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Predicting Substrate Exchange in Marine Diatom‐Heterocystous

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Predicting Substrate Exchange in Marine Diatom‐Heterocystous Environmental Microbiology (2020) 00(00), 00–00 doi:10.1111/1462-2920.15013 Minireview Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses Mercedes Nieves-Morión,1 Enrique Flores 2* and that given the importance of these symbioses in Rachel A. Foster 1* global N and C budgets, warrant future testing. 1Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, 106 91, Introduction Sweden. 2Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC In vast expanses of open ocean environments concentra- and Universidad de Sevilla, Américo Vespucio 49, tions of dissolved inorganic nitrogen are below analytical Seville, E-41092, Spain. detection. Here, microorganisms capable of reducing di- nitrogen (N2), which comprises 78% of the atmosphere, are at an advantage and typically dominate. The process of N2 Summary fixation, or the reduction of N2 to ammonium, is performed In the open ocean, some phytoplankton establish by a small and diverse group of bacteria and archaea fi symbiosis with cyanobacteria. Some partnerships (Young, 1992). Some N2 xing (diazotrophic) populations in involve diatoms as hosts and heterocystous cyano- the open ocean are symbiotic, with a few genera of micro- bacteria as symbionts. Heterocysts are specialized algae, specifically diatoms as hosts and heterocystous cya- cells for nitrogen fixation, and a function of the sym- nobacteria as symbionts (Foster and O’Mullan, 2008) biotic cyanobacteria is to provide the host with nitro- (Fig. 1A–C). Collectively these symbioses are referred to as gen. However, both partners are photosynthetic and diatom diazotrophic associations (DDAs). DDAs are globally capable of carbon fixation, and the possible metabo- distributed and are considered major contributors to both N lites exchanged and mechanisms of transfer are and carbon (C) cycles due to seasonal blooms with high N2 poorly understood. The symbiont cellular location and C fixation rates and rapid sinking (Mague et al., 1974; varies from internal to partial to fully external, and Venrick, 1974; Carpenter et al., 1999; Subramaniam et al., this is reflected in the symbiont genome size and 2008; Karl et al., 2016). Despite their recognition as globally content. In order to identify the membrane trans- significant, there still remain large gaps in our understanding porters potentially involved in metabolite exchange, of DDAs, especially how the partners interact and acquire we compare the draft genomes of three differently (and share) the elements necessary for metabolism and located symbionts with known transporters mainly growth. Here, we analysed the available DDA symbiont from model free-living heterocystous cyanobacteria. genomes to identify membrane transporters potentially The types and numbers of transporters are directly involved in interactions with the host partners. This allowed related to the symbiont cellular location: restricted in us to recognize target proteins for further investigation by the endosymbionts and wider in the external symbi- genetic approaches, for instance by the heterologous ont. Three proposed models of metabolite exchange expression and analysis of genes from the symbionts. are suggested which take into account the type of transporters in the symbionts and the influence of The diatom diazotrophic associations their cellular location on the available nutrient pools. These models provide a basis for several hypotheses 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 Received 17 January, 2020; revised 2 April, 2020; accepted 3 April, 2020. *For correspondence. E-mail efl[email protected]; Tel. nitrogenase reductase component (Fe protein) of the +34954489523; E-mail [email protected]; Tel. 4608161207. nitrogenase complex for N2 fixation, and the hetR gene, © 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. 2 M. Nieves-Morión, E. Flores and R. A. Foster 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 distin- guish 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. A chain of three Hemiaulus hauckii diatoms associated with Richelia intracellularis (RintHH01). C. A chain of >10 Chaetoceros compressus dia- toms 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. The CalSC01 isolate sev- eral months after isolation (imaged with blue excitation). Note the longer filaments when growing freely of its host diatom. Scale bars are approxi- mately 10 μm. which encodes a key regulator of cell differentiation in intracellularis RintHM01 (internal, associated with H. heterocystous cyanobacteria (Janson et al., 1999; Foster membranaceus), 2.21 Mbp; R. intracellularis RintRC01 and Zehr, 2006). Based on these phylogenies the symbi- (partial, associated with Rhizosolenia clevei), 5.4 Mbp; onts are related to other heterocystous cyanobacteria and Calothrix rhizosoleniae CalSC01 (external, associ- in the order Nostocales (e.g. Anabaena sp., Nostoc ated with Chaetoceros compresus), 5.97 Mbp (Hilton sp. and Calothrix sp.). Notably, the symbionts are more et al., 2013; Hilton, 2014). This shows that genome size related between them than to any other known heterocys- is directly related to the cellular location of the symbionts. tous cyanobacterium (Caputo et al., 2019). In spite of General features (size, GC content and percent coding) that, symbiont hetR and nifH sequences are relatively of the symbiont genomes are similar to other Nostocales, divergent (84% and 91% identity respectively) suggesting including free-living and facultative and obligate symbiotic a high specificity in the partnerships (Janson et al., 1999; strains of multicellular plants (e.g. Anabaena sp. PCC Foster and Zehr, 2006). In other words, one particular 7120, Nostoc punctiforme PCC 73102 and Nostoc symbiont strain associates with one particular host azollae 0708 respectively; PRJNA244, PRJNA216 and genus, and the driver of the specificity is currently PRJNA30807 respectively) (Meeks et al., 2001; Hilton unknown (Janson et al., 1999; Foster and Zehr, 2006). et al., 2013). The R. intracellularis RintHM01 draft Morphologically, the symbionts vary in terms of filament genome lacks several sequences expected of a full length and taper, and all possess terminal heterocysts. genome due to low sequencing coverage (Hilton et al., The symbionts vary in their cellular location: internal, 2013), and therefore it was not compared further. partial and external. ‘Internal’ symbionts have penetrated the diatom’s cytoplasm; ‘partial’ (or ‘periplasmic’) refers to The symbiotic diatoms symbionts that reside between the diatom’s cytoplasmic membrane and frustule (outer silicified cell wall of dia- Diatoms are single-celled eukaryotic plankton widely dis- toms); and ‘external’ are symbionts that are attached to tributed in aquatic environments that contribute signifi- the surface of the diatom (Villareal, 1989, 1990; Caputo cantly (20%) to global primary production (Field et al., et al., 2019). Importantly, the external symbiont (Calothrix 1998). Most diatoms dominate coastal environments, rhizosoleniae, CalSC01) can be grown independently of where dissolved nutrients are high, and like other micro- its host in the laboratory (Foster et al., 2010) (see algae, diatoms can utilize nitrate and ammonium Fig. 1D). Draft genomes are available for the heterocys- (Guillard and Kilham, 1977; Armbrust, 2009). In addition, tous symbionts: Richelia intracellularis RintHH01 (inter- diatoms possess a complete urea cycle (Allen et al., nal, associated with Hemiaulus hauckii), 3.24 Mbp; R. 2011). On the other hand, in oligotrophic marine © 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Transporters in diatom-cyanobacteria symbioses 3 environments, some diatoms form a symbiosis with cya- function from Anabaena sp. PCC 7120 (hereafter Ana- nobacteria (Villareal, 1992; Foster and O’Mullan, 2008). baena) as queries, although in some particular cases In general, we know far less about the symbiotic host dia- well-characterized proteins from other sources, mainly fil- toms compared with their respective symbionts. The host amentous cyanobacteria (Nostoc punctiforme ATCC diatoms differ dramatically in cell size (e.g. Hemiaulus 29133, Trichodesmium erythraeum IMS101) were used. hauckii,12–35 μm; H. membranaceus,30–70 μm; The symbionts are also predicted to have some mem- Rhizosolenia clevei,7–250 μm; Chaetoceros compre- brane proteins not found in other cyanobacteria, and ssus,7–40 μm). The Hemiaulus spp. and Chaetoceros such membrane
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