Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses

Minireview Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses Mercedes Nieves-Morión, 1 Enrique Flores 2 *and Rachel A. Foster 1 * 1 Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, 106 91, Sweden. 2 Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo Vespucio 49, Seville, E-41092, Spain. Summary In the open ocean, some phytoplankton establish symbiosis with cyanobacteria. Some partnerships involve diatoms as hosts and heterocystous cyanobacteria as symbionts. Heterocysts are specialized cells for nitrogen fixation, and a function of the symbiotic cyanobacteria is to provide the host with nitrogen. However, both partners are photosynthetic and capable of carbon fixation, and the possible metabolites exchanged and mechanisms of transfer are poorly understood. The symbiont cellular location varies from internal to partial to fully external, and this is reflected in the symbiont genome size and content. In order to identify the membrane transporters potentially involved in metabolite exchange, we compare the draft genomes of three differently located symbionts with known transporters mainly from model free-living heterocystous cyanobacteria. The types and numbers of transporters are directly related to the symbiont cellular location: restricted in the endosymbionts and wider in the external symbiont. Three proposed models of metabolite exchange are suggested which take into account the type of transporters in the symbionts and the influence of their cellular location on the available nutrient pools. These models provide a basis for several hypotheses that given the importance of these symbioses in global N and C budgets, warrant future testing. Introduction In vast expanses of open ocean environments concentrations of dissolved inorganic nitrogen are below analytical detection. Here, microorganisms capable of reducing dinitrogen (N 2 ), which comprises 78% of the atmosphere, are at an advantage and typically dominate. The process of N 2 fixation, or the reduction of N 2 to ammonium, is performed by a small and diverse group of bacteria and archaea (Young, 1992). Some N 2 fixing (diazotrophic) populations in the open ocean are symbiotic, with a few genera of microalgae, specifically diatoms as hosts and heterocystous cyanobacteria as symbionts (Foster and O’Mullan, 2008) (Fig. 1A–C). Collectively these symbioses are referred to as diatom diazotrophic associations (DDAs). DDAs are globally distributed and are considered major contributors to both N and carbon (C) cycles due to seasonal blooms with high N 2 and C fixation rates and rapid sinking (Mague et al., 1974; Venrick, 1974; Carpenter et al., 1999; Subramaniam et al., 2008; Karl et al., 2016). Despite their recognition as globally significant, there still remain large gaps in our understanding of DDAs, especially how the partners interact and acquire (and share) the elements necessary for metabolism and growth. Here, we analysed the available DDA symbiont genomes to identify membrane transporters potentially involved in interactions with the host partners. This allowed us to recognize target proteins for further investigation by genetic approaches, for instance by the heterologous expression and analysis of genes from the symbionts. The diatom diazotrophic associations The heterocystous cyanobacterial symbionts The heterocystous cyanobacterial symbionts of DDAs have been characterized by several genetic markers, including 16S rRNA, the nifH gene, which encodes the nitrogenase reductase component (Fe protein) of the nitrogenase complex for N 2 fixation, and the hetR gene, Received 17 January, 2020; revised 2 April, 2020; accepted 3 April, 2020. *For correspondence. E-mail efl[email protected]; Tel. +34954489523; E-mail rac[email protected]; Tel. 4608161207. © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Environmental Microbiology (2020) 22(6), 2027–2052 doi:10.1111/1462-2920.15013 which encodes a key regulator of cell differentiation in heterocystous cyanobacteria (Janson et al., 1999; Foster and Zehr, 2006). Based on these phylogenies the symbionts are related to other heterocystous cyanobacteria in the order Nostocales (e.g. Anabaena sp., Nostoc sp. and Calothrix sp.). Notably, the symbionts are more related between them than to any other known heterocystous cyanobacterium (Caputo et al., 2019). In spite of that, symbiont hetR and nifH sequences are relatively divergent (84% and 91% identity respectively) suggesting a high specificity in the partnerships (Janson et al., 1999; Foster and Zehr, 2006). In other words, one particular symbiont strain associates with one particular host genus, and the driver of the specificity is currently unknown (Janson et al., 1999; Foster and Zehr, 2006). Morphologically, the symbionts vary in terms of filament length and taper, and all possess terminal heterocysts. The symbionts vary in their cellular location: internal, partial and external. ‘Internal’symbionts have penetrated the diatom’s cytoplasm; ‘partial’(or ‘periplasmic’) refers to symbionts that reside between the diatom’s cytoplasmic membrane and frustule (outer silicified cell wall of diatoms); and ‘external’are symbionts that are attached to the surface of the diatom (Villareal, 1989, 1990; Caputo et al., 2019). Importantly, the external symbiont (Calothrix rhizosoleniae, CalSC01) can be grown independently of its host in the laboratory (Foster et al., 2010) (see Fig. 1D). Draft genomes are available for the heterocystous symbionts: Richelia intracellularis RintHH01 (internal, associated with Hemiaulus hauckii), 3.24 Mbp; R. intracellularis RintHM01 (internal, associated with H. membranaceus), 2.21 Mbp; R.intracellularis RintRC01 (partial, associated with Rhizosolenia clevei), 5.4 Mbp; and Calothrix rhizosoleniae CalSC01 (external, associated with Chaetoceros compresus), 5.97 Mbp (Hilton et al., 2013; Hilton, 2014). This shows that genome size is directly related to the cellular location of the symbionts. General features (size, GC content and percent coding) of the symbiont genomes are similar to other Nostocales, including free-living and facultative and obligate symbiotic strains of multicellular plants (e.g. Anabaena sp. PCC 7120, Nostoc punctiforme PCC 73102 and Nostoc azollae 0708 respectively; PRJNA244, PRJNA216 and PRJNA30807 respectively) (Meeks et al., 2001; Hilton et al., 2013). The R.intracellularis RintHM01 draft genome lacks several sequences expected of a full genome due to low sequencing coverage (Hilton et al., 2013), and therefore it was not compared further. The symbiotic diatoms Diatoms are single-celled eukaryotic plankton widely distributed in aquatic environments that contribute significantly (20%) to global primary production (Field et al., 1998). Most diatoms dominate coastal environments, where dissolved nutrients are high, and like other microalgae, diatoms can utilize nitrate and ammonium (Guillard and Kilham, 1977; Armbrust, 2009). In addition, diatoms possess a complete urea cycle (Allen et al., 2011). On the other hand, in oligotrophic marine Fig. 1. Epifluorescent images taken of wild populations (A–C) of the various Diatom Diazotroph Associations (DDAs) and the external symbiont Calothrix SC01 after isolation (D). Cells imaged in the field used a blue excitation filter (450–490 nm) to distinguish the heterocystous cyanobacterial symbionts (yellow-orange) from the diatom chloroplasts (red). A. One Rhizosolenia clevei-R. intracellularis (RintRC01) symbiosis. Note the longer filament of Richelia when associated with Rhizosolenia.B.Achainofthree Hemiaulus hauckii diatoms associated with Richelia intracellularis (RintHH01). C. A chain of >10 Chaetoceros compressus diatoms with Calothrix rhizosoleniae (CalSC01) attached to the outside. Note that the spines are not visible for the diatom and the taper of the Calothrix symbiont differs from that of Richelia.D.TheCalSC01isolate several months after isolation (imaged with blue excitation). Note the longer filaments when growing freely of its host diatom. Scale bars are approximately 10 μm. [Color figure can be viewed at wileyonlinelibrary.com] © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2028 M. Nieves-Morión, E. Flores and R. A. Foster environments, some diatoms form a symbiosis with cyanobacteria (Villareal, 1992; Foster and O’Mullan, 2008). In general, we know far less about the symbiotic host diatoms compared with their respective symbionts. The host diatoms differ dramatically in cell size (e.g. Hemiaulus hauckii,12–35 μm; H.membranaceus,30–70 μm; Rhizosolenia clevei,7–250 μm; Chaetoceros compressus,7–40 μm). The Hemiaulus spp. and Chaetoceros spp. hosts are capable of forming long chains (>50 cells), while R.clevei tend to be solitary. These observations raise questions on how/if symbiont metabolism (e.g. N 2 fixation) is influenced by host cell size, and whether/how substrates are also exchanged between symbiotic cells. The genetic identity of the hosts was only recently characterized for a few genetic markers and resulted in congruent phylogenies with the respective symbiont phylogenies suggesting co-evolution (Caputo et al., 2019). The symbiosis An important, interesting, and often challenging characteristic of any symbiosis is defining the function of each partner. In the DDAs the symbiont function is obvious, since only the symbiont can reduce N 2 , and the provision of fixed N to the diatom hosts has been shown on the cellular level (Foster et al., 2011), but the mechanism of N transfer is unstudied. In terrestrial symbioses involving heterocystous cyanobacteria, the symbiotic populations often reside in darkened cavities and rely (heterotrophically) on their host plants for reduced C substrates (Söderbäck and Bergman, 1993). In DDAs, both partners are photosynthetic, and hence capable of C fixation, but the possible exchange and transfer of C substrates is unknown. Nonetheless, interestingly, a recent cellular model for DDAs estimates that 25% of C fixed by the host is transferred to the symbiont, since the C requirement by the symbiont for N 2 fixation is higher than the fixation of C predicted from its own photosynthesis (Inomura et al., 2020). Since all DDAs have evaded long-term isolation (Villareal, 1989, 1990), it is difficult to study experimentally how the partners interact, share and potentially compete for substrates. Here, we have identified various candidate transporters in the symbiotic R.intracellularis/C.rhizosoleniae (hereafter Richelia/Calothrix) draft genomes by comparison mainly to those of model heterocystous cyanobacteria. We have focused on transporters for C, N and some other elements (e.g. iron, phosphorus and sulfur) that are important for the basis of the partnership. Note on methodology To identify particular gene products in the DDA symbionts, we have performed BLASTp analyses (Altschul et al., 1997). We generally used proteins of known function from Anabaena sp. PCC 7120 (hereafter Anabaena) as queries, although in some particular cases well-characterized proteins from other sources, mainly filamentous cyanobacteria (Nostoc punctiforme ATCC 29133, Trichodesmium erythraeum IMS101) were used. The symbionts are also predicted to have some membrane proteins not found in other cyanobacteria, and such membrane proteins were compared with transporters from other biological sources. To define orthologues, we have followed conservative criteria. Thus, generally, we checked that the symbiont’s protein was of approximately the same length as the query protein, which, combined with significant similarity, gave very low Expect values (e.g. <10 −50 to <10 −150 [indicated as <e-50 or <e-150] for proteins of 150–500 amino acid residues approximately). On the other hand, comparisons with Expect values of, e.g. >10 −20 (indicated as >e-20) were considered to denote similar but not necessarily orthologous proteins. This is especially common in membrane proteins that can belong to the same family of transporters but recognize different substrates. The alignments, using Clustal O (Madeira et al., 2019), of some examples of proteins that give significant similarity to be considered orthologues are presented in Figs. S1–S5. Outer membrane translocators Cyanobacteria are diderm bacteria, i.e. they contain an outer membrane (OM) outside of the cytoplasmic membrane (Hahn and Schleiff, 2014). The OM characteristically contains numerous proteins that take a β-barrel conformation, and the OM outer leaflet contains lipopolysaccharide (LPS) as a characteristic component. Although cyanobacterial LPS is not identical to that of the best-studied Gram-negative bacteria (Hahn and Schleiff, 2014), the genome of the heterocystous symbionts of DDAs encode a number of OM insertion proteins (BamA family proteins) as well as proteins involved in LPS synthesis and transport that collectively suggest the presence of a mature OM in the symbionts (Table S1). Hence, materials transferred between the diatom and the symbiont in DDAs must traverse the OM. Substrate translocation across the OM generally takes place through porins, which are trimeric β-barrel proteins (Yamashita and Buchanan, 2010). There are currently about 90 recognized families of β-barrel porins in the Transporter Classification Database [TCDB (Saier Jr et al., 2016); http://www.tcdb.org/], and cyanobacteria possess characteristic porins that constitute one of those families (TCDB #1.B.23). A characteristic feature of the cyanobacterial porins is the presence of an N-terminal domain with similarity to ‘S-layer homology’ domains that may connect the OM to the peptidoglycan © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 Transporters in diatom-cyanobacteria symbioses 2029 layer. These cyanobacterial porins show similarity to the OprB-type porins that mediate transfer of saccharides in bacteriasuchasPseudomonas aeruginosa (van den Berg, 2012). Indeed, one of these porins from the facultative symbiont N.punctiforme (Npun_R5320) has been shown to facilitate uptake of glucose and fructose into the cyanobacterium (Ekman et al., 2013). Nonetheless, it appears that these porins fulfil classical porin function generally permitting the transfer of small molecules and ions through the OM (Hahn and Schleiff, 2014). The Anabaena genome encodes seven OprB-type porins, of which All4499 and Alr4550 appear to be particularly abundant (Moslavac et al., 2007b; Nicolaisen et al., 2009). Using All4499 and Npun_R5320 as queries, we found four homologues in CalSC01 and two in each of the RintRC01 and RintHH01 genomes (Table 1). Thus, the external, facultative symbiont CalSC01 is more similar to Anabaena in number of porins than the partial and internal symbionts (RintRC01 and RintHH01, respectively). In the latter two, one of the porins (RintRC01_7172, RintHH01_240) may be additionally subjected to regulation by zinc (Zn), since the DNA sequence upstream of the encoding gene contains a possible binding site for the Zur transcription factor, which is involved in Zn homeostasis (Sein-Echaluce et al., 2015). The other OM porin (RintRC01_1265, RintHH01_6530), which shows the highest similarity to All4499 and Npun_R5320 (Table 1), may represent, therefore, the general porin of the endosymbionts. The Anabaena genome encodes an unusually high number of TonB-dependent OM transporters (Hahn and Schleiff, 2014), which mediate uptake through the OM of iron (Fe) complexes or vitamin B 12 . This uptake is energized by the interaction of a periplasmic domain of the TonB-dependent OM transporter with the inner membrane protein TonB (Yamashita and Buchanan, 2010). Neither RintRC01 nor RintHH01 appear to bear any TonB- dependent transporter, whereas CalSC01 shows two TonB-dependent Fe complex transporters (see Table 4 below) and one TonB-dependent vitamin B 12 transporter (Table 1). This points to a different strategy for trace element uptake in the symbiont strains (see below) that is likely influenced by the symbiont location: the nutrient pool of the external symbiont (CalSC01) is that of the Table 1. Outer membrane (OM) proteins encoded in the DDA symbiont genomes. Query CalSC01 (external) RintRC01 (partial) RintHH01 (internal) Protein/function All4499 2790721588 Ga0265390_12245 (1.5e-78) RintRC_1265 (0.0) RintHH _6530 (0.0) OM porin OprB 2790720937 Ga0265390_11703 (2.3e-81) RintRC_7172 Zn regulated (?) (1e-60) RintHH _240 Zn regulated (?) (1e-112) 2790720522 Ga0265390_113824 (6.5e-85) 2790722855 Ga0265390_13252 (6.5e-95) Npun_R5320 2790721588 Ga0265390_12245 (1e-129) RintRC_1265 (1e-126) RintHH_6530 (1e-128) OM porin OprB 2790720937 Ga0265390_11703 (1e-133) RintRC_7172 Zn regulated (?) (2e-54) RintHH_240 Zn regulated (?) (1e-107) 2790720522 Ga0265390_113824 (9e-134) 2790722855 Ga0265390_13252 (5e-127) Alr4028-Alr4029 2790719984 Ga0265390_110111 (2e-122; 2e-119) nd nd Vitamin B 12 transporter Alr2887 (HgdD) 2790721775 Ga0265390_124212 (0.0) RintRC_2765 (0.0) RintHH_21590* (0.0) TolC-like OM exporter ORFs from the symbionts (CalSC01, RintRC01, RintHH01) identified in BLASTp analysis (Expect values indicated in parenthesis) using the indicated protein from Anabaena (All, Alr) or Nostoc punctiforme (Npun) as a query. An asterisk indicates a possibly incomplete sequence; nd, not detected; (?), Zn-regulation is possible but not yet demonstrated. © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2030 M. Nieves-Morión, E. Flores and R. A. Foster surround, while the endosymbionts lack direct contact with the outside water column and are limited (if not reliant) to the host cytoplasm (RintHH01) or region between frustule and the host cytoplasmic membrane (RintRC01). TolC-type exporters are trimeric proteins that make an OM channel in the form of a β-barrel and a periplasmic channel formed by α-helixes (Yamashita and Buchanan, 2010). TolC exporters are commonly able to function together with several different plasma membrane exporters thus mediating the export of different substances from the cell. The Anabaena genome encodes only one TolC-like protein, HgdD (Alr2887), which can participate in the export of different substances including heterocyst-specific glycolipids (Moslavac et al., 2007a) and toxic compounds such as ethidium bromide (Hahn et al., 2012). Like Anabaena, each of the three symbionts has only one TolC-like protein (Table 1), which likely is involved in multiple export functions. Cytoplasmic membrane transporters: ABC and MFS transporters Cytoplasmic membrane transporters are currently classified into numerous phylogenetically distinct superfamilies and families (see TCDB [Saier Jr et al., 2016]; http:// www.tcdb.org/). Two such superfamilies with numerous protein members commonly referred to in this article are the ATP-Binding Cassette transporter superfamily (ABC; TCDB #3.A.1) and the Major Facilitator Superfamily (MFS; TCDB #2.A.1). The ABC transporter superfamily is one of the largest families among transport systems with a wide distribution in all three domains of life. ABC transporters can be divided into exporters, found in both eukaryotes and prokaryotes, and importers, which, with a few eukaryotic exceptions, are largely found in prokaryotes mediating the uptake of nutrients (Wilkens, 2015). ABC importers generally contain one periplasmic solute-binding protein (SBP), which binds the ligand in the periplasm for delivery to the appropriate membrane transporter complex; two integral membrane proteins (transmembrane domains [TMDs]) that form the solute-translocation pathway; and two nucleotide-binding proteins or domains (NBD) that hydrolyze ATP in the cytoplasm (Cui and Davidson, 2011). MFS proteins are the largest family of secondary transporters and allow the transport of a large variety of ions and solutes across membranes (Reddy et al., 2012). They comprise facilitators, symporters and antiporters, which move substrates across membranes via facilitated diffusion, co-transport or exchange respectively (Yan, 2015). MFS proteins are integral membrane proteins that generally possess 12 or 14 transmembrane segments (TMSs) (Reddy et al., 2012). C-compound transporters Cyanobacteria are mainly photoautotrophic, fixing carbon dioxide (CO 2 ), but many strains can also assimilate some organic compounds including sugars, mainly glucose, fructose and sucrose (Rippka et al., 1979). Diatoms are also predominantly photosynthetic and many are facultative heterotrophs (Hellebust and Lewin, 1977). In both diatoms and cyanobacteria, CO 2 fixation is catalysed by ribulose- 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Since both partners of DDAs possess RubisCO, a key question for the symbionts (especially for the internal and partial symbionts: RintHH01 and RintRC01) is how they obtain and/or compete for C, especially to cope with the high rate of N 2 fixation. Inorganic carbon Similar to many other autotrophs, both cyanobacteria and diatoms have developed C Concentration Mechanisms (CCMs) to increase the concentration of CO 2 around RubisCO (Raven et al., 2008). The CCMs of diatoms are considered genetically diverse and include high interspecies variations (Young et al., 2016; Shen et al., 2017). In general, cyanobacterial and diatom CCMs show functional and compositional similarities, and both possess subcellular compartments for housing RubisCO, i.e. carboxysomes in cyanobacteria and pyrenoids in diatoms, but their CCM genes are not homologous (Young and Hopkinson, 2017). The CCMs of diatoms and cyanobacteria are composed of bicarbonate (HCO 3 − ) transporters and carbonic anhydrases (CAs) that mediate the interconversion of CO 2 and HCO 3 − , and cyanobacteria possess, in addition, CO 2 transporters (Kaplan and Reinhold, 1999; Badger et al., 2006; Cameron et al., 2014). CAs vary in number, subtype (α,ß,γ, others) and localization in model diatoms such as Phaeodactylum tricornutum and Thalassiosira pseudonana (Hopkinson et al., 2016). The DDA symbionts contain the ccmKLMNO operon (with two copies of ccmK in each of RintHH01, RintRC01 and CalSC01) for β-carboxysomes and the gene for carboxysomal CA, which are essential for carboxysome functionality. The uptake systems for CO 2 and HCO 3 − species (inorganic C, Ci) in cyanobacteria include ABC and MFS transporters for HCO 3 − and a specialized type of NAD(P)H dehydrogenase (NDH-1; photosynthetic complex I) that functions in trapping CO 2 (Price, 2011; Schuller et al., 2020). In diatoms both CO 2 and HCO 3 − enter the cell from the environment. HCO 3 − is actively transported by membrane-embedded transporters, while CO 2 relies on a diffusive flux mediated by the ‘chloroplast pump’, the active pumping of HCO 3 − to the chloroplast (Hopkinson et al., 2011). © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 Transporters in diatom-cyanobacteria symbioses 2031 The Anabaena genome encodes two CO 2 uptake systems of the NDH-1 type, the NDH-1 3 and NDH-1 4 protein complexes, and three HCO 3 − importers, including one ABC transporter (high affinity) and two sodium-dependent transporters (SbtA, high affinity; BicA, low affinity) (Herrero and Flores, 2019), whereas the symbiont’sCi uptake systems appear to be influenced by the diatom host. The chloroplast pump mechanism and subsequent distribution of Ci is particularly interesting in relation to symbiont cellular location and their respective genome content for Ci transport. Thus, none of the endosymbionts has genes encoding high-affinity HCO 3 − transporters, whereas the external symbiont CalSC01 has a gene encoding an MFS high-affinity HCO 3 − transporter, SbtA (Table 2; Fig. 2A). The absence of the high-affinity HCO 3 − transporters in the internal symbionts may reflect the influence of the ‘chloroplast pump’and CA mechanisms of its host, which drive high HCO 3 − concentrations within the vicinity of RintRC01 and RintHH01 symbionts. Low HCO 3 − concentrations are common in the surface ocean and could drive the retention of the high-affinity HCO 3 − transporter (SbtA) in the external symbiont, CalSC01. Thus, an open and important question in each symbiosis is the uptake kinetics for Ci, and how the symbiont cellular location influences the activity and partner interaction. Interestingly, the three symbionts bear genes encoding proteins similar to the SulP-family HCO 3 − transporter BicA. CalSC01 and RintRC01 each shows two genes encoding BicA-like proteins, whereas RintHH01 shows the possible bicA gene fragmented in four consecutive sequences, perhaps reflecting incomplete sequencing coverage or, alternatively, that it is a gene in the process of degeneration (Table 2, see also Table 5 below). Because SulP transporters for HCO 3 − and sulfate are very similar to each other, experimental tests are needed to define the actual substrate of those proteins. Additionally, each of the three symbionts encodes the components of an NDH-1 type (NDH-1 3 )ofCO 2 uptake system. Whereas CalSC01 is expected to fixCO 2 (at least when growing independently of the diatom), the presence of RubisCO, carboxysome genes, an NDH-1 complex and, likely, a BicA transporter in the internal and partial symbionts support the idea that they can also fixCO 2 , which is consistent with high rates of C fixation measured in field populations (Carpenter et al., 1999; Subramaniam et al., 2008). Organic carbon Despite the fact that the main trophic mode of cyanobacteria is photoautotrophy, some strains, including some heterocystous types that engage in symbioses with terrestrial plants, have the capacity of sugar uptake supporting heterotrophic growth. Thus, GlcP (MFS transporter for glucose) and Frt (ABC transporter for fructose) in symbiotically competent N.punctiforme (Ekman et al., 2013) and Frt in A.variabilis (Ungerer et al., 2008) are well-known examples of transporters for some kind of heterotrophic growth (Wolk and Shaffer, 1976; Summers et al., 1995). Additionally, mixotrophic growth with different C sources (Malatinszky et al., 2017) and the identification of components of ABC glucoside transporters involved in sugar-stimulated growth in Anabaena have been recently reported (Nieves-Morión and Flores, 2018). Surprisingly, no homologues to GlcP or Frt proteins are encoded in any of the diatom symbionts. However, the three symbionts may encode the ABC transporter Gls (Table 2; Fig. 2A), which in Anabaena functions in the uptake of glucosides (Nieves-Morión and Flores, 2018). CalSC01 bears two homologous proteins to each GlsQ and GlsP (TMDs), which might suggest the function of two ABC-glucoside transporters (Fig. 2A) as it has been reported in Anabaena (Nieves-Morión and Flores, 2018). In contrast, due to the low similarity of the putative SBP of either Richelia strain to the Anabaena SBP GlsR (Table 2), experimental confirmation of their substrates is required. The Anabaena Alr3705 MFS symporter is a possible glycoside transporter, although it could not experimentally transport glucosides in Anabaena (Nieves-Morión et al., 2017). Whereas RintHH01 and RintRC01 bear a homologous protein, CalSC01 contains two possible homologues for Alr3705 (Table 2; Fig. 2A). The presence of these transporters raises the possibility that the symbionts can assimilate a sugar such as sucrose, which in Anabaena has a principal role in the transfer of reduced C from vegetative cells to heterocysts (López-Igual et al., 2010; Nürnberg et al., 2015). Consistently, the three symbionts encode invertases, which are enzymes that irreversibly split sucrose into glucose and fructose (Table 2). In cyanobacteria, sucrose is synthesized by the combined action of sucrose-phosphate synthase (Sps) and sucrose-phosphate phosphatase (Spp), which constitute an irreversible pathway of sucrose biosynthesis, or by the reversible enzyme sucrose synthase (Sus) (Salerno and Curatti, 2003). Whereas RintRC01 and CalSC01 bear genes encoding Sps and Spp, RintHH01 lacks these enzymes or the alternative sucrose synthase. Hence, RintHH01 has the capacity to hydrolyze but not synthesize sucrose. Therefore, a mixotrophic growth, fixing CO 2 and assimilating sugars provided by the host, is possible for the symbionts, and this could be especially relevant in the case of the internal symbiont RintHH01. While CalSC01 and RintRC01 encode invertases InvA and InvB, the only invertase present in RintHH01 is most similar to Anabaena InvB, which is heterocyst-specific (López-Igual et al., 2010). It would, therefore, be of great © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2032 M. Nieves-Morión, E. Flores and R. A. Foster Table 2. Carbon uptake transporters and related proteins encoded in the DDA symbiont genomes. Query CalSC01 (external) RintRC01 (partial) RintHH01 (internal) Protein/function Alr2877 (CmpA) Alr2878 (CmpB) Alr2879 (CmpC) Alr2880 (CmpD) nd nd nd ABC Bicarbonate transporter: CmpA (SBP) CmpB (TMD) CmpC (NBD) CmpD (NBD) All2134 2790721,442 Ga0265390_12075 (3e-178) nd nd High-affinity bicarbonate:Na + symporter, SbtA BicA from Synechococcus sp. PCC 7002 2790719465 Ga0265390_10577 (0.0) RintRC_3892 (0.0) RintHH_3960# (4e-12) RintHH_3970# (1e-103) RintHH_3980# (3e-21) RintHH_3990# (5e-32) Low-affinity, SulP-family bicarbonate: Na + symporter, BicA (two similar proteins in Anabaena: All1304, Alr1635)2790720482 Ga0265390_11353 (0.0) RintRC_4851 (0.0) Alr4156 (NdhF) 2790722617 Ga0265390_130615 (0.0) Downstream genes: 2790722616 2790722615 RintRC_4170 (0.0) Downstream genes: RintRC_4169 RintRC_4168 RintHH_18810 (0.0) Downstream genes: RintHH_18800 RintHH_18780 Three-gene clusters that encode an NDH-1 type (NDH-1 3 )ofCO 2 uptake complex Alr2532 (GlsQ) 2790723713 Ga0265390_139111 (TMD, 3e-61) 2790721923 Ga0265390_12555 (TMD, 6.9e-56) RintRC_0814 (TMD, 9e-62) RintHH_290 (TMD, 8e-52) RintHH_15070 (TMD, 9e-47) Glucoside ABC transporter 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) RintRC_6152 (SBP, 4e-11) RintHH_17430 (SBP, 9e-21) Alr4781 (GlsC) 2790719311 Ga0265390_10456 (NBD, 8e-118) RintRC_6050 (NBD, 1e-92) RintHH_19540 (NBD, 1e-166) All1823 (GlsD) 2790721447 Ga0265390_12083 (NBD, 2e-117) Alr3705 2790723960 Ga0265390_14405 (1.3e-71) 2790720797 Ga0265390_11606 (3.18e-169) RintRC_2529 (0.0) RintHH_3250** (1e-39) RintHH_3260** (1e-174) Glycoside-Pentoside-Hexuronide: Cation MFS symporter Alr1521 2790722378 Ga0265390_12903 (0.0) RintRC_4014 (0.0) nd Invertase InvA Alr0819 2790722061 Ga0265390_12672 (0.0) RintRC_3156 (0.0) RintHH_3860 (0.0) Invertase InvB (heterocyst-specificin Anabaena) All3028 (SBP) 2790720991 Ga0265390_11733 (SBP, 0.0) RintRC_2147 (SPB, 1e-134) RintHH_12690 (SBP, 0.0) TRAP carboxylate transporter. Consists of SBP, small TMD (DctQ), and large TMD (DctM) Alr3026 (small TMD) 2790722400 Ga0265390_12928 (TMD small, 8e-59) RintRC_3100 (TMD small, 8e-93) RintHH_15260 (TMD small, 1e-83) Alr3027 (large TMD) 2790722399 Ga0265390_12927 (TMD large, 0.0) RintRC_3101 (TMD large, 0.0) RintHH_15250 (TMD large, 0.0) (Continues) © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 Transporters in diatom-cyanobacteria symbioses 2033 interest to locate and determine the functionality of InvB in the endosymbiont strains RintRC01 and RintHH01. Tripartite ATP-independent periplasmic (TRAP) transporters are generally carboxylate transporters (Mulligan et al., 2011). Anabaena contains three genes encoding a TRAP transporter that mediates the uptake of pyruvate and other monocarboxylate 2-oxoacids (Pernil et al., 2010). Genes encoding homologues to the Anabaena TRAP transporter proteins can be detected in the three symbionts (Table 2; Fig. 2A). A recent field investigation reported the co-expression of the RintRC01 gene for the TRAP solute receptor and EAMA-like transporters in the diatom host Rhizosolenia (Harke et al., 2018). EAMA transporters belong to the Drug/Metabolite Transporter superfamily (TCDB #2.A.7), are associated with triosephosphate translocators located on the plastid membrane, and function in the model diatom P.tricornutum to export carbohydrates derived from photosynthesis (Moog et al., 2015). Hence, it was suggested that the diatom host was supplying sugar substrates to the symbiont (Harke et al., 2018). However, considering that the RintRC01 resides outside the host diatom cytoplasm in the periplasmic space, an additional transport mechanism across the host cell membrane is required. Finally, if indeed reduced C substrates are transported from the host, other candidates for simple organic molecule transport are a predicted acetate permease present in CalSC01 and RintRC01, and a predicted proline transporter in CalSC01 and RintHH01 (Table 2). Identifying the substrate(s) of these transporters would be of great interest to understand the physiology of the DDAs, especially in the case of the internal symbiont (RintHH01), in which substrate(s) can be taken up directly from the host diatom’s cytoplasm. N-compound transporters Cyanobacteria and diatoms have the ability to assimilate inorganic and simple organic N compounds, and some cyanobacteria fix atmospheric N 2 . However, the environments where the DDAs are reported are notoriously N deplete, since concentrations of inorganic and organic N are below analytical detection. Hence, the host diatoms are dependent on the symbionts for N. Primary N sources Nitrogenase, the enzyme responsible for N 2 fixation, is inactivated in the presence of O 2 , and therefore N 2 -fixing cyanobacteria have to separate spatially or temporally N 2 fixation and oxygenic photosynthesis (Flores et al., 2015). Under conditions of combined-N deprivation, cyanobacteria of the order Nostocales (including, e.g. Anabaena,Calothrix,Nostoc and Richelia) produce differentiated cells called heterocysts where N 2 fixation takes place. A common observation in terrestrial-based symbioses with heterocystous cyanobacteria is that heterocyst frequency (normally about 7%-10%) increases (e.g. to 17%–60%) when the symbionts are living in symbioses rather than in a free-living state (Meeks, 2009). The Richelia/Calothrix symbionts, however, are unique symbionts in that they possess terminal heterocysts, and therefore cannot increase the number of heterocysts, but rather maintain a high ratio of heterocysts to vegetative cells if the number of vegetative cells is small. In RintHH01 and CalSC01 filaments the latter is true, since they typically have 1–3 vegetative cells, however, filaments tend to be longer in the case of RintRC01 (Fig. 1; see also Villareal, 1992). Longer filaments in RintRC01 and notably in free-living CalSC01 imply more vegetative cells performing C fixation and hence a higher C supply from their own photosynthesis. It is currently unknown how filament length is regulated in these organisms. Heterocyst formation involves morphological and metabolic changes that allow the expression and function of nitrogenase (Flores et al., 2019b). One of these morphological changes consists in the deposition of two envelope layers outside of the OM: the glycolipid layer (HGL) Table 2. Continued Query CalSC01 (external) RintRC01 (partial) RintHH01 (internal) Protein/function ActP of Rh.capsulatus a 2790722390 Ga0265390_129110 (e-45) RintRC_5151 (e-49) nd Cation/acetate permease PutP of B.subtilis b 2790719613 Ga0265390_10674 (4e-61) nd RintHH_18700 (e-67) Nutritional high-affinity sodium/proline permease PutP a.Rh., Rhodobacter. b.Bacillus. ORFs from the symbionts (CalSC01, RintRC01, RintHH01) identified in BLASTp analysis (Expect values indicated in parenthesis) using the indicated protein from Anabaena (unless indicated otherwise) as a query. Double asterisks designate genes that are split; Hash indicates genes that are fragmented; nd, not detected. © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2034 M. Nieves-Morión, E. Flores and R. A. Foster that reduces the permeation of O 2 , and the polysaccharide layer (HEP) that provides protection (Nicolaisen et al., 2009). The HGL is composed of fatty alcohols glycosidically linked to sugar residues. Whereas HGLs made of C6 sugar have been found in freshwater freeliving cyanobacteria and some strains of benthic Calothrix (Bauersachs et al., 2009), Richelia (RintHH01, RintRC01) contains novel HGLs with a C5 sugar, ribose, rather than a C6 sugar (Schouten et al., 2013; Bale et al., 2015). Recently, a novel HGL was characterized for a new Calothrix sp. CCY1611 isolated from the North Atlantic (Bale et al., 2018); it is unknown if CCY1611 is similar and/or genetically related to CalSC01, and the HGLs of CalSC01 are uncharacterized. The C5 sugar found in RintRC01 and RintHH01 might be explained by an adaptation of the endosymbiont to the high O 2 concentration within the diatom host (Walsby, 1985). This adaptation illustrates the importance of N 2 fixation in the DDAs, and details of the production of the heterocyst envelope in the symbionts will merit specific research in the future. In addition to N 2 , heterocystous cyanobacteria can assimilate from the environment various sources of N including ammonium, nitrate, nitrite, urea and some amino acids (Herrero and Flores, 2019). The intracellular conversion of these compounds to ammonium is required for N incorporation into carbon skeletons to produce organic N compounds. Ammonium is a preferred N source for many organisms and its uptake in environments with low external concentrations involves the Amt Fig. 2. Upset diagram showing the intersection of each set of transporters in the model cyanobacterium Anabaena sp. PCC 7120 and the DDA symbionts Calothrix CalSC01, Richelia RintRC01, and Richelia RintHH01 for (A) carbon, (B) nitrogen, (C) iron and (D) phosphorus. The C, N, Fe and P transporter content in CalSC01 is similar to Anabaena and higher in number than transporters present in the endosymbionts RintRC01 and RintHH01. A. Note the presence of SbtA, Gls_2 and MFS_2 in the external symbiont and their absence in the endosymbionts. TRAP carboxylate transporter, NDH-1 3 and the Gls_1 are present in the three symbionts. CalSC01 bears two homologous proteins each to GlsQ and GlsP (Table 2) suggesting the presence of two glucoside ABC transporters (Gls_1 and Gls_2) as in Anabaena. We refer to the second transporter as that containing Ga0265390_12555 and _117612. Two possible MFS transporters for glycosides are identified in CalSC01: MFS_1 (Ga0265390_14405) and MFS_2 (Ga0265390_11606) (see Table 2). B. Note the ammonium transporter (Amt) and nitrate/nitrite MFS transporter are only present in the external symbiont. The three symbionts bear a possible amino acid permease (APC superfamily), which is not present in Anabaena. C. The three symbionts bear the ferric iron ABC transporter and the ferrous iron permease EfeU, as in Anabaena, whereas uncertainty of SchE in the symbionts is designated with an asterisk (see the text). D. Note the presence of the phosphonate ABC transporter and a second phosphate ABC transporter (Phosphate ABC transporter 2) in the external symbiont CalSC01. We refer to the second phosphate ABC transporter 2 as that containing Ga0265390_11512, _12441, _12442, _12443, _11047 (see Table 5). © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 Transporters in diatom-cyanobacteria symbioses 2035 Stevanovic et al., 2011). Once Fe(III) is bound to the siderophore in the medium, the complex is taken up by the OM protein SchT (Alr0397), a TonB-dependent transporter (Nicolaisen et al., 2008), and then translocated into the cytoplasm by the ABC transporter FhuBCD (Stevanovic et al., 2011; Rudolf et al., 2016). Additionally, Anabaena expresses other TonB-dependent transporters involved in Fe and copper (Cu) uptake including IutA2 (Alr2581; Rudolf et al., 2016) and IacT (All4026; Nicolaisen et al., 2010). Only the external symbiont CalSC01 contains homologues to siderophore biosynthesis genes (see Table S3), although its genome encodes a protein with only low similarity to the SchE exporter (Table 4; Fig. 2C). Because bacteria often express receptors for siderophores that they do not produce or for Fe contained in their hosts (Braun and Killman, 1998; Ratledge and Dover, 2000), CalSC01 homologues to schizokenin receptor SchT and TonB- dependent transporters IutA2 and IacT (Table 4; Fig. 2C) might be involved in the uptake of siderophores secreted by CalSC01 itself as well as by other bacteria. CalSC01 bears also the ABC-transporter FhuBCD required for Fe 3+ -siderophore acquisition (Table 4; Fig. 2C). Because siderophores are secreted in response to low dissolved Fe concentration (Wilhelm and Trick, 1994), these transporters may allow CalSC01 to maintain Fe homeostasis in a low Fe concentration environment. Moreover, CalSC01 is expected to acquire Fe through other pathways, as supported by the presence of another Fe +3 ABC transporter (homologue to E.coli FutABC), a Fe 3+ or ferricdicitrate ABC transporter (homologue to E.coli FecBCDE), ahigh-affinity Fe 2+ /Pb 2+ permease, and (possibly) a FeoB-like ferrous iron transporter (Table 4; Fig. 2C). Finally, although eukaryotes cannot produce siderophores, some, including diatoms, can access the bound Fe from siderophores (Amin et al., 2009; Kazamia et al., 2018); perhaps a similar strategy occurs in the Chaetoceros- Calothrix symbiosis and the host diatom uses its symbiont’s siderophore synthesis for its own Fe acquisition. RintHH01 and RintRC01 symbionts lack any TonB- dependent transport system and the ABC transporter FhuBCD required for ferric-siderophore acquisition, but their genomes encode homologues of a ferric iron ABC transporter (FutABC homologue), the high-affinity Fe 2+ /Pb 2+ permease and only in RintRC01, possibly the FeoB-like transporter (Table 4; Fig. 2C). Residing inside the diatom, the concentration of Fe might be sufficient and apparently, both RintHH01 and RintRC01 symbionts evolved to incorporate inorganic or organic forms of Fe(III) and Fe(II) by pathway(s) different to the siderophore uptake systems, as also reported for Synechocystis sp. PCC 6803 and T.erythraeum IMS101 (Roe and Barbeau, 2014; Jiang et al., 2015). The presence of proteins similar to schizokinen exporter SchE in the three DDM symbionts (Table 4) should be taken with caution, since those proteins are indeed most similar to bacterial AmpG (Expect values <e-100), which is involved in the uptake of cell wall degradation products. In summary, whereas endosymbionts RintRC01 and RintHH01 appear to be non-siderophore-utilizing cyanobacteria having other pathways for Fe homeostasis, CalSC01, living externally attached, appears to be able to use siderophores and have more strategies for Fe uptake. Sulfate ABC transporter and sulfate permease Sulfate is a macronutrient, which is required for photosynthetic organisms. Sulfur is present in proteins, lipids, electron transport components and many cellular metabolites. The limited intracellular storage of S implies the uptake, mainly in the form of sulfate anion, from the environment through specific transporters. The main prokaryotic transporter involved in sulfate uptake is in the SulT group of ABC transporters (TCDB #3.A.1.6). This transporter, Sbp/CysPTWA, is responsible for sulfate and thiosulfate uptake in prokaryotes (Saier Jr et al., 1999). It is composed of two TMD proteins, CysT and CysW, the NBD CysA protein (homodimer-forming CysA) and the sulfatebinding (Sbp) and thiosulfate-binding (CysP) periplasmic SBPs (Hryniewicz et al., 1990; Sirko et al., 1990). Table 5. Continued Query CalSC01 (external) RintRC01 (partial) RintHH01 (internal) Protein/function (NBD, 7e-169) _113152 (TMD) 2790720875 Ga0265390_11654 (TMD, 4e-153) _113151 (TMD) 2790720874 Ga0265390_11653 (TMD, 1e-165) ORFs from the symbionts (CalSC01, RintRC01, RintHH01) identified in BLASTp analysis (Expect values indicated in parenthesis) using the indicated protein from Anabaena (unless indicated otherwise) as a query. Expect values in Anabaena that correspond to comparison to the E.coli proteins are provided in parenthesis. An asterisk indicates a possibly incomplete sequence; nd, not detected. © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2042 M. Nieves-Morión, E. Flores and R. A. Foster Anabaena and the three symbionts bear the genes that encode the components of this transporter (Table 5), which supports the idea that they can take up sulfate. Proteins of the SulP family are permeases that can transport several different anions including sulfate (TCDB #2.A.53). Anabaena and the three symbionts contain several proteins of this family, some of which could be sulfate transporters, whereas others could be bicarbonate transporters as they are most similar to the bicarbonate transporter BicA from oceanic cyanobacteria (Price et al., 2004) mentioned earlier (Tables 2 and 5). The identification of the substrate transported by each particular SulP-family protein will be of much interest and requires further investigation. Phosphate and phosphonate transporters Similar to Fe, phosphorus (P) is often a limiting nutrient in marine systems since it can be found at low concentrations or even in forms not biologically available (Schindler, 1977; Karl et al., 2001). The phosphorus compounds and strategies for acquisition under P limitation have been characterized in some model systems, and some progress has recently been made on marine phytoplankton (reviewed in Lin et al., 2016). The preferred form of P for phytoplankton is inorganic phosphate (Pi). However, under the chronically low Pi conditions typical of the oligotrophic ocean where DDAs reside, some phytoplankton utilize dissolved organic phosphorus (DOP) substrates (Cui et al., 2015) and/or reduce their cellular P quotas by using non-P lipids in their lipid membranes (Van Mooy et al., 2009). DOP utilization has been less studied in diatoms, and most work to date has used alkaline phosphatase activity assays as indicative of DOP utilization or, recently, quantitative profiling of transcripts and proteins under P deficient conditions (Perry, 1976; Dyhrman et al., 2006, 2012). In the DDA hosts, it is not known how or which P substrates are utilized. Cyanobacteria have evolved mechanisms and regulatory adaptations to acquire Pi since it is a crucial requirement to fixN 2 (Bardin et al., 1996) and maintain growth. Two major transporters for Pi acquisition in bacteria are the PhoT and PiT systems. The PhoT transporters (Phosphate Uptake Transporter Family; TCDB #3.A.1.7) are high-affinity ABC transporters that are normally expressed in cells growing under low-Pi concentrations (Rao and Torriani, 1990). The E.coli PhoT system Pst comprises a periplasmic SBP (PstS), two TMD proteins (PstA and PstC) and the NBD protein (PstB). On the other hand, proteins in the PiT (Inorganic Phosphate Transporter) family (TCDB #2.A.20) normally consist of 10–12 TMSs and mediate transport of Pi—complexed with a metal divalent cation—in a symport mechanism with H + or Na + ions (Jackson et al., 2008). In E.coli, PitA and PitB are low-affinity transport systems that function when the external Pi concentration is higher than 20 μM, which represses the Pst system (Rao and Torriani, 1990). Anabaena contains two PhoT (All4575 to All4572 and All0911 to All0907) and two PiT (Alr2336 and Alr3096) transporters. Each of the three symbionts contains the components of at least one PhoT ABC transporter that is most similar to the Anabaena All4575 to All4572 transporter (Table 5; Fig. 2D). Whereas RintRC01 and RintHH01 bear homologues to the Anabaena PiT transporter All2336, CalSC01 lacks this Pi transporter, as do several freshwater strains and most marine picocyanobacteria (Su et al., 2007; Scanlan et al., 2009) (Table 5; Fig. 2D). Nevertheless, only the CalSC01 symbiont contains an extra gene cluster and two independent genes that together may encode another PhoT family ABC transporter (Table 5; Fig. 2D), resembling the duplicate or multiple Pst systems found in some freshwater and terrestrial cyanobacterial strains (Pitt et al., 2010; Hudek et al., 2016). Some cyanobacteria can also utilize phosphonates, as first demonstrated for the marine N 2 -fixing cyanobacterium T.erythraeum IMS101 (Dyhrman et al., 2006). Phosphonates are organic molecules containing a covalent bond between atoms of P and C, which are derived from the degradation of glycolipids, glycoproteins, antibiotics or phosphonolipids (Kolowith et al., 2001). Bacteria, some eukaryotes, and plants are known to synthesize phosphonates (Horigushi, 1984; Kugler et al., 1990), however, only prokaryotes and some fungi are capable of acquiring phosphonates as a source of P, N or C (Kononova and Nesmeyanova, 2002). In E.coli, the ABC transporter PhnCDE mediates the uptake of phosphonates. Importantly, assimilation of phosphonates also requires the hydrolysis of the C─P bond, which in E. coli and many other bacteria is carried out by the enzymes of the C-P lyase pathway (Hove-Jensen et al., 2014). Trichodesmium erythraeum contains the genes encoding the phosphonate ABC transporter (Table 5; Fig. 2D) and the phnG to phnM genes encoding the C-P lyase pathway (Dyhrman et al., 2006). We, therefore, used the T.erythraeum genes to look for phosphonate utilization genes in the DDA symbionts [although Anabaena also seems to contain the phosphonate utilization pathway (Hove-Jensen et al., 2014), it has not been experimentally characterized]. Among the DDA symbionts, only CalSC01 bears the phosphonate transporter (Table 5; Fig. 2D) and some homologues for genes encoding the C-P lyase pathway (although the pathway gene cluster appears to be incompletely sequenced). In summary, the endosymbionts RintHH01 and RintRC01 contain high-affinity and low-affinity phosphate transporters, whereas the external symbiont CalSC01 contains high-affinity phosphate transporters and a phosphonate utilization pathway. Concentrations of Pi are © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 Transporters in diatom-cyanobacteria symbioses 2043 often low in the regions in which the DDAs thrive (i.e. 10-year average, 25-m depth in North Pacific subtropical gyre: 70 39 nmol L −1 ; Björkman et al., 2018); hence the absence of the low-affinity PiT transporter in the external symbiont (CalSC01) is not surprising. Micronutrient transporters in RintHH01 Given the cytoplasmic location of RintHH01 within the diatom, we checked the presence of possible transporters for micronutrients in this endosymbiont. RintHH01 contains possible transporters for soft metals, including a Na + :H + exchanger (RintHH_10180; TCDB #2.A.36), a K + channel (RintHH_22210; TCBD #1.A.1), the KtrAB transporter for K + (RintHH_14160, _14170; TCBD #2.A.38.4) and a possible transporter for Mg 2+ (RintHH_14380; TCDB #1. A.26). RintHH01 also contains possible ABC transporters for Mn 2+ and/or Zn 2+ (RintHH_220, _230, _21250 and RintHH_5870, _5880, _5890; both belonging to TCDB #3. A.1.15), Ni 2+ and/or Co 2+ (RintHH_19750, _19760, _19770; TCDB #3.A.1.23) and molybdate (RintHH_20340, _20350; TCDB #3.A.1.8). Finally, possible folate-biopterin transporters (RintHH_9790, _1390; TCDB#2.A.71) are also present. The presence of these transporters in RintHH01 identifies some essential micronutrients that the endosymbiont can take up actively from the diatom’s cytoplasm. Proposed C and N fixation and metabolite exchange (C, N, Fe, S and P) model in the diatomcyanobacterium symbioses Based on the main knowledge of the symbiosis between diatoms and diazotrophic cyanobacteria and the new insights introduced in this article regarding different transport mechanisms in three symbionts, we propose the following models. In each model, the symbiont cellular location and how this might favour or limit the symbiont acquisition and transport of a particular substrate is considered. Model for RintHH01 Although RintHH01 resides inside its host, it is expected that the symbiont also performs CO 2 fixation because of the presence of RubisCO, carboxysome genes and an NDH-1 complex (Fig. 3A). (The presence of a bicarbonate transporter, BicA, in RintHH01 is unsure). Of particular interest is the expected concentration gradient of Ci within the host diatom cytoplasm, in which HCO 3 − concentration is predicted to be several-fold higher nearer to the chloroplast (Hopkinson et al., 2016; Young and Hopkinson, 2017). RintHH01 is commonly observed surrounded by the host chloroplast (Caputo et al., 2019). Interestingly, a competition for C uptake, based on an extra C requirement for N 2 fixation in Richelia, could exist between the partners. This competition might rely on the uptake of C compounds from the host mediated by Richelia transporters, including the uptake of glucosides by ABC (Gls) or MFS transporters and the uptake of carboxylates such as 2-OG by the TRAP transporter. Moreover, since RintHH01 appears to have the capability of hydrolyzing but not synthesizing sucrose, this disaccharide would provide reduced C to the symbiont. Indeed, an invertase (InvB) is present in RintHH01 that potentially functions in the cleavage into glucose and fructose of sucrose taken up from the host (Fig. 3A). RintHH01 contains the complete suite of genes to carry out N 2 fixation but lacks any Amt ammonium transporter and the nitrate/nitrite and urea utilization pathways, which defaults the exchange of N-related compounds to the diatom. Therefore, the reduced N compounds must be exchanged by other transporters, such as amino acid ABC transporters N-I and N-II (taking up glutamate and other amino acids), an oligopeptide transporter (possible glutathione transporter), the polyamine ABC transporter, a proline permease, the possible ammonium/amino acid permease RintHH_4450, and the DME permease (with an incomplete sequence) (Tables 2 and 3; Fig. 3A). Due to the lack of GOGAT in RintHH01, we propose the uptake of glutamate from the diatom with the participation of amino acid transporter(s) such as N-I and N-II. Glutamate could be exchanged with glutamine in a process resembling the Gln for Glu exchange between heterocysts and vegetative cells in free-living heterocystous cyanobacteria. Additionally, the presence of cyanophycin metabolism genes but absence of arginine catabolism genes in RintHH01 strongly supports the transfer of arginine from the symbiont to the host. Hence, for N supply to the diatom, glutamine and arginine are candidate vehicles. Regarding Fe, S and P acquisition, RintHH01 presents a low number of transporters with respect to Anabaena and CalSC01 (Fig. 2). RintHH01 might acquire Fe through the Fe 3+ ABC transporter FutABC and the Fe 2+ permease EfeU. Sulfate transport is identical in all three symbionts and Anabaena and characterized by the Sbp/CysPTWA ABC transporter and a SulP permease. RintHH01 is limited to Pi, using a high-affinity Pst (PhoT) ABC transporter and the low-affinity PiT transporter. Model for RintRC01 Although RintRC01 resides between the cytoplasmic membrane and the frustule of the diatom, it has C and N compound uptake capabilities similar to those in RintHH01. For example, genes encoding Amt ammonium transporters or nitrate/nitrite and urea assimilation pathways are also missing. On the other hand, genes © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2044 M. Nieves-Morión, E. Flores and R. A. Foster Fig. 3. Proposed models of metabolite exchange and transport mechanisms in the DDAs. The filament of the symbionts is composed of a single terminal heterocyst and a variable number of vegetative cells. Some diatom hosts develop chains. A. Model for the Hemiaulus hauckii-Richelia intracellularis (RintHH01) symbiosis. From two to four filaments of Richelia per diatom can reside in the host cytoplasm (Bustos-Díaz et al., 2019; Caputo et al., 2019). Note the white areas showing the spaces between two single diatoms in a chain. B. Model for the Rhizosolenia clevei-Richelia intracellularis (RintRC01) symbiosis. The symbiont is located between the diatom plasma membrane and the frustule with the terminal heterocyst close to the diatom valve (Taylor, 1982). Note that the number of vegetative cells in the symbiont is higher than in the two other symbionts. C. Model for Chaetoceros compressus-Calothrix rhizosoleniae (CalSC01) symbiosis. The diatom host contains spines to which the symbiont attaches transversely with the heterocyst (Norris, 1961). Symbiont transporters and related proteins are shown framed. 2OG, 2-oxoglutarate; aa(s), amino acid(s); Amm/aa permease, RintHH_4450/ RintRC_1364/ Ga0265390_112922 proteins; DME, Drug/Metabolite Exporter; EAMA, Drug/Metabolite Transporter superfamily protein; Gls, ABC glucoside transporter; MFS, Major Facilitator Superfamily; N-I and N-II, amino acid ABC transporters; TRAP, Tripartite ATP-independent periplasmic Transporter. Other transporters are indicated by their formal names: BicA, NDH-1 3 , OppABCD, PotADB and SbtA. © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 Transporters in diatom-cyanobacteria symbioses 2045 encoding transporters for the uptake of glucosides, amino acids, peptides (possibly glutathione) and polyamines are present, as well as genes encoding possible amino acid exporters including a DME protein and RintRC_1364 that might also transport ammonium. Significant differences with respect to RintHH01 are the presence in RintRC01 of the genes encoding sucrose biosynthesis proteins, a second invertase (InvA) and an acetate permease instead of the proline permease (Fig. 3B). Additionally, the presence of a BicA transporter in RintRC01 is more likely than in RintHH01. On the other hand, RintRC01 lacks cyanophycin making the exchange of arginine less likely than in RintHH01. Iron, S and Pi uptake capabilities are also similar between RintRC01 and RintHH01. Thus, other than some specific metabolites that may be exchanged (e.g. arginine and proline in RintHH01/H. hauckii; bicarbonate and acetate in RintRC01/R.clevei), the main difference between the symbiotic associations involving RintRC01 and RintHH01 must reside in the physiology of and/or dependency on the host diatom. Whereas RintHH01 takes up nutrients directly from the diatom’s cytoplasm, RintRC01 takes up nutrients from the ‘periplasmic space’, implying that materials that the cyanobacterium obtains from the diatom are exported through the diatom’s cytoplasmic membrane. The specific study of export mechanisms in the diatom will be therefore of much interest. The possibility that RintRC01 takes up nutrients more from the external medium (ocean) than from the diatom is not supported by our findings, which show a remarkably similar array of transporters in RintRC01 and RintHH01. Model for CalSC01 CalSC01 resides external to the frustule of the diatom and can grow as a free-living organism, thus it is a facultative symbiont (Foster et al., 2010). Consistently, the membrane transporter repertoire of this symbiont is more similar to that of typical free-living heterocystous cyanobacteria, including high-affinity bicarbonate transporter (SbtA) and ammonium (Amt) and nitrate/nitrite permeases (Fig. 3C). Additionally, in contrast to the endosymbionts, CalSC01 contains a phosphonate utilization pathway and, notably, has the capability of incorporating Fe with a complete concourse of siderophores. Thus, ammonium, nitrite/nitrate, phosphonate and ferric ironsiderophore complexes are most likely obtained from the surrounding marine medium. On the other hand, the external symbiont (CalSC01) has several transporters similar to those of the endosymbionts (Fig. 3), suggesting that it may facultatively use resources obtained from the host’s phycosphere (nutrient-rich area surrounding phytoplankton cells), thus broadening its growth options. Compounds obtained from the diatom host could include glucosides, amino acids, peptides (possibly glutathione), polyamines and carboxylates. Regarding N nutrition of the host, CalSC01 might behave similarly to the endosymbionts providing arginine and glutamine (as we hypothesize for RintHH01) or glutamine (which is possible for RintRC01). Additionally, the presence of Amt proteins in CalSC01 suggests the possibility of an exchange of ammonium, which would be more likely in CalSC01 than in the endosymbionts. Given that CalSC01 is capable of growth in the laboratory with reported evidence on solid growth medium (Foster et al., 2010), it could potentially be a model system to test the functionality of several of the transporters mentioned here. Concluding remarks In this article, we have focused on the membrane transporters encoded in the genomes of globally distributed and biogeochemically significant N 2 -fixing symbionts of diatoms with special emphasis on the acquisition of macronutrients. Our findings show that the endosymbionts, either internal or partial (RintHH01 and RintRC01 respectively), contain a similar array of transporters, suggesting a similar dependence on the diatom host physiology, whereas the external symbiont (CalSC01) has transporters similar to those of the endosymbionts and additionally other transporters useful for life in a dilute ocean. Based on current knowledge, the only role of the cyanobacterial symbionts is to provide their host diatoms with fixed N. Heterocystous cyanobacteria that engage in terrestrial symbioses such as Nostoc azollae and N. punctiforme release N in the form of ammonium to their plant partners, whereas another Nostoc symbiont releases organic N, mainly citrulline and glutamine (reviewed in Meeks and Elhai, 2002). The amount of N 2 - derived ammonium released to terrestrial host plants varies tremendously (40%–90% of fixed N) and is largely controlled by the GS activity of the respective symbionts (Meeks, 2009). Currently, the amount of N and the chemical form in which N is transferred in the DDAs is unknown. GS and N 2 fixation activities in the various DDAs are expected to vary given the absence of GOGAT in RintHH01 and retention in the other symbionts. It will be of great interest to determine which form of N (ammonium, amino acids) is transferred from the symbiont and the role of the host in potentially providing glutamate or other C skeletons to influence the extent of N 2 fixation in the symbionts. A possible role of Amt transporters has been discussed in the transfer of ammonium in various symbioses including the N.azollae –Azolla symbiosis (Roy et al., 2020). Here, we have hypothesized transfer of glutamine and arginine in DDAs, but the presence of Amt proteins specifically in CalSC01 and of an NKCC1-type cation transporter that might transport © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,22, 2027–2052 2046 M. Nieves-Morión, E. Flores and R. A. Foster ammonium in the three symbionts make ammonium a possible N vehicle in at least some DDAs. In this article, we have proposed a number of transporters encoded in the DDA genomes as responsible for the transfer of specific compounds between symbiont and host. The substrates of many of those transporters can be predicted with reasonable certainty from sequence analysis, but for some other transporters, only the general chemical nature of the substrate can be anticipated. In addition to performing experimental research to corroborate the activity of those transporters whose substrates cannot be defined with certainty, further research is needed. In particular, future work should also address the composition of the diatom’s membrane transporter systems, which will be important to understand its nutritional physiology in the marine environment as well as its peculiar symbiosis with the partial and external symbionts, which are likely provided with nutrients by the diatom host. ACKNOWLEDGEMENTS We are grateful to Daniel Lundin (Linnaeus University, Sweden) for support in data analysis and presentation, Ignacio Luque (CSIC, Seville, Spain) for analysis of the promoter sequences of Zn-regulated porins, and Martin Ekman (Stockholm University, Sweden) for discussion on cyanobacteria in symbiosis. We also thank two anonymous reviewers for their helpful criticisms of this article. 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