/B978-0-12-
814667-5.00002-7.
Cai, Y., and Wolk, C.P. (1997) Anabaena sp. s ain PCC
7120 esponds o ni ogen dep i a ion wi h a cascade-like
sequence o ansc ip ional ac i a ions. J Bac e iol 179:
267–271.
Came on, J.C., Su e , M., and Ke eld, C.A. (2014) The
ca boxysome: unc ion, s uc u e and cellula dynamics. In
The Cell Biology o Cyanobac e ia, Flo es, E., and
He e o, A. (eds): No olk: Cais e Academic P ess,
pp. 171–188.
Capu o, A., Nylande , J.A.A., and Fos e , R.A. (2019) The
gene ic di e si y and e olu ion o dia om-diazo oph
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2047

associa ions highligh ing ai s a o ing symbion in eg a-
ion. FEMS Mic obiol Le 366: 2. h ps://doi.o g/10.1093/
emsle/ ny297.
Ca pen e , E.J., Mon oya, J.P., Bu ns, J.P., Mulholland, M.,
Sub amaniam, A., and Capone, D.G. (1999) Ex ensi e
bloom o a N
2
-fixing dia om/cyanobac e ial associa ion in
he T opical A lan ic Ocean. Ma Ecol P og Se 185:
273–278.
Chappell, P.D., Mo e , J.W., Hynes, A.M., and Webb, E.A.
(2012) Molecula e idence o i on limi a ion and a ailabili y in
he global diazo oph T ichodesmium.ISME J 6:1728–1739.
Cui, J., and Da idson, A.L. (2011) ABC solu e impo e s in
bac e ia. Essays Biochem 50:85–99.
Cui, Y., Lin, X., Zhang, H., Lin, L., and Lin, S. (2015) PhnW-
PhnX pa hway in dinoflagella es no unc ional o u ilize
ex acellula phosphona es. F on Ma Sci 2: 120.
Day, R.M., and Suzuki, Y.J. (2005) Cell p oli e a ion, eac i e
oxygen and cellula glu a hione. Dose–Response 3:
425–442. h ps://doi.o g/10.2203/dose- esponse.003.03.010.
Dyh man, S.T., Chappell, P.D., Haley, S.T., Mo e , J.W.,
O cha d, E.D., Wa e bu y, J.B., and Webb, E.A. (2006)
Phosphona e u iliza ion by he globally impo an ma ine
diazo oph T ichodesmium.Na u e 439:68–71.
Dyh man, S.T., Jenkins, B.D., Rynea son, T.A., Sai o, M.A.,
Me cie , M.L., Alexande , H., e al. (2012) The ans-
c ip ome and p o eome o he dia om Thalassiosi a
pseudonana e eal a di e se phospho us s ess
esponse. PLoS One 7: e33768.
Ekman, M., Picossi, S., Campbell, E.L., Meeks, J.C., and
Flo es, E. (2013) A Nos oc punc i o me suga anspo e
necessa y o es ablish a cyanobac e ium-plan symbiosis.
Plan Physiol 161: 1984–1992.
Escude o, L., Ma iscal, V., and Flo es, E. (2015) Func ional
dependence be ween sep al p o ein SepJ om Anabaena
sp. s ain PCC 7120 and an amino acid ABC- ype up ake
anspo e . J Bac e iol 197: 2721–2730.
Field, C.B., Beh en eld, M.J., Rande son, J.T., and
Falkowski, P.G. (1998) P ima y p oduc ion o he bio-
sphe e: in eg a ing e es ial and oceanic componen s.
Science 281: 237–240.
Finzi-Ha , J.A., Pe -Ridge, J., Webe , P.K., Popa, R.,
Fallon, S.J., Gunde son, T., e al. (2009) Fixa ion and a e
o C and N in he cyanobac e ium T ichodesmium using
nanome e -scale seconda y ion mass spec ome y. P oc
Na l Acad Sci USA 106: 6345–6350 E a um in: P oc Na l
Acad Sci USA 106: 9931.
Flo es, E., A é alo, S., and Bu na , M. (2019a) Cyanophycin
and a ginine me abolism in cyanobac e ia. Algal Res 42:
101577.
Flo es, E., F ías, J.E., Rubio, L.M., and He e o, A. (2005)
Pho osyn he ic ni a e assimila ion in cyanobac e ia. Pho-
osyn h Res 83: 117–133.
Flo es, E., López-Lozano, A., and He e o, A. (2015) Ni o-
gen fixa ion in he oxygenic pho o ophic p oka yo es (cya-
nobac e ia): he figh agains oxygen. In Biological
Ni ogen Fixa ion, Vol. 2, De B uijn, F.J. (ed),
pp. 879–889. Hoboken, NJ: JohnWiley & Sons, Inc.
Flo es, E., Picossi, S., Vallada es, A., and He e o, A.
(2019b) T ansc ip ional egula ion o de elopmen in
he e ocys - o ming cyanobac e ia. Biochim Biophys Ac a
–Gene Regul Mech 1862: 673–684.
Fos e , R.A., Goebel, N.L., and Zeh , J.P. (2010) Isola ion o
Calo h ix hizosoleniae s ain SC01 om Chae oce os
spp. dia oms o he sub opical N. Pacific Ocean. J Phycol
46: 1028–1037.
Fos e , R.A., Kuype s, M.M.M., Vagne , T., Pae l, R.W.,
Musa , N., and Zeh , J.P. (2011) Ni ogen fixa on and
ans e in open ocean dia om-cyanobac e ial symbioses.
ISME J 5: 1484–1493.
Fos e , R.A., and O’Mullan, G.D. (2008) Ni ogen-fixing and
ni i ying symbioses in he ma ine en i onmen . In Ni o-
gen in he Ma ine En i onmen , Capone, D.G., B onk, D.
A., Mulholland, M.R., and Ca pen e , E.J. (eds). London:
Academic P ess, pp. 1197–1218.
Fos e , R.A., and Zeh , J.P. (2006) Cha ac e iza ion o
dia om-cyanobac e ia symbioses on he basis o ni H,
he R, and 16S RNA sequences. En i on Mic obiol 8:
1913–1925.
Fos e , R.A., and Zeh , J.P. (2019) Di e si y genomics, and
dis ibu ion o phy oplank on-cyanobac e ia single-cell
symbio ic associa ions. Annu Re Mic obiol 73: 435–456.
Guilla d, R.R.L., and Kilham, P. (1977) Ecology o ma ine
plank onic dia oms. In The Biology o Dia oms, We ne , D.
(ed): New Yo k: Uni e si y o Cali o nia P ess,
pp. 372–469.
Hahn, A., and Schlei , E. (2014) The cell en elope. In The
Cell Biology o Cyanobac e ia, Flo es, E., and He e o, A.
(eds). No olk, UK: Cais e Academic P ess, pp. 29–87.
Hahn, A., S e ano ic, M., Mi us, O., and Schlei , E. (2012)
The TolC-like p o ein HgdD o he cyanobac e ium Ana-
baena sp. PCC 7120 is in ol ed in seconda y me aboli e
expo and an ibio ic esis ance. J Biol Chem 287:
41126–44138.
Hamana, K., Miyagawa, K., and Ma suzaki, S. (1983) Occu -
ence o sym-homospe midine as he majo polyamine in
ni ogen-fixing cyanobac e ia. Biochem Biophys Res
Commun 112: 606–613.
Ha ke, M.J., Fischko n, K.R., Haley, S.T., Aylwa d, F.O.,
Zeh , J.P., and Dyh man, S.T. (2018) Pe iodic and coo di-
na ed gene exp ession be ween a diazo oph and i s dia-
om hos . ISME J 13: 118–131.
Hellebus , J.A., and Lewin, J. (1977) He e o ophic nu i ion.
In The Biology o Dia oms, We ne , D. (ed). Be keley: Uni-
e si y o Cali o nia P ess, pp. 169–197.
He e o, A., and Flo es, E. (2019) Gene ic esponses o ca -
bon and ni ogen a ailabili y in Anabaena.En i on
Mic obiol 21:1–17.
He e o, A., Flo es, E., and Impe ial, J. (2019) Ni ogen
assimila ion in bac e ia. In Encyclopedia o Mic obiology,
4 h ed. Ams e dam: Else ie . h ps://doi.o g/10.1016/
B978-0-12-809633-8.20680-8.
Hil on, J.A. (2014) Ecology and E olu ion o Dia om-
Associa ed Cyanobac e ia h ough Gene ic Analyses. Dis-
se a ion Thesis, San a C uz CA: Uni e si y o Cali o nia.
Hil on, J.A., Fos e , R.A., T ipp, H.J., Ca e , B.J., Zeh , J.P.,
and Villa eal, T.A. (2013) Genomic dele ions dis up ni o-
gen me abolism pa hways o a cyanobac e ial dia om
symbion . Na Commun 4: 1767.
Hopkinson, B.M., Dupon , C.L., Allen, A.E., and Mo el, F.M.
M. (2011) E ficiency o he CO
2
-concen a ing mechanism
o dia oms. P oc Na l Acad Sci U S A 108: 3830–3837.
h ps://doi.o g/10.1073/pnas.1018062108.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2048 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
Hopkinson, B.M., Dupon , C.L., and Ma suda, Y. (2016) The
physiology and gene ics o CO
2
concen a ing mecha-
nisms in model dia oms. Cu Opin Plan Biol 31:51–57.
h ps://doi.o g/10.1016/j.pbi.2016.03.013.
Ho igushi, M. (1984) Occu ence, iden ifica ion and p ope ies o
phosphonic and phosphinic acids. In Biochemis y o Na u al
C-P Compounds, Ho i, T., Ho igushi, M., and Hayashi, A.
(eds). Shiga, Japan: Japanese Associa ion o Resea ch on
he Biochemis y o C-P Compounds, pp. 24–52.
Ho e-Jensen, B., Zechel, D.L., and Jochimsen, B. (2014)
U iliza ion o glyphosa e as phospha e sou ce: biochemis-
y and gene ics o bac e ial ca bon-phospho us lyase.
Mic obiol Mol Biol Re 78: 176–197.
H yniewicz, M., Si ko, A., Palucha, A., Bock, A., and
Hulanicka, D. (1990) Sul a e and hiosul a e anspo in
Esche ichia coli K-12: iden ifica ion o a gene encoding a
no el p o ein in ol ed in hiosul a e binding. J Bac e iol
172: 3358–3366.
Hudek, L., P emachand a, D., Webs e , W.A., and B äu, L.
(2016) Role o phospha e anspo sys em componen
Ps B1 in phospha e in e naliza ion by Nos oc punc i o me.
Appl En i on Mic obiol 82: 6344–6356.
Inomu a, K., Folle , C.L., Masuda, T., Eichne , M., P ášil, O.,
and Deu sch, C. (2020) Ca bon ans e om he hos dia-
om enables as g ow h and high a e o N
2
fixa ion by
symbio ic he e ocys ous cyanobac e ia. Plan s (Basel) 9:
E192. h ps://doi.o g/10.3390/plan s9020192.
Jack, D.L., Yang, N.M., and Saie , M.H., J . (2001) The
d ug/me aboli e anspo e supe amily. Eu J Biochem
268: 3620–3639.
Jackson, R.J., Bine , M.R., Lee, L.J., Ma, R., G aham, A.I.,
McLeod, C.W., and Poole, R.K. (2008) Exp ession o he
Pi A phospha e/me al anspo e o Esche ichia coli is
esponsi e o zinc and ino ganic phospha e le els. FEMS
Mic obiol Le 289: 219–224.
Janson, S., Rai, A.N., and Be gman, B. (1995) In acellula
cyanobion Richelia in acellula is: ul as uc u e and
immuno-localisa ion o phycoe y h in, ni ogenase,
Rubisco and glu amine syn he ase. Ma Biol 124:1–8.
Janson, S., Wou e s, J., Be gman, B., and Ca pen e , E.J.
(1999) Hos specifici y in he Richelia-dia om symbiosis
e ealed by he R gene sequence analysis. En i on
Mic obiol 1: 431–438.
Jeanjean, R., Tall, E., La ifi, A., Ha aux, M., Janicki, A., and
Zhang, C.C. (2008) A la ge gene clus e encoding pep ide
syn he ases and polyke ide syn hases is in ol ed in p o-
duc ion o side opho es and oxida i e s ess esponse in
he cyanobac e ium Anabaena sp. s ain PCC 7120. En i-
on Mic obiol 10: 2574–2585. h ps://doi.o g/10.1111/j.
1462-2920.2008.01680.x.
Jiang, H.B., Lou, W.J., Ke, W.T., Song, W.Y., P ice, N.M.,
and Qiu, B.S. (2015) New insigh s in o i on acquisi ion by
cyanobac e ia: an essen ial ole o ExbB-ExbD complex
in ino ganic i on up ake. ISME J 9: 297–309. h ps://doi.
o g/10.1038/ismej.2014.123.
Kaplan, A., and Reinhold, L. (1999) CO
2
concen a ing
mechanisms in pho osyn he ic mic oo ganisms. Annu Re
Plan Physiol Plan Mol Biol 50: 539–570.
Ka l, D.M., Bjö kman, K.M., Do e, J.E., Fujieki, L., Hebel, D.
V., Houlihan, T., e al. (2001) Ecological ni ogen- o-
phospho us s oichiome y a s a ion ALOHA. Deep-Sea
Res II 48: 1529–1566. h ps://doi.o g/10.1016/S0967-0645
(00)00152-1.
Ka l, D.M., Chu ch, M.J., Dio e, J.E., Le elie , R.M., and
Maha ey, C. (2016) P edic able and e ficien ca bon
seques a ion in he No h Pacific Ocean suppo ed by
symbio ic ni ogen fixa ion. P oc Na l Acad Sci U S A 109:
1842–1849.
Kazamia, E., Su ak, R., Paz-Yepes, J., Do ell, R.G.,
Viei a, F.R.J., Mach, J., e al. (2018) Endocy osis-
media ed side opho e up ake as a s a egy o Fe acquisi-
ion in dia oms. Sci Ad 4: eaa 4536. h ps://doi.o g/10.
1126/sciad .aa 4536.
Kolowi h, L.C., Ingall, E.D., and Benne , R. (2001) Composi-
ion and cycling o ma ine o ganic phospho us. Limnol
Oceanog 46: 309–320. h ps://doi.o g/10.4319/lo.2001.
46.2.0309.
Konono a, S.V., and Nesmeyano a, M.A. (2002) Pho-
sphona es and hei deg ada ion by mic oo ganisms.
2002. Biochemis y 67: 184–195.
K öge , N., and Poulsen, N. (2008) Dia oms- om cell wall
biogenesis o nano echnology. Annu Re Gene 42:
83–107.
Kugle , M., Loe fle , W., Rapp, C., Ke n, A., and Jung, G.
(1990) Rhizoc icin A, an an i ungal phosphono-
oligopep ide o Bacillus sub ilis ATCC 6633: biological
p ope ies. A ch Mic obiol 153: 276–281.
Kus ka, A.B., Sañudo-Wilhelmey, S.A., Ca pen e , E.J.,
Capone, D., Bu ns, J., and Sunda, W.G. (2003) I on
equi emen s o di-ni ogen- and ammonium-suppo ed
g ow h in cul u es o T ichodesmium (IMS 191): compa i-
son wi h ni ogen fixa ion a es and i on:ca bon a ios o
field popula ions. Limnol Oceang 48: 1869–1884.
Lin, S., Li ake , R.W., and Sunda, W.G. (2016) Phospho us
physiological ecology and molecula mechanisms in
ma ine phy oplank on. J Phycol 52:10–36. h ps://doi.o g/
10.1111/jpy.12365.
López-Igual, R., Flo es, E., and He e o, A. (2010) Inac i a-
ion o a he e ocys -specific in e ase indica es a p incipal
ole o suc ose ca abolism in he e ocys s o Anabaena sp.
J Bac e iol 192: 5526–5533.
Luque, I., and Fo chhamme , K. (2008) Ni ogen assimila ion
and C/N balance sensing. In The Cyanobac e ia: Molecu-
la Biology Genomics and E olu ion, He e o, A., and
Flo es, E. (eds). No olk, UK: Cais e Academic P ess,
pp. 335–382.
Madei a, F., Pa k, Y.M., Lee, J., Buso, N., Gu , T.,
Madhusoodanan, N., e al. (2019) The EMBL-EBI sea ch
and sequence analysis ools APIs in 2019. Nucleic Acids
Res 47: W636–W641.
Mague, T.H., Wea e, N.M., and Holm-Hansen, O. (1974)
Ni ogen-fixa ion in he No h Pacific gy e. Ma Biol 24:
109–119.
Mala inszky, D., S eue , R., and Jones, P.R. (2017) A com-
p ehensi ely cu a ed genome-scale wo-cell model o he
he e ocys ous cyanobac e ium Anabaena sp. PCC 7120.
Plan Physiol 173: 509–523.
Ma in, J.H., Go don, R.M., and Fi zwa e , S.E. (1991) The
case o i on. Limnol Oceanog 36: 1793–1802.
Ma ín-Figue oa, E., Na a o, F., and Flo encio, F.J. (2000)
The GS-GOGAT pa hway is no ope a i e in he he e o-
cys s. Cloning and exp ession o glsF gene om he
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2049
cyanobac e ium Anabaena sp. PCC 7120. FEBS Le 476:
282–286.
Meeks, J.C. (2009) Physiological adap a ions in ni ogen-
fixing Nos oc-plan symbio ic associa ions. Mic obiol
Monog 8: 181–205.
Meeks, J.C., and Elhai, J. (2002) Regula ion o cellula di e -
en ia ion in filamen ous cyanobac e ia in ee-li ing and
plan -associa ed symbio ic g ow h s a es. Mic obiol Mol
Biol Re 66:94–121.
Meeks, J.C., Elhai, J., Thiel, T., Po s, M., La ime , F.,
Lame din, J., e al. (2001) An o e iew o he genome o
Nos oc punc i o me, a mul icellula symbio ic cyanobac e-
ium. Pho osyn h Res 70:85–106.
Michael, A.J. (2016) Biosyn hesis o polyamines and polyamine-
con aining molecules. Biochem J 473: 2315–2329.
Mon esinos, M.L., Mu o-Pas o , A.M., He e o, A., and
Flo es, E. (1998) Ammonium/me hylammonium pe me-
ases o a cyanobac e ium. Iden ifica ion and analysis o
h ee ni ogen- egula ed am genes in Synechocys is
sp. PCC 6803. J Biol Chem 273: 31463–31470.
Moog, D., Rensing, S.A., A chibald, J.M., Maie , U.G., and
Ull ich, K.K. (2015) Localiza ion and e olu ion o pu a i e
iose phospha e ansloca o s in he dia om
Phaeodac ylum ico nu um.Genome Biol E ol 7:
2955–2969.
Moo e, J.K., Doney, S.C., Glo e , D.M., and Fung, I.Y.
(2001) I on cycling and nu ien -limi ing pa e ns in su ace
wa e s o he wo ld Ocean. Deep-Sea Res Pa II 49:
463–507.
Mosla ac, S., Nicolaisen, K., Mi us, O., Dehni, F.A.,
Pe nil, R., Flo es, E., e al. (2007a) A TolC-like p o ein is
equi ed o he e ocys de elopmen in Anabaena
sp. s ain PCC 7120. J Bac e iol 189: 7887–7895.
Mosla ac, S., Reisinge , V., Be g, M., Mi us, O., Vosyka, O.,
Plösche , M., e al. (2007b) The p o eome o he he e o-
cys cell wall in Anabaena sp. PCC 7120. Biol Chem 388:
823–829.
Mulligan, C., Fische , M., and Thomas, G.H. (2011) T ipa i e
ATP-independen pe iplasmic (TRAP) anspo e s in bac-
e ia and a chaea. FEMS Mic obiol Re 35:68–86.
Mu o-Pas o , M.I., Reyes, J.C., and Flo encio, F.J. (2001)
Cyanobac e ia pe cei e ni ogen s a us by sensing in a-
cellula 2-oxoglu a a e le els. J Biol Chem 276:
38320–38328.
Mu o-Pas o , M.I., Reyes, J.C., and Flo encio, F.J. (2005)
Ammonium assimila ion in cyanobac e ia. Pho osyn h Res
83: 135–150.
Nicolaisen, K., Hahn, A., and Schlei , E. (2009) The cell wall
in he e ocys o ma ion by Anabaena sp. PCC 7120.
J Basic Mic obiol 49:5–24.
Nicolaisen, K., Hahn, A., Valdebeni o, M., Mosla ac, S.,
Sambo ski, A., Maldene , I., e al. (2010) The in e play
be ween side opho e sec e ion and coupled i on and cop-
pe anspo in he he e ocys - o ming cyanobac e ium
Anabaena sp. PCC 7120. Biochim Biophys Ac a 1798:
2131–2140.
Nicolaisen, K., Mosla ac, S., Sambo ski, A.,
Valdebeni o, M., Han ke, K., Maldene , I., e al. (2008)
Al 0397 is an ou e memb ane anspo e o he side-
opho e schizokinen in Anabaena sp. s ain PCC 7120.
J Bac e iol 190: 7500–7507.
Nie es-Mo ión, M., and Flo es, E. (2018) Mul iple ABC glu-
coside anspo e s media e suga -s imula ed g ow h in
he he e ocys - o ming cyanobac e ium Anabaena
sp. s ain PCC 7120. En i on Mic obiol Rep 10:40–48.
Nie es-Mo ión, M., Lechno-Yosse , S., López-Igual, R.,
F ías, J.E., Ma iscal, V., Nü nbe g, D.J., e al. (2017) Spe-
cific glucoside anspo e s influence sep al s uc u e and
unc ion in he filamen ous he e ocys - o ming cyanobac e-
ium Anabaena sp. s ain PCC 7120. J Bac e iol 199:
e00876–e00816.
No is, R.E. (1961) Obse a ions on phy oplank on o gan-
isms collec ed on he N.Z.O.I. Pacific C uise, Sep embe
1958. N Z J Sci 4: 162–168.
Nü nbe g, D.J., Ma iscal, V., Bo nikoel, J., Nie es-
Mo ión, M., K auß, N., He e o, A., e al. (2015) 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. MBio 6:
e02109.
Paz-Yepes, J., Me ino-Pue o, V., He e o, A., and Flo es, E.
(2008) The am gene clus e o he he e ocys - o ming
cyanobac e ium Anabaena sp. s ain PCC 7120.
J Bac e iol 190: 6534–6539.
Pe nil, R., He e o, A., and Flo es, E. (2010) A TRAP ans-
po e o py u a e and o he monoca boxyla e 2-oxoacids
in he cyanobac e ium Anabaena sp. s ain PCC 7120.
J Bac e iol 192: 6089–6092.
Pe nil, R., Piccossi, S., Ma iscal, V., He e o, A., and
Flo es, E. (2008) ABC- ype amino acid up ake ans-
po e s Bg and N-II o Anabaena sp. s ain PCC 7120
sha e an ATPase subuni and a e exp essed in ege a i e
cells and he e ocys s. Mol Mic obiol 67: 1067–1080.
Pe nil, R., Picossi, S., He e o, A., Flo es, E., and
Ma iscal, V. (2015) Amino acid anspo e s and elease o
hyd ophobic amino acids in he he e ocys - o ming cyano-
bac e ium Anabaena sp. s ain PCC 7120. Li e 5:
1282–1300.
Pe y, M.J. (1976) Phospha e u iliza ion by an oceanic dia-
om in phospho us-limi ed chemos a cul u e and in oligo-
ophic wa e s o cen al No h-Pacific. Limnol Oceang
21:88–107.
Pi , F.D., Maza d, S., Humph eys, L., and Scanlan, D.J.
(2010) Func ional cha ac e iza ion o Synechocys is
sp. s ain PCC 6803 ps 1 and ps 2 gene clus e s e eals
a no el s a egy o phospha e up ake in a eshwa e cya-
nobac e ium. J Bac e iol 192: 3512–3523.
P ice, G.D. (2011) Ino ganic ca bon anspo e s o he
cyanobac e ial CO
2
concen a ing mechanism. Pho osyn h
Res 109:47–57.
P ice, G.D., Woodge , F.J., Badge , M.R., Howi , S.M., and
Tucke , L. (2004) Iden ifica ion o a SulP- ype bica bona e
anspo e in ma ine cyanobac e ia. P oc Na l Acad Sci U
SA101: 18228–18233. h ps://doi.o g/10.1073/pnas.
0405211101.
P ihoda, J., Tanaka, A., de Paula, W.B.M., Allen, J.F.,
Ti ichine, L., and Bowle , C. (2012) Chlo oplas -
mi ochond ia c oss- alk in dia oms. J Exp Bo 63:
1543–1557.
Rao, N.N., and To iani, A. (1990) Molecula aspec s o
phospha e anspo in Esche ichia coli.Mol Mic obiol
4: 1083–1090. doi.o g/10.1111/j.1365-2958.1990.
b00682.x.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
2050 M. Nie es-Mo ión, E. Flo es and R. A. Fos e
Ra ledge, C., and Do e , L.G. (2000) I on me abolism in
pa hogenic bac e ia. Annu Re Mic obiol 54: 881–941.
Ra en, J.A., Cockell, C.S., and De la Rocha, C.L. (2008) The
e olu ion o ino ganic ca bon concen a ing mechanisms in
pho osyn hesis. Philos T ans R Soc Lond B Biol Sci 363:
2641–2650. h ps://doi.o g/10.1098/ s b.2008.0020.
Reddy, V.S., Shlyko , M.A., Cas illo, R., Sun, E.I., and
Saie , M.H. (2012) The majo acili a o supe amily (MFS)
e isi ed. FEBS J 279: 2022–2035.
Rippka, R., De uelles, J., Wa e bu y, J.B., He dman, M., and
S anie , R.Y. (1979) Gene ic assigmen s, s ain his o ies
and p ope ies o pu e cul u es o cyanobac e ia. J Gen
Mic biol 111:1–61.
Roe, K.L., and Ba beau, K.A. (2014) Up ake mechanisms o
ino ganic i on and e ic ci a e in T ichodesmium
e y h aeum IMS101. Me allomics 6: 2042–2051. h ps://
doi.o g/10.1039/c4m 00026a.
Roy, R., Reinde s, A., Wa d, J.M., and McDonald, T.R.
(2020) Unde s anding anspo p ocesses in lichen,
Azolla-cyanobac e ia, ec omyco hiza, endomyco hiza,
and hizobia-legume symbio ic in e ac ions. 9(F1000Res):
39. h ps://doi.o g/10.12688/ 1000 esea ch.19740.1.
Rudol , M., S e ano ic, M., K anzle , C., Pe nil, R.,
Ke en, N., and Schlei , E. (2016) Mul iplici y and specific-
i y o side opho e up ake in he cyanobac e ium Anabaena
sp. PCC 7120. Plan Mol Biol 92:57–69. h ps://doi.o g/
10.1007/s11103-016-0495-2.
Saie , M.H., J ., Eng, B.H., Fa d, S., Ga g, J., Hagge y, D.
A., Hu chinson, W.J., e al. (1999) Phylogene ic cha ac e -
iza ion o no el anspo p o ein amilies e ealed by
genome analyses. Biochim Biophys Ac a 1422:1–56.
Saie , M.H., J ., Reddy, V.S., Tsu, B.V., Ahmed, M.S., Li, C.,
and Mo eno-Hagelsieb, G. (2016) The anspo e classifi-
ca ion da abase (TCDB): ecen ad ances. Nucleic Acids
Res 44: D372–D379.
Sale no, G.L., and Cu a i, L. (2003) O igin o suc ose
me abolism in highe plan s: when, how and why? T ends
Plan Sci 8:63–69.
Scanlan, D.J., Os owski, M., Maza d, S., Du esne, A.,
Ga cza ek, L., Hess, W.R., e al. (2009) Ecological geno-
mics o ma ine picocyanobac e ia. Mic obiol Mol Biol Re
73: 249–299.
Schindle , D.W. (1977) E olu ion o phospho us limi a ion in
lakes. Science 195: 260–262. h ps://doi.o g/10.1126/
science.195.4275.260.
Schou en, S., Villa eal, T.A., Hopmans, E.C., Me s, A.,
Swanson, K.M., and Dams e, J.S.S. (2013) Endosymbio ic
he e ocys ous cyanobac e ia syn hesize di e en he e o-
cys glycolipids han ee-li ing he e ocys ous cyano-
bac e ia. Phy ochemis y 85: 115–121.
Schulle , J.M., Sau a, P., Thiemann, J., Schulle , S.K.,
Gamiz-He nandez, A.P., Ku isu, G., e al. (2020) Redox-
coupled p o on pumping d i es ca bon concen a ion in
he pho osyn he ic complex I. Na Commun 11: 494.
h ps://doi.o g/10.1038/s41467-020-14347-4.
Sein-Echaluce, V.C., González, A., Napoli ano, M.,
Luque, I., Ba ja, F., Pelea o, M.L., and Filla , M.F. (2015)
Zu (Fu B) is a key ac o in he con ol o he oxida i e
s ess esponse in Anabaena sp. PCC 7120. En i on
Mic obiol 17: 2006–2017.
Shen, C., Dupon , C.L., and Hopkinson, B.M. (2017) The
di e si y o CO
2
-concen a ing mechanisms in ma ine dia-
oms as in e ed om hei gene ic con en . J Exp Bo 68:
3937–3948.
Simpson, F.B., and Neilands, J.B. (1976) Side och omes in
cyanophyceae: isola ion and cha ac e iza ion o
schizokinen om Anabaena sp. J Phycol 12:44–48.
h ps://doi.o g/10.1111/j.1529-8817.1976. b02824.x.
Si ko, A., H yniewicz, M., Hulanicka, D., and Böck, A. (1990)
Sul a e and hiosul a e anspo in Esche ichia coli K-12:
nucleo ide sequence and exp ession o he cysTWAM
gene clus e . J Bac e iol 172: 3351–3357.
Söde bäck, E., and Be gman, B. (1993) The Nos oc-
Gunna a symbiosis: ca bon fixa ion and ansloca ion.
Physiol Plan 89: 125–132.
S e ano ic, M., Hahn, A., Nicolaisen, K., Mi us, O., and
Schlei , E. (2011) The componen s o he pu a i e i on
anspo sys em in he cyanobac e ium Anabaena
sp. PCC 7120. En i on Mic obiol 14: 1655–1670. h ps://
doi.o g/10.1111/j.1462-2920.2011.02619.x.
Su, Z.C., Olman, V., and Xu, Y. (2007) Compu a ional p edic ion
o Pho egulons in cyanobac e ia. BMC Genomics 8:156.
Sub amaniam, A., Yage , P.L., Ca pen e , E.J., Maha ey, C.,
Bjö kman, K., Cooley, S., e al. (2008) Amazon i e
enhances diazo ophy and ca bon seques a ion in he opi-
cal No h A lan ic Ocean. P oc Na l Acad Sci U S A 105:
10460–10465. h ps://doi.o g/10.1073/pnas.0710279105.
Summe s, M.L., Wallis, J.G., Campbell, E.L., and Meeks, J.
C. (1995) Gene ic e idence o a majo ole o glucose-
6-phospha e dehyd ogenase in ni ogen fixa ion and da k
g ow h o he cyanobac e ium Nos oc sp. s ain ATCC
29133. J Bac e iol 177: 6184–6194.
Sunda, W.G., and Hun sman, S.A. (2015) High i on equi e-
men o g ow h, pho osyn hesis, and low-ligh acclima ion in
he coas al cyanobac e ium Synechococcus bacilla is.F on
Mic obiol 6: 561. h ps://doi.o g/10.3389/ micb.2015.00561.
Suzuki, H., Koyanagi, T., Izuka, S., Onishi, A., and
Kumagai, H. (2005) The yliA, -B, -C, and -D genes o
Esche ichia coli K-12 encode a no el glu a hione impo e
wi h an ATP-binding casse e. J Bac e iol 187: 5861–5867.
Taylo , F.J.R. (1982) Symbioses in ma ine mic oplank on.
Ann Ins Oceanog 58:61–90.
Thomas, J., Meeks, J.C., Wolk, C.P., Sha e , P.W., and
Aus in, S.M. (1977) Fo ma ion o glu amine om [
13
N]
ammonia, [
13
N]dini ogen, and [
14
C]glu ama e by he e o-
cys s isola ed om Anabaena cylind ica.J Bac e iol 129:
1545–1555.
Thompson, A.W., Fos e , R.A., K upke, A., Ca e , B.J.,
Musa , N., Vaulo , D., e al. (2012) Unicellula cyanobac e-
ium symbio ic wi h a single-celled euka yo ic alga. Sci-
ence 337: 1546–1550.
T ipp, H.J., Bench, S.R., Tu k, K.A., Fos e , R.A., Desany, B.
A., Niazi, F., e al. (2010) Me abolic s eamlining in a
open-ocean ni ogen-fixing cyanobac e ium. Na u e 464:
90–94. h ps://doi.o g/10.1038/na u e08786.
Tu k-Kubo, K.A., Fa nelid, H.M., Shilo a, I.N., Henke, B.,
and Zeh , J.P. (2017) Dis inc ecological niches o ma ine
symbio ic N
2
-fixing cyanobac e ium Candida us
A elocyanobac e ium halassa sublineages. J Phycol 53:
451–461. h ps://doi.o g/10.1111/jpy.12505.
© 2020 The Au ho s. En i onmen al Mic obiology published by Socie y o Applied Mic obiology and John Wiley & Sons L d.,
En i onmen al Mic obiology,22, 2027–2052
T anspo e s in dia om-cyanobac e ia symbioses 2051