Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses

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. 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