Uncultured Marine Cyanophages Encode for Active Nbla, Phycobilisome Proteolysis Adaptor Protein

Home , Chroococcidiopsis, Galdieria sulphuraria, Hapalosiphonaceae, Microcoleaceae, Planktothrix agardhii

1 1 1 1 2 Omer Nadel , Andrey Rozenberg , José Flores-Uribe , Shirley Larom , Rakefet Schwarz ,

1 and Oded Béjà *

1 2 Faculty of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel; The

Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan

5290002, Israel

*Corresponding author: Oded Béjà, Tel: +972-4-8293961; E-mail: [email protected]

Running title: Marine cyanophages encode active NblA proteins

Originality-Significance Statement:

This is the rst study to examine the distribution and function of nblA genes of marine cyanophage origin. We describe as well the distribution of nblA-like genes in marine cyanobacteria using bioinformatic methods.

Phycobilisomes (PBS) are large water-soluble membrane-associated complexes in cyanobacteria and some chloroplasts that serve as a light-harvesting antennas for the photosynthetic apparatus. When short of nitrogen or sulfur, cyanobacteria readily degrade their phycobilisomes allowing the cell to replenish the vanishing nutrients. The key regulator in the degradation process is NblA, a small protein (∼6 kDa) which recruits proteases to the PBS. It was discovered previously that not only do cyanobacteria possess nblA genes but also that they are encoded by genomes of some freshwater cyanophages.

A recent study, using assemblies from oceanic metagenomes, revealed genomes of a novel uncultured marine cyanophage lineage which contain genes coding for the PBS degradation protein. Here, we examine the functionality of nblA-like genes from these marine cyanophages by testing them in a freshwater model cyanobacterial nblA knockout. One of the viral NblA variants could complement the non-bleaching phenotype and restore

PBS degradation. Our findings reveal a functional NblA from a novel marine cyanophage lineage. Furthermore, we shed new light on the distribution of nblA genes in cyanobacteria and cyanophages.

Introduction

Phycobilisomes (PBS) are large light-harvesting protein supercomplexes (3-7 MDa) that are widely distributed in cyanobacteria, glaucocystophytes, and rhodophytes (Watanabe and Ikeuchi 2013; Adir 2005). These PBS antennas form water soluble protein-pigment complexes which are anchored to photosynthetic membranes. A typical PBS comprises several peripheral rods that project radially from the central core subcomplex. The central core consists of disks of allophycocyanin (AP) trimers and the peripheral rod subcomplexes consist of disks of phycobiliproteins [e.g. phycocyanin (PC),

PBS composition and the amount of cellular phycobiliproteins. For instance, the PBS can be adapted to changes in the ambient light by modifying the PBS pigment composition, a process called complementary chromatic acclimation (Gutu and Kehoe 2012; Montgomery 2017). Furthermore, nutrient limitation might lead to a complete degradation of the PBS (Collier and Grossman 1994). This is observed when cyanobacteria face nitrogen or sulfur starvation and results in cell bleaching. Currently, two biological functions are attributed to

PBS degradation. First, dismantling of the PBS releases nutrients in the form of amino acids; second, reduced cellular metabolism under starvation might lead to over-excitation and therefore, degradation and uncoupling of the PBS serve as a mechanism to reduce electron transfer rate under such conditions. Multiple studies of PBS degradation have provided insight into the mechanism underlying this process (Forchhammer and Schwarz 2018). Although the knowledge of the molecular mechanisms of PBS disassembly is very fragmentary, the key proteins involved in the process have been already known for two decades. In a genetic screen, mutants of Synechococcus elongatus were identified that were unable to degrade their PBS under nitrogen starvation (Collier and Grossman 1994). These non-bleaching mutants (nbl) retained their greenish color during nitrogen stress while the wild type displayed the typical yellowish bleached phenotype. Among the identified genes were nblA (Collier and Grossman 1994) and nblB (Dolganov and Grossman 1999). NblA is a small protein of ~60 aa with homologs found in many cyanobacteria, as well as in red algae (Nakamura et al. 2013; Stoebe et al. 1998). Biochemical assays, supported by the solved crystal structure, revealed that the functional form of NblA is a dimer (Bienert et al. 2006; Dines et al. 2008). NblA binds to the

bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4 α-subunits of PC and PEC (Karradt et al. 2008) and the β-subunit of PC (Nguyen et al. 2017), and in addition, it interacts with ClpC to recruit the Clp protease and induce proteolytic degradation of pigment proteins. Accordingly, it was suggested that NblA serves as a proteolysis adaptor, namely it interacts with the pigment and designates it to degradation (Dines et al. 2008; Karradt et al. 2008). Additionally, based on the close association of NblA with PBS complexes while attached to the photosynthetic membranes,

NblA was also assigned a role in PBS disassembly (Sendersky et al. 2014; Sendersky et al. 2015). Although the cyanobacteria-derived chloroplasts of red algae also undergo bleaching under nitrogen starvation, they do not seem to recruit the plastid homologe of nblA, ycf18, for this function (Kawakami et al. 2009), although no experiments were performed on the other nblA homologe encoded in their nuclear genomes (Nakamura et al. 2013). Over the last decade it became clear that many genes involved in photosynthesis are encoded in the genomes of viruses that infect cyanobacteria (cyanophages) and corresponding sequences were also identified in metagenomic assemblies originating from phages [see (Puxty et al. 2015) for review]. These auxiliary metabolic genes (AMGs) (Breitbart et al. 2007) seem to increase phage fitness during infection by supporting the growing metabolic demand needed to produce phage progeny (Lindell et al. 2005; Bragg and Chisholm 2008; Hellweger 2009). Analogously, genes encoding proteins required for bilin biosynthesis are also frequently found in cyanophages (Puxty et al. 2015). Moreover, it appears that cyanophage genomes might carry genes for PBS degradation, and nblA-like genes were described in genomes of some cyanophages infecting freshwater cyanobacteria (Gao et al. 2012; Ou et al. 2015). At the same time, nblA-like genes were not reported from marine cyanophages till now. This does not come as a surprise as their hosts are reported to lack nblA genes as well (Palenik et al. 2003; Karradt et al. 2008). Yet, this is contradicted by the fact that a number of nblA genes are actually annotated in

In the current paper, we report on the discovery of putative nblA genes from marine cyanophages with a focus on two genes found in a cyanophage genome obtained from metagenomic data. By expressing cyanophage-derived nblA genes in a freshwater model cyanobacterium, we show that one of the two proteins is able to restore the wild-type phenotype in such an assay. In addition, a systematic inventorization of nblA genes in annotated genomes reveals the existence of other marine cyanophages with nblAs and indicates that most PBS-containing cyanobacteria contain nblA-like genes in their genomes. We also highlight the fact that multiplicity of nblA genes per genome, while rather exceptional for cyanophages, is in reality a widespread phenomenon in cyanobacteria, and discuss the implications of these observations in the context of NblA complex structure.

Results and discussion

Using metagenomic assembled genomes (MAGs) from the Red Sea (Philosof et al. 2017) and the Tara Oceans metagenomes (Brum et al. 2015; Sunagawa et al. 2015), we recently identified a novel uncultured oceanic cyanophage lineage (Flores-Uribe et al. 2018). Genomes of cyanophages belonging to this lineage contain common cyanophage genes, as well as genes coding for integrases, split DNA polymerases and NblA-like proteins (Flores-Uribe et al. 2018). Some of these phages appear to contain two different nblA-like genes (Figure 1a).

In order to check if the NblA-like proteins encoded by cyanophage MAGs were indeed bona fide NblAs, we took advantage of a S. elongatus PCC 7942 NblA knockout

bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 6 strain (Sendersky et al. 2015; Collier and Grossman 1994). This mutant does not degrade its PBSs under nitrogen starvation and hence does not show the bleaching phenotype. We attempted to complement this mutant using the cyanophage NblA-like proteins under the control of the native S. elongatus nblA promoter. We chose two nblA genes (G1 and G1b) from a single cyanophage MAG (Figure 1a) for the complementation experiment. While in the G1-NblA variant degradation of PC was observed upon nitrogen starvation, the variant

G1b-NblA was unable to restore the phenotype (Figure 1b).

To test whether the viral NblAs can potentially form dimers as cyanobacterial NblAs

(Dines et al. 2008), we predicted their monomer and dimer structures using pyDockWEB (see Experimental Procedures for details). The structures of G1-NblA and G1b-NblA monomers were homology-modelled and their ability to form dimers was examined using pyDockWEB’s default parameters. G1-NblA demonstrated a homodimeric structure similar to cyanobacterial NblAs when the two monomers were docked (Figure S1). In contrast, the nonfunctional G1b-NblA did not form a homodimer, nor did the G1/G1b heterodimer. As a control we performed the same analysis for the reference cyanobacterial NblAs (PDB accessions 3CS5, 2Q8V and 1OJH), as well as for the two NblAs from Synechocystis sp. PCC 6803 that are known to form a heterodimer but were not crystallized (Baier et al. 2001; Baier et al. 2014). All of the known cyanobacterial dimers could be replicated using this de novo approach as formed the expected heterodimer for the two NblAs from Synechocystis (see Figure S1). These observations suggest that while G1-NblA is able to function as a homodimer as confirmed in-silico, G1b-NblA might require a different NblA to form a heterodimer, which can explain its inability to cause bleaching. One can tentatively hypothesize that this second monomer is not G1-NblA but an NblA provided by the yet unknown host of the virus.

(see Fig. 2c and Suppl. Fig. S2), therefore we collected a broader sample of NblAs and

NblA-like proteins to understand their diversity. Combining protein profile with blast analysis and applying relaxed E-value thresholds to obtain divergent protein sequences in a taxonomy-independent manner, we collected protein sequences that were either annotated explicitly as NblA, NblA-like, Ycf18 or as proteins of unknown function from freshwater and marine cyanobacteria, red algae, and most intriguingly marine cyanophages alongside metagenomic assemblies (Fig. 2, Suppl. File 1 and 2 and Fig. S4).

Eleven marine cyanophages, mostly cultured, appeared to contain single copies of nlbA, none of which is currently annotated as such (see Suppl. File 1 and Fig. S4). Moreover, we discovered four of these genes among unannotated ORFs. The additional metagenomic assembled contigs are of special interest, since they are annotated in GenBank as various unrelated bacterial (non-cyanobacterial) groups. A closer examination of the gene content and blast matches for the corresponding sequences revealed that all of them are in fact fragments of cyanophage genomes, except for one of likely cyanobacterial origin.

Comparison of protein amino-acid profiles between marine phages and marine cyanobacteria revealed a higher overall diversity of the cyanophage NblAs, with most of the positions conserved in cyanobacteria conserved to a similar degree in their potential viruses (see Fig. 2a,b). Yet, some positions moderately or highly conserved in marine cyanobacteria (most prominently, Q6 in Fig. 2, cf. also Fig. 1 in (Dines et al. 2008)) allow more variability in marine cyanophages. These discrepancies between the two profiles explain the low scoring of marine cyanophage NblAs, including G1 and G1b, when matched against the NblA protein profile from Pfam (see Fig. S2) and their dissimilarity to the previously studied NblAs (see Fig. 2c).

bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 8 Interestingly, unlike cyanophages, multiplicity of nblA genes seems to be the rule for cyanobacteria. Analysis of phycobilisome-containing cyanobacteria with chromosome-level genome assemblies indicated that most of them contain multiple nblAs (two and up to seven), with some nblA genes not being predicted or annotated, apparently due to their short sequence and high divergence (Suppl. Fig. S3).

Notwithstanding the short protein sequences and the corresponding alignment, we reconstructed a moderately supported phylogenetic tree for the protein family (see Suppl.

Fig. S4). It should be stressed that due to the short alignment length, high sequence divergence and selection, the phylogenetic reconstruction is expected to be biased and is used here merely as a guide. Phage NblAs group into eight or nine putative clades, some of which are nested within cyanobacterial sequences indicating recent gene transfer from the corresponding hosts (e.g. Phormidium and Planktothrix phages) while others appear to branch deeply. The pairs of nblA genes from our cyanophage MAGs systematically belong to different divergent clades without apparent affinity to cyanobacterial NblAs (Figure 2).

All of the NblAs from the apparently mis-classified phage contigs from metagenomic assemblies cluster with cyanophage NblAs. In line with the earlier observations by

(Nakamura et al. 2013), the NblA-like proteins from red algae form two independent clades corresponding to the well-sampled plastid Ycf18 proteins and the smaller group of nuclear-encoded proteins (see Suppl. Fig. S4).

Our results show that, similarly to their freshwater relatives, nblA genes are carried by marine cyanophages. Moreover, one of the variants was successfully expressed in the heterologous S. elongatus model-system and was shown to be active. This implies that cyanophage NblAs play a role in the degradation of their hosts’ PC. As S. elongatus does not contain PE, future expression experiments with the viral G1 and G1b nblA gene variants in marine Synechococcus hosts containing both PC and PE are currently sought.

Experimental Procedures

Strains and culture conditions. Synechococcus elongatus PCC 7942 and derived strains were grown as described previously (Schatz et al. 2013). The cells were grown in the presence of antibiotics, and starvation was induced as described in Sendersky et al.

(Sendersky et al. 2014).

Marine cyanophage NblA detection. NblAs in the metagenomic assembled genomes were found with Hidden Markov Models trained on NblA amino acid sequences of freshwater cyanobacteria using HMMER and further analyzed employing HHblits in a pipeline based on HHpred (Remmert et al. 2011).

Absorbance measurements. Absorbance was measured as described previously

(Sendersky et al. 2014), with the exception that no integrating sphere was employed. Absorbance spectra were normalized at 680 nm.

Cloning of nblA-like genes into pES57. Synthesized nblA-like genes (Hylabs, Rehovot, Israel), G1 and G1b were digested with BamHI and NcoI, cloned into pES57 and transformed into Escherichia coli DH10B. PCR with 5’-TGCAAGGCGATTAAGTTGG-3’ (forward) and 5’-TTGTGTGGAATTGTGAGCG-3’ (reverse) primers was used to verify the desired clones’ sequences.

bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 10 Structural predictions. Structural homology models for NblA proteins were built with SWISS-MODEL (Bienert et al. 2017). The pyDockWEB server (Jiménez-García et al. 2013) was used for in silico docking of the obtained NblA monomer structures. Three available crystal structures of cyanobacterial NblAs (PDB accessions 3CS5, 2Q8V and 1OJH) were recruited for reference. All structures were visualized in PyMOL (https://pymol.org/).

Alignment and phylogenetic analysis. Sequences for phylogenetic analysis were collected by searching the Uniref90 database (https://www.uniprot.org/uniref/) with the Pfam NblA profile PF04485 (http://pfam.xfam.org/family/PF04485) using hmmsearch v. 3.1b2 (http://hmmer.org). According to empirical criteria, protein sequences with hits covering no less than 84% of the profile’s length were sampled with the default relaxed e-value threshold. The matching Uniref90 sequences, as well as remaining sequences assigned to the InterPro family IPR007574 and all Uniprot proteins with 50% identity to them were used as queries in a blastp v. 2.7.1+ (Altschul et al. 1990) search against Uniref90 with an e-value threshold of 0.03. The final dataset included all Uniprot KB sequences assigned to the matching Uniref90 and corresponding Uniref50 clusters, as well as the NblAs from the cyanophage MAGs analyzed here. In addition, a focused search for more divergent and/or unannotated nblA-genes was performed for all ORFs longer than 150 bp from cyanophage genomes available from Uniprot proteomes using the same strategy but with a blastp e-value threshold of 0.0001. All protein sequences (872 in total) were aligned with mafft v. 7.313 (Katoh and Standley 2013) in L-INS-i mode, trimmed with TrimAl v. 1.4.rev15 (Capella-Gutiérrez et al. 2009) (final alignment length of 55 positions) and the gene phylogeny was reconstructed with FastTree v. 2.1.9 (Price et al. 2010) under the LG-G model. One sequence (A0A2K8SZX4) had to be excluded due to

Gene number analysis. nblA gene numbers were inferred from cyanobacterial genomes tagged as complete in Genbank using the same strategy as described for the phylogenetic analysis above. As input, all predicted proteins and all ORFs at least 100 bp were taken and analyzed together.

Acknowledgments

We thank Faris Salama for help with the spectroscopic measurements. This work was funded by a European Commission ERC Advanced Grant (no. 321647), Israel Science

Foundation grant (143/18) and the Louis and Lyra Richmond Memorial Chair in Life

Sciences (to O.B.).

Author Contributions

R.S. devised the initial idea for the project. O.N., S.L., R.S. and O.B conceived the experiments, J.F.-U. and A.R. performed bioinformatic analyses, and O.N. conducted the molecular biology and biophysical experiments. O.B. wrote the manuscript with contributions from all authors to data analysis, figure generation, and the final manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

a G1 G1b nblA nblA AMG psbA AMGsnrd exo virE DNA Pol

MCP Portal TerL Integrase

b PCC 7942 PCC 7942/nblAΩ PCC 7942/nblAΩ::G1-nblA PCC 7942/nblAΩ::G1b-nblA

PC PC Chl Chl PC PC Chl Chl

0 hrs

-N 24 hrs

-N 48 hrs Absorbance, au

550 600 650 700 750 550 600 650 700 750 550 600 650 700 750 550 600 650 700 750 Wavelength, nm

Figure 1. An uncultured marine cyanophage with two different nblA-like genes. a. Genome map of cyanophage IUI_154509. b. Absorbance spectra of S. elongatus PCC 7942 nblA knockout (PCC 7942/nblAΩ), Wild Type PCC 7942, PCC 7942 knockout complemented with phage G1 NblA (PCC 7942/nblAΩ::G1-nblA), and PCC 7942 knockout complemented with phage G1b NblA (PCC 7942/ nblAΩ::G1b-nblA). Absorption spectra were recorded at the onset of nitrogen starvation time (0 h, blue line), and following 24 h (green line) and 48 h (orange line) nitrogen starvation (n=5). PC and chlorophyll-a (Chl) absorbance peaks are indicated. bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a 3.42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Marine 1.71 cyanobacteria M Q V Q W W Q E F D L Q Q Q LM A T E S K E K Q D AK L L R L K H L Q S R M L K Y E L YRK L KE n = 115 E EK L L E Q QE E F E Y Q E Q R DA DS R A A QA D F RN Q I MGS KVQ I K T KA DD E VK M I I NT KQV I Q K I L E S NT V A D RD H T NS T V E A Information content (bits) Information S T A Y R I D V L R S E Q L A L A SL V V I A A Q L N P I R I M E K V D T S D N V QQ L E T S DS M S QN I E K M H NN H F V N S I T L E V H V D K A L MRA V K K N KVM Q A S A T A V T I V I A K S SA M S E Y I E K S A S S QH A K T R R N L F R KDA S A T DA KAQ S I QV T M HAA I K T L E T T SRR L AA T K KE L T S RH HS E T R T R F NGG A E V PN A R S S V N KM M S E L S T Q T NQ RAR NN R A F E R T N T A S N F AD D K QA I D T K T AE E H NNA A T I F F S E S R T T S K S G Y N T G T M D T C R N T DVHHD S T R C RN I T GH I C VHRR L NE I Q I DM A VQ GH Q LQ MN MA S F V T A G T HPH G H L G G DH T ANG R RWM V D R GMT F LM C M H F V MH T I I I Q T N Y T LWG L L L L L G G G T H V S D E D V H T G F R G R G K Y N V P V FT HV L G L V V S I P GR Q F F EKMH G F H V S C V M P F M T A A V S MY 0 HA L AVQP PWV Y V E SD L GSH L Y L G L I Y V L S Y S L SRGGCRK L GMH C V P Y S G L N Occupancy 1.00 1.00 1.00 1.00 1.00 1.00 1.00 F1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.92 Insert probability 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Insert length 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 0

b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

2.70 Marine

1.35 cyanophages T F C M V W I I A Q Q N Q QE K DDD L L QK N H N L N L R E L F L K L H E E M D R L E D R K L D T L A L L L n = 50 A R RE D K AK Y AR L Q K K L D T S E KR V K P K K E E ES K I I E V E L A L P I S L L E L G E E E M S A Q A A I T M R R V L L R A I R Y Q QQ Q K A I L E T K M K I R Y S K S Q K A Q P I V S A T N S A A E V V T A D SM K R Y V S Q K L KWRQ QSQ NC T V E E RK E R S Information content (bits) Information A E A M S F V HR S R I S D I Q I S D N T KD M F Y E Y R S S L N G T Y I T T R Y A D T T D L N G K M I I S R R S RV Y AV Q VM I C TM DM M Y Q V A S F I V KV F Q N E R K M ENS A A S E T I G G S M T K Y GK VQ T S N T V L GR Y N Y D Q HRVNS R NH T N D N T CN I HM L S R H I N W S V N Y T A YA L R V L K E S Q I A S NQ V I A Q P V C T I AMR A AGM ADAE E N A Q V NAY GMHDEM L R T V G G L T T AMN F V T Y YQ T T AA AMT C F HV K V L S A T V G S I AV CG F D T P MVH V T DV A F A T HRMT L R T T I T HS L N P CT S G I F T D AM A KR E I S S D MV V T N M S F A V T C R F M F W A A G 0 I A F I T YDSHN QQ T Y S PQNN F D L S S T F ND SQK V K L T F F I N Occupancy 0.76 0.81 0.84 0.97 0.99 0.99 1.00 L1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.98 0.99 0.99 0.99 1.00 1.00 1.00 1.00 1.00 1.00 0.91 0.94 0.96 0.97 Insert probability 0.00 0.00 0.00 0.00 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.16 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Insert length 1.9 1.9 1.9 1.9 2.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 2 1.9 1.9 1.9 1.9 1 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 0 c Phage 1 gene G1 D 2I P 3L 4 E G Q LR LRV IAD E INE C SD I D E LRKQ L IV S AKL V M TYQ 41N I LNR I L KEQ Phage 1 gene G1b D L T I E QR FA LQR ARY E VSR M SRH E L ER T A VK L LKSR MQ QK N G V QN V LLQN Planktothrix phage PaV-LD Q L T M E Q E FK LAI IKRN VDI L TL E E AR E Y I IE L VRLD M I K D D L I KN W M RMK Thermosynechococcus vulcanus str. Copeland D L S F E Q E FQ MRV MEE Q VSA M SL Q E AR E L L LQ A SRL L M M K D N V I RS L V KRA Synechococcus elongatus PCC 7942 S L S V E Q Q FD LQK YRQ Q VRD I SR ED L E D L F IE V VR QK M AH E N I F K G M I RQ G Synechocystis sp. PCC 6803 NblA1 D L T I E QM FE FRR MQDA TAN I SQ E Q AL E L L VQ A SRL L M I KS N V I RD L M RQA Synechocystis sp. PCC 6803 NblA2 N L S L E QK FQ LQC LQQ E YQE LDR E Q TVN Y L LE T MQ Q I M V R D N L I RD L M KNS Nostoc sp. PCC 7120 E L S L E Q Q FS IRS FAT Q VQN M SH D Q AK D F L VK L YE Q M V V R EA T Y QE L L KHQ

Figure 2. Protein profiles for the conserved region of NblA proteins. a. Protein profile for 115 NblA proteins from marine cyanobacteria. b. Protein profile for 50 NblA proteins derived from marine cyanophages (including metagenomic sequences erroneously annotated as various microbes). c. Fragment of the whole alignment of 871 NblA and NblA-like proteins showing chosen representatives: two marine cyanophage proteins investigated here (marked with blue diamonds) and NblAs studied in detail before, including a freshwater cyanophage (cyan diamond). All three panels are aligned to each other and trimmed to the boundaries of the Pfam NblA profile (PF04485). Amino acid properties and conservation relative to the corresponding profiles or whole alignment are highlighted according to the ClustalX color scheme. bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 14 Supplementary Figures (see below): Supplementary Figure S1. Structural predictions for NblA dimers from Synechocystis sp. PCC 6803 (P73890 and P73891 heterodimer), Synechococcus elongatus PCC 7942 (P35087 homodimer) and G1-NblA (homodimer) obtained with pyDockWEB. Supplementary Figure S2. Distribution of e-values and alignment lengths of hmmsearch matches for the Uniref90 database and phage MAG when searched with the Pfam NblA profile PF04485. Uniref90 hits based on records outside of the Uniprot KB database are not shown.

Supplementary Figure S4. Phylogenetic tree of NblA, Ycf18 and NblA-like sequences. Branches with bootstrap support values of at least 80% are indicated with circles. G1-NblA and G1b-NblA are indicated with asterisks. Interactive representation of the tree is available at https://itol.embl.de/tree/13268108169281151544017427. The taxonomy assigned to the entries is left unchanged except in the following cases: all non-cyanobacterial metagenomic assemblies are grouped into one category; L8AI55 and L8AJF3 are derived from a Synechocystis-Bacillus chimera; A0A2R7TRJ3 is apparently from a short fragment of a Synechococcus genome mis-classified as Pseudomonas sp. HMWF031.

Supplementary File 1 (spreadsheet). Metadata for the 872 NblA and NblA-like proteins collected by us for the phylogenetic analysis and protein profiles, including marine cyanophages analyzed here.

Supplementary File 2 (fasta file). Untrimmed alignment of the NblA and NblA-like proteins.

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Synechocystis sp. Synechococcus elongatus G1-NblA PCC6803 PCC7942

Supplementary Figure S1. Structural predictions for NblA dimers from Synechocystis sp. PCC 6803 (P73890 and P73891 heterodimer), Synechococcus elongatus PCC 7942 (P35087 homodimer) and G1-NblA (homodimer) obtained with pyDockWEB.

Category

Annotated in InterPro Phage -10 log e-value Other sequences

-20

10 20 30 40 50 Alignment length

Supplementary Figure S2. Distribution of e-values and alignment lengths of hmmsearch matches for the Uniref90 database and phage MAG when searched with the Pfam NblA profile PF04485. Uniref90 hits based on records outside of the Uniprot KB database are not shown. bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

GenBank annotations 40 Predicted proteins All ORFs

20 Number of genomes

0 1 2 3 4 5 6 7 Number of nblA genes

Supplementary Figure S3. Frequency distribution of numbers of nblA genes in 126 complete genomes of cyanobacteria. Excluded are 13 Prochlorococcus genomes none of which encodes for phycobilisome proteins or nblAs. The counts provided correspond to three sets of annotations: explicit annotations from GenBank, predictions made by us for annotated proteins and predictions made for all ORFs of at least 100 bp long. Taxono mic groups bioRxiv preprint doi: https://doi.org/10.1101/494369; this version posted December 13, 2018. The copyright holder for this preprint (which Cyanoba cteriawas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Cyanosarcina cf. burmensis CCALA 770

Chroococcidiopsis thermalis PCC 7203

Chroococcidiopsis cubana CCALA 043

Oscillatoriales cyanobacterium JSC-12

Hydrococcus rivularis NIES-593

filamentous cyanobacterium CCP3 Nostoc sp. 'Lobaria pulmonaria

filamentous cyanobacterium CCT1

Calothrix parasitica NIES-267 Aphanocapsa montana BDHKU210001

Pleurocapsa sp. CCALA 161

filamentous cyanobacterium CCP3 Pleurocapsa sp. CCALA 161 Leptolyngbya sp. Heron JIsland

filamentous cyanobacterium CCT1 Pleurocapsa sp. PCC 7327

Leptolyngbya sp. 7375 PCC

Lyngbya confervoides BDU141951

Rivularia sp. PCC 7116

Leptolyngbya sp. PCC 7375

cyanobacterium TDX16

"Gammaproteobacteria bacterium TMED243" cyanobacterium TDX16 Rhodop hyta filamentous cyanobacterium CCP5

Phormidium tenue NIES-30

uncultured phage MedDCM-OCT-S09-C28

"Flavobacteriaceae bacterium" Phormidesmis priestleyi Ana

Phormidesmis priestleyi Synechococcus phage S-EIVl

Leptolyngbya sp. "Balneolaceae bacterium"

Cyanobium gracile ATCC 27147 / PCC 6307 "Flavobacteriales bacterium" "Euryarchaeota archaeon"

"Euryarchaeota archaeon"

Aphanothece cf. minutissima CCALA 015 "Parvibaculum sp." Synechococcus sp. TMED169Synechococcus sp. RCC307

Calothrix sp. HK-06

Chroogloeocystis siderophila 5.2 s.c.1

Gloeocapsa sp. PCC 7428

Neosynechococcus sphagnicola sy1

Leptolyngbya frigida ULC18

Leptolyngbya sp.

Leptolyngbya antarctica

Leptolyngbya sp.

Neosynechococcus sphagnicola sy1

Leptolyngbya sp. NIES-2104 Synechococcus sp. MED-G71 Leptolyngbya sp. NIES-3755 Leptolyngbya boryana NIES-2135

cyanobacterium TDX16

Cyanosarcina cf. burmensis CCALA 770

Synechococcus sp. LanierSynechococcus lacustris Chroococcidiopsis cubana CCALA 043 Synechococcus sp. TMED19 Chroococcidiopsis thermalis PCC 7203

Calothrix sp. PCC 7507

Scytonema sp. HK-05

Scytonema sp. NIES-4073

filamentous cyanobacterium CCP2

Calothrix elsteri CCALA 953

Synechococcales bacterium UBA10510 Calothrix sp. NIES-2100 cyanobacterium BACL30 MAG-120619-bin27 Fischerella sp. NIES-4106

Cyanobium sp. CACIAM 14 Calothrix sp. PCC 6303

Nostoc sp. 'Peltigera membranacea cyanobiont' 213

Synechococcus sp. WH 5701 Nostoc sp. 'Peltigera membranacea cyanobiont' N6 mis-classified fragment "Pseudomonas sp. HMWF031" Nostoc punctiforme NIES-2108 Nostoc flagelliforme CCNUN1

Nostoc punctiforme ATCC 29133 / PCC 73102

Cyanobium sp. PCC 7001 Nostoc sp. 'Peltigera membranacea cyanobiont' 210A Nostoc sp. 'Peltigera malacea cyanobiont' DB3992

Cyanobium usitatum str. Tous Cyanobium sp. NIES-981 Nostoc punctiforme ATCC 29133 / PCC 73102

A0A2L2NRT8 A0A1Z4LNC8 Nostoc commune NIES-4072 A0A1U7HTL9 A0A2T1CVY3

A0A2T1GW23 A0A2T1CG25 Nostoc commune HK-02 A0A254W8N0 A0A2T1FCS7

A0A254W7A2

A0A2T2W7K7 Nostoc punctiforme NIES-2108 Cyanobium sp. A0A2P8W8S1 "Propionibacteriaceae bacterium" Synechococcus sp. KORDI-49 Nostoc sp. 'Peltigera membranacea cyanobiont' 210A Cyanobium sp. Baikal-G2

K9TC99 Nostoc sp. 'Peltigera malacea cyanobiont' DB3992 K9RJ04 A0A2T2VXR1

K9U173 A0A2P8W4J5 Scytonema sp. NIES-4073 A0A2W6Z631 K9EPW8 Synechococcus sp. MIT S9504 A0A0C1W8N0 Trichormus variabilis NIES-23 A0A2W4WQW4 A0A1U7J020 U9W8U9 Nostoc sp. PCC 7120 / SAG 25.82 / UTEX 2576 A0A0C1UUI1 K8GQX7 Vir uses K9F440 Nostoc sp. KVJ20 A0A2P8WHR5 A0A0P8DFA6 Synechococcus sp. EAC657 Nostoc punctiforme NIES-2108

A0A359MBR0 A0A1Q4RAT3 A0A1U7HAR3 K9XFX4

A0A098TMA9

A0A2T1E7K0

A0A2W7A059 A0A2D8XM82 A0A2W4XMD5 Synechococcus sp. WH7803 A0A2W7ATX9 Nostoc punctiforme NIES-2108 A0A2W6ZU80

A0A098TRK6

A0A0P4UNR2 A0A359E2P8 A0A0S3UCZ7 A0A1Z4JL25 Nostoc sp. ATCC 53789 A0A0C4K651 A0A254W539 A0A2T1FCM5 A0A2E5B6D2 A0A2T1GWB1 Nostoc punctiforme NIES-2108 Synechococcus sp. TMED90 K9U030 A0A350M1X0 K9PRM1 A0A2E7WBT9 A0A1Z4IMG4 Nostoc sp. 'Peltigera membranacea cyanobiont' N6

A0A1Z4PR29 Synechococcus sp. WH7805 A0A2T1DR43 Nostoc sp. 'Lobaria pulmonaria A0A1Z9R652 A0A2A2TP57 A0A1Z4GWV3 Nostoc commune NIES-4072 Synechococcus sp. RS9916 A0A1Z4TRW6 K9UWF2 Synechococcales bacterium UBA8138 Nostoc commune HK-02 A0A235HPF5 A0A1Z9JBD0 A0A2L2NBI5 A0A2P7EHR8 Nostoc sp. ATCC 53789 A0A367RZK6 Synechococcus sp. NAT40 D6PG83 A0A2K8T5T8 Synechococcus sp. RS9917 A0A251Z8Z4 A5GQX1 Nostoc minutum NIES-26 B2IXJ1 A0A368CH60 A0A235IH05 Nostoc linckia z16 A0A1Z8ND19 A0A2D0HCW3

Synechococcus sp. XM-24 B2J792 Nostoc minutum NIES-26

A0A2R5FEK6 A0A2T1ET24 Nostoc sp. KVJ20 A0A2Z6DBH1 A0A081GLQ4 A0A367RRP7 Nostoc flagelliforme CCNUN1 A0A326QGI7 K9P6Y6 A0A235I565 Synechococcus lacustris A0A2D0HDZ5 Cylindrospermum stagnale PCC 7417 Synechococcus sp. Lanier A0A1Z4PQE1 Nostoc azollae 0708 Cyanobium sp. CACIAM 14 A0A1Z4KXF1 A0A182ATG2 Synechococcus sp. TMED20 Q8YK25 Anabaena cylindrica ATCC 27899 / PCC 7122 Synechococcus sp. KORDI-100 A3YW23 A0A354AYC5 A0A1E2WM53 Calothrix sp. NIES-2098 A0A2P7MTE7 A0A367RTM8 B5IL66 A0A2G4G1E2 A0A367S1H2 Calothrix brevissima NIES-22

A0A367QYF0 Synechococcus sp. RS344 A0A0R2U3T6 Calothrix sp. NIES-2100 A0A367S356 A0A076HBK7 Acaryochloris sp. RCC1774 A0A2L2N4Z0

Synechococcus sp. KORDI-100 A0A2E0E997 A0A2L2NZI7 Limnothrix sp. P13C2 Synechococcus sp. CPC35 A0A163YC68 A0A2R5G4A3 Cyanobium sp. NAT70 Limnothrix sp. CACIAM 69d A0A2D8U0Y7 A0A2Z6DN20 Hydrocoleum sp. CS-953 Synechococcus sp. WH8103 A0A367R5L7 Metagen omic assem blies A0A367S0Z5 Oscillatoria acuminata PCC 6304 A0A2R7TRJ3 A0A2C6VRE1 Synechococcus sp. CPC35 A0A1Z9WA43 A5GKC6 Cyanobacteria bacterium UBA9273 Synechococcus sp. WH8103 A0A367RXU7 Cyanobacteria bacterium UBA8803 Synechococcus sp. ARS1019 A0A1E2WDN3 A0A2K8SMJ2 Moorea producens PAL-8-15-08-1

A4CTT0 K9X014 Synechococcus sp. RS9916 A0A2E0KI01 Moorea producens 3L Q05R15 D7E489 Synechococcus sp. CC9311 Moorea producens JHB A0A316JM73 K9ZI29 Synechococcus sp. WH 8016 Moorea bouillonii PNG A0A358BZY7 A0A1Z4G4J1 Synechococcus sp. MIT S9508 A3Z503 A0A2P7EBP5 A0A1Z4K2W3 Gloeomargarita lithophora Alchichica-D10 Synechococcus sp. MIT S9504 A0A1Z4H7R7 A0A251Z8A2 Alkalinema sp. CACIAM 70d A0A2W1J8Y3 Leptolyngbya sp. NIES-2104 A0A081GI73 Synechococcus sp. WH7803 A0A1C0V194 A0A1Z8PF49 A0A372DFK3 Leptolyngbya sp. NIES-3755 Synechococcus sp. WH7805 A0A076H298 A0A261KT45 Leptolyngbya boryana NIES-2135 Synechococcus sp. WH 8109 A0A2E7LY54 K9TTD6 filamentous cyanobacterium CCP2 A0A2E0AGH7 Synechococcus sp. KORDI-52 A0A352AFB3 Chamaesiphon minutus ATCC 27169 / PCC 6605 A0A2D4YWD7 Oscillatoriales cyanobacterium USR001 A0A355HTX4 A0A076GWZ4 [Oscillatoria] sp. PCC 6506 A0A1D8U180 Pseudanabaena biceps SynechococcusPCC 7429 sp. XM-24 A0A0H5PUN8 F4XRL8 Microcoleaceae bacterium UBA11344

A0A2D4YYM7 A0A1D9G8D0 Tychonema bourrellyi FEM_GT703

Stanieria cyanosphaera ATCC 29371 / PCC 7437 A0A1U7N7E5 Pseudanabaena sp. ABRG5-3 A0A0H5PS55 filamentous cyanobacterium Phorm 46 Cyanophage S-TIM5 A0A1J0ACS2 A0A2D6Y678 filamentous cyanobacterium Phorm 6 A0A251WBJ3 Microcoleaceae bacterium UBA10368 A0A0P4UUJ8 Microcoleus vaginatus FGP-2 Pseudanabaena sp. PCC 7367 Q05RZ2 A0A0S3UA85

Pseudanabaena frigida A0A1Z4JCS6 Oscillatoria nigro-viridis PCC 7112 A0A164D4P8 Q0I980 G4FLU8 A0A2T1D0K1 uncultured Phormidium sp. A0A163YVV7 A0A1C0VWH8 Oscillatoriales cyanobacterium JSC-12 Lyngbya aestuarii BL J Pseudanabaena sp. ABRG5-3 D8FVC6 Stanieria sp. NIES-3757 Lyngbya sp. PCC 8106 A0A349JNW6 A5GK08 Limnoraphis robusta CS-951 A4CXQ4 A0A2G4EUG6 "Crocinitomicaceae bacterium" Arthrospira platensis NIES-39 / IAM M-135 A0A076HIZ6W0GZG9 A0A2T1D496 Arthrospira sp. O9.13F A0A316JT77 A0A2T1G2U5 "Phycisphaerae bacterium" A0A355CGG2 Arthrospira maxima CS-328

H6WG80 F5UMZ3 Arthrospira sp. PCC 8005 "Rickettsiales bacterium" K9VG16 A0A2Z5X1P1 Arthrospira platensis C1 L8MZ58 W0FFT8 Planktothrix serta PCC 8927 "Blastopirellula sp." A0A2W4W271 U7QDH3 K9UDX9 Planktothrix tepida PCC 9214 A0YZ32 Planktothrix rubescens A0A0F5YET4 Synechococcus sp. ATCC 27167 / PCC 6312 K9SLP2 Habitat s D4ZWQ8 Planktothrix sp. PCC 11201 A0A140KD08 K9XR04 Phage 1 A0A328V6Q0 Planktothrix sp. UBA10369 Scytonema sp. NIES-4073 A0A2Z5WX36 Phage 4 B5W238 uncultured Oscillatoria sp.

Phage 3 H1WK52 Planktothrix agardhii NIVA-CYA 126/8 A0A2E1FMP7 Scytonema sp. HK-05 K8GMX4 "Alphaproteobacteria bacterium" K1VYX4 Phage 2 Planktothrix agardhii A0A2D9C987 A0A1J1KLF0 A0A2D9KP98 Planktothrix paucivesiculata PCC 9631 A0A1J1LJD2 A0A2E0ATB0 Oscillatoriales cyanobacterium CG2_30_40_61 A0A1J1KZ27 "bacterium TMED88" Planktothrix agardhii NIES-204 "Rickettsiales bacterium TMED131" A0A1T4ZGW8 "Phycisphaerae bacterium" "bacterium TMED88" Aphanocapsa montana BDHKU210001 gene 1 A0A354WLZ7 Lyngbya confervoides BDU141951 gene 4 W0FFW3 "Planctomycetes bacterium" A0A073CFW4 Roseofilum reptotaenium AO1-A Synechococcus phage S-RIP2 gene 3 A0A1Z4PQZ9 A0A1J1J9W9 gene 2 Cyanobacteria bacterium UBA11691

A0A1Z4IL96 A0A1J1KGP4 filamentous cyanobacterium CCP3

Cyanophage KBS-P-1A A0A1J5GMR8 Halomicronema hongdechloris C2206 A0A2E0M480K9RQX5 A0A2Z6C999 Mari ne filamentous cyanobacterium CCT1 Synechococcus"Rhodobacteraceae T7-like virus S-TIP37 bacterium" A0A1Z9BPX8 A0A0C1WE15 "Halieaceae bacterium" filamentous cyanobacterium CCP5 A0A0C1Y5B6 "Haliea sp." A0A1Z9B0Y3 filamentous cyanobacterium CCT1 A0A2D9C3K4 A0A1L9QTC2 Leptolyngbya sp. HQ317389 29283 29008A0A1Z9F4H2 A0A357A5B0 Cyanophage KBS-S-1A Phormidium tenue NIES-30 A0A2D7LZZ3 A0A2T2W439 Prochlorococcus phage P-SSP7 Leptolyngbya antarctica Cyanophage P-SSP2 A0A1V8NI40 A0A2P8VIN1 Leptolyngbya sp. "Euryarchaeota archaeon" A0A2P8WCD1 Moorea producens PAL-8-15-08-1 "Rhodospirillaceae bacterium" A0A2D5F8A2M4SN19 A0A2P8W9V9 A0A2D7JAX6 Moorea producens JHB "Flavobacteriales bacterium" MH540083 12266 12703 A0A2W6ZP09 A0A2E0E7N2 Moorea bouillonii PNG "Candidatus Poribacteria bacterium" A0A1U7J640 "Candidatus Marinimicrobia bacterium" Moorea producens 3L "Coraliomargarita sp." A0A2W4VYD0 Rubidibacter lacunae KORDI 51-2 "Candidatus Poribacteria bacterium" A0A2W7BTD9 G8EXI1 Cyanobacteria bacterium QH_9_48_43 A0A1D8TRQ7 Bracki shwater E3SQL8 Cyanobacteria bacterium QS_8_64_29 A0A1D9FZQ0 A0A2D9X1T3I3RYR3 Halothece sp. PCC 7418 SynechococcusCyanophage phage S-CBP4 NATL1A-7 A0A1U7MWJ6 A0A2E7VC62 Cyanobacteria bacterium SW_9_44_58 Synechococcus phage S-CBP1 F4XJS2 A0A2D7KSS7 Dactylococcopsis salina PCC 8305 U5DLJ8 A0A2E0NDY3 A0A2T2RND8 Leptolyngbya sp. PCC 7376 Synechococcus"Chloroflexi phage S-RIP1bacterium" A0A2E8RP01 A0A2T2S2B9 Limnothrix rosea IAM M-220 Synechococcus phage S-CBP3 A0A2E7W1Q2 GU071102 12728 12447 K9Y825 Synechococcus sp. PCC 7003 Prochlorococcus phage P-GSP1 A0A2E8E067 A0A2T2RHM3 Synechococcus sp. PCC 7117 Synechococcus sp. ATCC 27144 / PCC 6301 / SAUG 1402/1 K9YUC2 Synechococcus sp. NIES-970

K9Q097 Synechococcus sp. ATCC 27264 / PCC 7002 / PR-6 SynechococcusGeminocystis elongatus sp.PCC NIES-3709 7942 M1PXY9 A0A096VKE1 A0A1Q4R1A5 HQ317388 34578 34946 Phormidesmis priestleyi ULC007 Phage 5 A0A2E5T1E3 A0A1B1TQ72 Neosynechococcus sphagnicola sy1 Salin e/alkalin e lakes A0A1B1TZH3 Acaryochloris marina MBIC 11017 A0A1Q2U042 I3ULV4 Cyanobacteria bacterium UBA11370 "Blastopirellula sp." B1XQM5 M1UH18 Cyanobacteria bacterium UBA11162 A0A2T1D6K6 gene 5 Cyanobacteria bacterium UBA11148 "bacterium TMED88" A0A0D6ARR6 A0A098TPU0 Phage 3 Desertifilum sp. IPPAS B-1220 A8ZMV5 "Gammaproteobacteria bacterium TMED234" P35087 Cyanobacteria bacterium UBA9273 A0A0H3KBA2 A0A350Y7S7 Phage 4 "Rickettsiales bacterium" A0A2E0B578 A0A355DRT1 Cyanobacteria bacterium UBA8803 Phage 1 A0A359NJC1 Cyanobacteria bacterium UBA11367 gene 3b A0A1Z9B2U3 A0A1E5QIR3 Cyanobacteria bacterium UBA11366

A0A352AFS6 Cyanobacteria bacterium UBA11166 gene 4b A0A355HIK2 Cyanobacteria bacterium UBA11159 Freshw ater/terr estrial gene 1b A0A2D9KQL6 A0A352XHM9 Cyanobacteria bacterium UBA11153

A0A1Z9PHL7 A0A353A8W4 Cyanobacteria bacterium UBA11149 A0A354ZNU5 Coleofasciculus chthonoplastes PCC 7420 A0A357GKS9 Aphanothece hegewaldii CCALA 016 A0A357P3G4 Chondrocystis sp. NIES-4102 A0A358YKC5 Pleurocapsa sp. CCALA 161 B4VRF8 Gloeocapsa sp. PCC 73106 A0A2T1M1M9 Stanieria sp. NIES-3757 A0A1Z4RRB9 Stanieria cyanosphaera ATCC 29371 / PCC 7437 A0A2T1CIS6 Microcoleus sp. PCC 7113 L8LPF1 Cyanobacterium stanieri ATCC 29140 / PCC 7202 A0A140K300

K9XZG2 Cyanobacterium sp. IPPAS B-1200

K9WFG0 Cyanobacterium sp. HL-69

K9YL59 Geminocystis sp. NIES-3709

A0A1E5QWD5 Geminocystis sp. NIES-3708

A0A2K8WLY8 Cyanobacterium aponinum IPPAS B-1201

A0A0D6ASN3 Cyanobacterium aponinum PCC 10605

A0A0D6AFD1 Oscillatoriales cyanobacterium JSC-12 A0A2G3PF51 Leptolyngbya frigida ULC18 K9Z6R7 Leptolyngbya sp. K8GML8 Cyanothece sp. PCC 7424 A0A2T1E6G3 Cyanothece sp. PCC 7822 A0A2W7A0W7 Hydrococcus rivularis NIES-593 B7KIR0 Pleurocapsa sp. PCC 7327 Periphykon beckeri E0UGH9 Gredgaria maugeana Microcystis aeruginosa NIES-44 A0A1U7HHD4 Microcystis aeruginosa NIES-2549 Dictyomenia sonderi K9T5H7 Symphyocladia dendroidea A0A1Z1M3L1 Microcystis panniformis FACHB-1757 A0A0A1VSZ7 A0A1Z1MM83 Microcystis aeruginosa NIES-88 Polysiphonia scopulorum A0A0F6U6W0 A0A1Z1MSK0 Microcystis aeruginosa NIES-98 A0A0K1RVA0 Polysiphonia stricta A0A1Z1M7T6 A0A139GN67 Microcystis aeruginosa KW Polysiphonia sp. A0A1Z1MHJ1 Dipterosiphonia australica A0A1E4QGE8 Microcystis aeruginosa PCC 7806SL A0A1Z1MCK0 Ophidocladus simpliciusculus A0A1V4BZJ2 Microcystis aeruginosa NIES-298 A0A1Z1M3A0 A0A1X9LBJ0 Microcystis aeruginosa NIES-87 Polysiphonia sp. A0A1Z1ML41 A0A2H6BVC1 Microcystis sp. 0824 Thaumatella adunca A0A1Z1MIQ3 A0A2H6L4Q4 Microcystis sp. MC19 Herposiphonia versicolor A0A1Z1MUC5 A0A2L2XUZ5 Microcystis aeruginosa Sj Rhodomela confervoides A0A1Z1MNS8 A0A2P1UKU4 Microcystis aeruginosa NIES-1211 A0A1Z1MFH3 A0A2Z6UTZ3 Chondria sp. Microcystis aeruginosa PCC 7806 A0A1Z1MA05 A0A2Z6V1R9 Chondria sp. Microcystis aeruginosa NIES-843 A0A1Z1MDT2 A8YAR4 Chondria sp. Microcystis aeruginosa PCC 9432 Lophocladia kuetzingii A0A1Z1MC65 B0JM57 Microcystis aeruginosa PCC 9717 Osmundaria fimbriata A0A1Z1MQA7 I4FC48 Microcystis aeruginosa PCC 9443 Kuetzingia canaliculata A0A1Z1MP30 I4FJF3 Microcystis aeruginosa PCC 7941 Bryothamnion seaforthii A0A1Z1M4T5 I4GAL9 Microcystis aeruginosa PCC 9806 A0A1Z1MPP3 I4GLK5 Palisada sp. Microcystis aeruginosa PCC 9807 A0A1Z1MDF7 I4GXR3 Laurenciella marilzae Microcystis aeruginosa PCC 9808 A0A1Z1MSC3 I4H8H1 Laurencia snackeyi Microcystis aeruginosa PCC 9809 A0A1Z1M1H2 I4HGE7 Laurencieae sp. Microcystis sp. T1-4 A0A0G4KBA5 I4HUY6 Digenea simplex Microcystis aeruginosa PCC 9701 A0A1Z1M2X3 I4IAD9 Cliftonaea pectinata Microcystis aeruginosa TAIHU98 A0A1Z1MUZ0 I4IR44

Sonderella linearis A0A1Z1MQE9 L7E219 Microcystis aeruginosa DIANCHI905 Bostrychia simpliciuscula A0A1Z1MLM5 L8NQ26 Microcystis aeruginosa SPC777

Bostrychia tenella A0A1Z1M8G0 S3K2S2 Leptolyngbya sp. O-77

Bostrychia moritziana A0A1Z1M5C2 A0A0X8WWI5 Phormidium sp. OSCR

Dasyclonium flaccidum A0A1Z1M6U5 A0A0P8A4Z1 Crinalium epipsammum PCC 9333 K9VZ14 Vertebrata lanosa A0A1Z1MKH5 Chamaesiphon polymorphus CCALA 037 A0A2T1GIH8 Vertebrata australis A0A0B5W3L8 Chamaesiphon minutus ATCC 27169 / PCC 6605 K9UJ05 Vertebrata isogona A0A1Z1MJ30 Merismopedia glauca CCAP 1448/3 A0A2T1CA33 Vertebrata thuyoides A0A1Z1MF83 Synechococcus sp. JA-2-3B'a Q2JH66 A0A1Z1MB18 Polysiphonia elongata Synechococcus sp. 60AY4M2 A0A2G8PDT8 A0A1Z1MBV7 Synechococcus sp. JA-3-3Ab Polysiphonia brodiei Q2JXW9 A0A1Z1MAB7 Polysiphonia schneideri Phormidium ambiguum IAM M-71 A0A1U7IU08 A0A1Z1MSY9 Cyanobacteria bacterium UBA11372 Melanothamnus harveyi A0A350XGR8 A0A1Z1MHV2 Cyanobacteria bacterium UBA11371 Polysiphonia infestans A0A351KYA7 A0A1Z1MER9 Cyanobacteria bacterium UBA11368 Polysiphonia sertularioides A0A352YHY3 A0A1Z1M905 Cyanobacteria bacterium UBA11369 Tolypiocladia glomerulata A0A352ZN58 A0A1Z1MUE2 Nostoc flagelliforme CCNUN1 Corynoplastis japonica A0A2K8T9C7 A0A1X9PTR6 cyanobacterium TDX16 Polysiphonia sertularioides A0A254WA60 A0A1Z1MG31 Chroococcidiopsis sp. CCALA 051 Caloglossa beccarii A0A2P8QXV2 A0A1Z1M912 A0A2T1FIB6 Cyanosarcina cf. burmensis CCALA 770 Caloglossa intermedia A0A1Z1M6E0 A0A2T1GER9 Chroococcidiopsis cubana CCALA 043 Caloglossa monosticha A0A1Z1M5F3 K9TTQ3 Chroococcidiopsis thermalis PCC 7203 Thuretia quercifolia A0A1Z1MKH3 A0A0D8ZWS4 Aliterella atlantica CENA595 Platysiphonia delicata A0A1Z1M0F5 A0A2T1EAQ0 Chlorogloea sp. CCALA 695 Dasya naccarioides A0A1Z1MHA4 A0A2G7GHB4 Chroococcales cyanobacterium IPPAS B-1203 Dipterocladia arabiensis A0A1Z1M060 A0A1U7HIL1 Chroogloeocystis siderophila 5.2 s.c.1 Dasya binghamiae A0A1C8XSB1 A0A2S6V987 Chroococcidiopsis sp. TS-821 Membranoptera tenuis A0A1L1YA43 K9X979 Gloeocapsa sp. PCC 7428 A0A1L1WAH1 Membranoptera weeksiae A0A1Z4QR72 Cylindrospermum sp. NIES-4074 A0A1I9KQX5 Membranoptera platyphylla K9WWV7 Cylindrospermum stagnale PCC 7417 A0A1Z1MRS5 Taenioma perpusillum A0A0C1XVN3 Hassallia byssoidea VB512170 A0A1P8D6J7 Gracilaria firma A0A218QGC8 Tolypothrix sp. NIES-4075 A0A2Z2JMY5 Gracilaria changii K9ZBC0 Anabaena cylindrica ATCC 27899 / PCC 7122 A0A345U6I5 Gracilaria caudata A0A1Z4NML7 Calothrix sp. NIES-3974 A0A345U740 A0A0V7ZJR3 Gracilaria ferox W8DXF9 Mastigocoleus testarum BC008 K9PJF3 Gracilaria salicornia A0A345U7P4 Calothrix sp. PCC 7507 A0A0C2M5D0 Gracilaria gracilis A0A345U8V3 Scytonema tolypothrichoides VB-61278 A0A0T7BM90 Gracilaria vermiculophylla Calothrix sp. 336/3 A0A141SEM2 A0A139X8G9 Gracilaria chilensis Q6B8M8 Scytonema hofmannii PCC 7110 K9UYG1 Calothrix sp. PCC 6303 Gracilaria tenuistipitata var. liui A0A345U897 A0A1Q4RY16

Hydropuntia rangiferina A0A1C9CF09 A0A1Z4R6X7 Calothrix sp. HK-06

Gracilariopsis chorda A0A0C5DZM8 A0A0C1QY31 Calothrix sp. NIES-4101 H3JUF7 Gracilariopsis lemaneiformis A0A2A2TDG3 Tolypothrix bouteillei VB521301

Gracilariopsis chorda A0A345UA10 A0A1Z4SP35 Calothrix elsteri CCALA 953

A0A345U9F7 Gracilariopsis mclachlanii A0A2H2Y5P6 Calothrix sp. NIES-4105 A0A344V693 D7E598 Calothrix sp. NIES-4071 Gracilariopsis longissima A0A1C9C9W3 A0A1Z4UPD3 Nostoc azollae 0708 Gracilariopsis heteroclada A0A1C9C7I5 D4TMX9 Sebdenia flabellata Sphaerospermopsis kisseleviana NIES-73 K4PXY9 A0A1X4G2R7 Raphidiopsis brookii D9 Rhodymenia pseudopalmata A0A173FZY2 A0A1Z4VGD3 Sparlingia pertusa Cylindrospermopsis raciborskii CENA303 A0A1C9CD81 A0A0R0M3J9 Raphidiopsis curvata NIES-932 Coeloseira compressa A0A1B0TIE7 A0A1S1ZVX0 Cylindrospermopsis sp. CR12 Ceramium japonicum A0A345UAL3 D4TH90 Cylindrospermopsis raciborskii MVCC14 Ceramium sungminbooi P48446 A0A2S6CT17 Cylindrospermopsis raciborskii CS-505 Melanthalia intermedia A0A1C9C8P4 A0A1Z4SFG8 Cuspidothrix issatschenkoi CHARLIE-1 Aglaothamnion neglectum A0A2H4FRJ8 A0A367Q2E6 Nostoc sp. NIES-4103 A0A1Z4LH84 Schimmelmannia schousboei A0A141SE20 Nostoc minutum NIES-26 Kappaphycus alvarezii A0A141SDH1 K9RKW3 Calothrix parasitica NIES-267 Gelidium vagum A0A0B0QN88 A0A1C9CHQ6 Rivularia sp. PCC 7116 A0A0K2LX35 Gelidium elegans A0A1C9C838 Aphanizomenon flos-aquae 2012/KM1/D3 A0A1B7V2K7 Plocamium cartilagineum A0A1C9C9B9 Anabaena sp. WA102 A0A1B7V5L7 Riquetophycus sp. A0A1C9CC84 A0A1B7WY42 Anabaena sp. LE011-02 Schizymenia dubyi A0A342RZQ5 A0A1Z4V7E8 Anabaena sp. AL09 A0A1Z1M246 Asparagopsis taxiformis M4ITT3 K7WU75 Anabaena sp. MDT14b Mastocarpus papillatus A0A1B7WQY3 Dolichospermum compactum NIES-806 A0A2Z2KXU5 Acrosorium ciliolatum A0A1B7VJG2 Anabaena sp. 90 A0A1C9CCT7 Calliarthron tuberculosum A0A1B7VRR2 Anabaena sp. CRKS33 A0A141SCX2 Porolithon onkodes A0A1B7X5S3 Aphanizomenon flos-aquae LD13 A0A1C9CB03 Bangiopsis subsimplex M5DC07 A0A1Z4L8G1 Anabaena sp. WA113 Sporolithon durum A0A2C6W0B9 J7M8C6 Aphanizomenon flos-aquae WA102 Ahnfeltia plicata I4DEE2 A0A367QBX7 Nostoc linckia NIES-25 Chondrus crispus M1WNW5 A0A1Z1MK15 Nostoc linckia z16 Mazzaella japonica R9XWF2 M1WZJ3 Nostoc sp. ATCC 43529 Chondrus yendoi A0A1U7IKI3 A0A2S1FXE9 Richelia intracellularis HM01 A0A235IDQ1 Spyridia filamentosa A0A1G4P0D4 Richelia intracellularis HH01 A0A2D0HH71 Grateloupia taiwanensis A0A1G4NU14 Nostoc calcicola FACHB-389 A0A2L2NVA1 Grateloupia filicina A0A1G4NZB7 Nostoc sp. 'Peltigera membranacea cyanobiont' 210A A0A367R6U3 A0A1G4NY30 Nostoc sp. 'Peltigera malacea cyanobiont' DB3992 Trichogloeopsis pedicellata A0A1E2WJT2 A0A1G4NZV0 Nostoc sp. 'Lobaria pulmonaria A0A235ID81 A0A1G4NV81 HommersandiophycusHelminthora borowitzkae furcellata A0A235J3B3 Nostoc punctiforme NIES-2108 A0A1G4NUJ4 Nostoc sp. KVJ20 Titanophycus setchellii A0A2L2N7J9

Liagora brachyclada A0A1G4NWW2 A0A2R5FQ87 Nostoc sp. 'Peltigera membranacea cyanobiont' 213 A0A1G4NTB7 Liagora harveyana A0A2Z6D665 Nostoc sp. 'Peltigera membranacea cyanobiont' 232 A0A1G4NRL4 Izziella formosana A0A367QJC3 Nostoc sp. 'Peltigera membranacea cyanobiont' N6 A0A1G4NYJ1 Neoizziella asiatica B2J4V4 Nostoc commune NIES-4072 A0A1G4NVM8 X5JS84 Nostoc commune HK-02 Helminthocladia australis A0A1G4NT79 A0A252DF43 Dermonema virens Nostoc sp. ATCC 53789 A0A1G4NS76 A0A252DVE5 Nostoc punctiforme ATCC 29133 / PCC 73102 Yamadaella caenomyce A0A1C9CH37 A0A252E8J6 Richelia intracellularis Liagoropsis maxima A0A1G4NXD4 A0A1Z4HDN5 Galaxaura rugosa Nostoc sp. RF31Y A0A1G4NWE7 A0A0F6PMW0 Nostoc sp. 106C Dichotomaria marginata A0A1C9CAF6 A0A0P1BTT2 Palmaria palmata Nostoc sp. T09 A0A1C9CGL2 A0A218PZU8 Scinaia undulata Calothrix sp. NIES-2100 A0A1C9CBK1 A0A166KAL5 Chrysosporum ovalisporum ILC-164 Nemalion sp. H.1444 A0A1C9CFJ9 A0ZKT4 Thorea hispida Chrysosporum ovalisporum A0A1C9CG19 A0A1D2PDK2 Nodularia sp. NIES-3585 A0A1C9CE57 A0A1Y0RP61 Kumanoa americana A0A1Z4IP12 Nodularia spumigena CENA596 Apophlaea sinclairii A0A1X9PUV0 A0A1Z1XB58 A0A0C2LQP4 Nodularia spumigena CCY9414 Hildenbrandia rivularis A0A1Y9TLR6 A0A1Z4QAY6 Scytonema millei VB511283 Hildenbrandia rubra A0A126G4B3 A0A0D6KWE3 Nostocales cyanobacterium HT-58-2 Erythrotrichia carnea A0A059XLT0 A0A0M3V5B0 Scytonema sp. HK-05 Rhodochaete parvula A0A059XM83 A0A1C0UYG7 Tolypothrix campylonemoides VB511288 Q1XDC1 A0A1Z4FU91 Scytonema sp. NIES-4073 Compsopogon caeruleus M4QKR9 Boldia erythrosiphon A0A1Z4GBL7 Tolypothrix sp. PCC 7601 M9PQR1 A0A1Z4HVL0 Nostoc piscinale CENA21 Wildemania schizophylla A0A1Z4JWV0 Pyropia kanakaensis A0A1U6ZGL0 Nostoc sp. MBR 210 Pyropia fucicola A0A1Z4ML15 A0A059XH17 Calothrix sp. NIES-2098 Pyropia yezoensis A0A1Z4MSZ2 A0A023HRF9 Anabaenopsis circularis NIES-21 A0A1Z4U130 Pyropia yezoensis A0A141SF51 J7F9M0 Nostoc carneum NIES-2107 A0A2H6LEK2 Pyropia haitanensis Calothrix brevissima NIES-22 A0A2Z6CVQ6 A0A0F6VXM6 Pyropia endiviifolia P51366 Fremyella diplosiphon NIES-3275 K9QF25 Pyropia perforata P48447 A0A2I8A4V6 Tolypothrix tenuis PCC 7101 Pyropia perforata K9QZE6 Aulosira laxa NIES-50 A0A075W3T5 Porphyra pulchra Q85G54 A0A0S3PDY4 Nostoc cycadae WK-1

A0A1W5CMP0 Porphyra umbilicalis Nostoc sp. HK-01 A0A060A4U1O19900 A0A1Z4I8N3 Nostoc sp. PCC 7107 Bangia fuscopurpurea W0RYI8 A0A1Z4KMG5 Porphyra purpurea Nostoc sp. CENA543 A0A358TIR2 Nostoc sp. ATCC 29411 / PCC 7524 Galdieria sulphuraria A0A1C9CDZ3 Q3M7P6 Nostoc sp. NIES-3756 A0A1X9PUD8 Galdieria sulphuraria K9T8W1 Q7ATT3 Anabaena sp. 39858 Q8YNP7 A0A1U7H7J9 Nostoc sp. NIES-2111 A0A1Z4TJI3 Cyanidioschyzon merolae 10D Trichormus variabilis NIES-23 A0A0D6ARB3 A0A2N6KCU4 CyanidiaceaeCyanidium sp. MX-AZ01 caldarium Nostoc sp. UBA8866 A0A0D6ADV1K9Z5A3 A0A0D6YNH2

Porphyridium purpureum A0A0M0SUE0 Anabaena variabilis ATCC 29413 / PCC 7937 Porphyridium sordidum A0A2G3P7N7K9YMU7 A0A0S3TIX7 Anabaena variabilis Flintiella sanguinaria L8LRF9 A0A1U7GW00 Nostoc sp. PCC 7120 / SAG 25.82 / UTEX 2576

K9EMS1 A0A2N6K0J5 Fischerella sp. NIES-4106 Pleurocapsa sp. PCC 7327 A0A2N6KTM5 Fischerella thermalis CCMEE 5268 A0A2N6LGF2 A0A2T1CFX9 Mastigocladus laminosus UU774 Hydrococcus rivularis NIES-593 A0A1Z4RUU5L8LXA0 A0A2N6M0G4 Geminocystis sp. NIES-3709 Hapalosiphon sp. MRB220 A0A2N6M9L9 Geminocystis sp. NIES-3708 Fischerella sp. NIES-3754 A0A2T1C3P7L8M1V5 A0A2N6MCS3 Fischerella major NIES-592 K9XUV5 A0A2N6N0K3 Fischerella muscicola CCMEE 5323 A0A328I6E4 Cyanobacterium aponinum PCC 10605 Fischerella thermalis CCMEE 5273 A0A140K8X3B2J480 A0A370CMT1 Fischerella thermalis CCMEE 5318 Gloeocapsa sp. PCC 73106 G6FQV7 Cyanobacterium aponinum IPPAS B-1201 A0A139X8A9 Fischerella thermalis CCMEE 5319 Leptolyngbya sp. PCC 7375 A0A2L2NT03 A0A0C1MXE9 Fischerella thermalis CCMEE 5330 Pleurocapsa sp. CCALA 161 A0A2D0HNS2 Cyanobacterium stanieri ATCC 29140 / PCC 7202 A0A2N6K7C9 Fischerella thermalis CCMEE 5205 A0A367RYH9 Chondrocystis sp. NIES-4102 M2WV85 Fischerella thermalis CCMEE 5201 Xenococcus sp. PCC 7305 A0A367R6X9 A0A1X6PFB5 Hapalosiphonaceae cyanobacterium JJU2 A0A2L2NGC3 A0A2V3INN9 A0A235J3X4 Mastigocladus laminosus WC112 Xenococcus sp. PCC 7305 M1V7P9 A0A235IKX2 Merismopedia glauca CCAP 1448/3 Fischerella thermalis A0A075BUP2 A0A367PRZ2 Scytonema hofmannii PCC 7110 Stanieria sp. NIES-3757 A0A7B9

A0A1U7HYB6 A0A068NZJ6 Tolypothrix bouteillei VB521301

A0A1E2WR77 A0A068NZU2 Fischerella muscicola CCMEE 5323

A0A2C6VXM8 A0A068P089 Galdieria sulphuraria Nostoc sp. 'Lobaria pulmonaria A0A1Z4L0A9 Stanieria cyanosphaera ATCC 29371 / PCC 7437 A0A068P093 Porphyra umbilicalis A0A1Z4R7I3 A0A068NZM4 Gracilariopsis chorda Nostoc sp. ATCC 53789 A0A0V7ZI40 Nostoc punctiforme ATCC 29133 / PCC 73102 K9UWY5 A0A068NZZ7 Nostoc punctiforme NIES-2108 Cyanidioschyzon merolae 10D A0A068NZT2 Microcystis phage MaMV-DC A0A2H2XTI3 A0A068NZU6 Microcystis virus Ma-LMM01 A0A1Z4TB45 A0A068NZW7 Nostoc sp. 'Peltigera malacea cyanobiont' DB3992 A0A068P039 Myoviridae environmental sample A0A1Q4S4Q0 Nostoc sp. ATCC 43529 F4XQV4 Myoviridae environmental sample A0A1Z4NHJ6 A0A068NZJ0 Nostoc sp. KVJ20 A0A068NZY7 Myoviridae environmental sample Nostoc linckia z16 Nostoc calcicola FACHB-389 A0A068NZL6 Myoviridae environmental sample Nostoc sp. 'Peltigera membranacea cyanobiont' N6 A0A1U7N8R1 A0A068NZH9 A0A1D9G7P0 Myoviridae environmental sample Nostoc sp. 'Peltigera membranacea cyanobiont' 232 A0A068NZH3 Nostoc linckia NIES-25 A0A1D8U001 Myoviridae environmental sample A0A068P042 A0A357A7E7 Nostoc sp. 'Peltigera membranacea cyanobiont' 213 Myoviridae environmental sample Calothrix sp. NIES-4101 A0A068NZE6 A0A1L9QNB3 A0A068NZE4 Myoviridae environmental sample A0A358Y987 Calothrix sp. PCC 6303 A0A068NZF9 Myoviridae environmental sample A0A357PE56 Mastigocoleus testarum BC008 A0A2T1CCH0 Myoviridae environmental sample Calothrix sp. NIES-4071 A0A357GD25 A0YM89 Myoviridae environmental sample A0A354ZT71 Calothrix sp.Calothrix NIES-4105 sp. HK-06 U7QJ99 Myoviridae environmental sample A0A353A6D5 A0A166TWP0 U9W2E5 Myoviridae environmental sample A0A352X8R6 A0A0P7YTB2 Calothrix sp. NIES-3974 K9FME4 Moorea producens 3L A0A1L2BX41 Myoviridae environmental sample U9VWJ6 A0A1U7H1H4 Myoviridae environmental sample A8HTL9 Moorea bouillonii PNG A0A1D8U2M2 Myoviridae environmental sample L8M1R6 Moorea producens JHB Myoviridae environmental sample A0A140K6K1 A0A1B1TZ86 Myoviridae environmental sample K9XU38 A0A1Q2TYQ9 K9Q1L1 A0A2T1CKL8 Myoviridae environmental sample A0A1B1TQG6 A0A1Z4RQX1 Pleurocapsa sp. CCALA 161

K9XYQ0 L8LUA4 Moorea producens PAL-8-15-08-1 Lyngbya sp. PCC 8106 A0A1Q4R1P8 B7KCK5 L8LZH2 Lyngbya aestuarii BL J E0UIK6 CyanobacteriaRoseofilum bacterium reptotaenium UBA11691 AO1-A A0A140KA12 K9EPX3 A0A251WMK9 Leptolyngbya valderiana BDU 20041

A0A2T1GGS1 Phormidesmis priestleyi Ana

K9UHV5 Cyanobacteria bacterium UBA11149 B4WGK0 Bacteriophage vB_NpeS-2AV2 A0A350XIR7 A0A2W5A114 Fischerella major NIES-592 Cyanobacteria bacterium UBA11153 A0A351LH44

A0A352YFA2 Moorea producens PAL-8-15-08-1 Cyanobacteria bacterium UBA11159 A0A0P7Z4B7 B4WKJ3 A0A352ZPS7 Xenococcus sp. PCC 7305 W0FJC0 Cyanobacteria bacterium UBA11166 A0A2W4U0M4 A0A1Z4SA27 Stanieria sp. NIES-3757 A0A367QSY9

Cyanobacteria bacterium UBA11366 A0A1Q8ZEJ9 Stanieria cyanosphaera ATCC 29371 / PCC 7437 Leptolyngbya sp. PCC 7375 Leptolyngbya sp. Heron Island J A0A2N5JPH1 A0A354WHV9 Pleurocapsa sp. CCALA 161 Cyanobacteria bacterium UBA11367 A0A1J1L7J2 A0A2W7AMX2 Chondrocystis sp. NIES-4102 K9WKW6 A0A1J5GQZ6 A0A2I8AFP0 Xenococcus sp. PCC 7305 Leptolyngbya sp. Heron Island J A0A1J1KC59 A0A1Z4HHJ9 A0A1T4ZP47 Cyanothece sp. PCC 7424 A0A1Z4N0R8 Synechococcus sp. PCC 7117 Cyanothece sp. PCC 7822 A0A2Z6CDB0 A0A1Z4UC06 A0A1J1JJU3 Synechococcus sp. NIES-970 A0A0D6KQM2 Alkalinema sp. CACIAM 70d

A0A073CBN1 A0A1Z4M9W1 U7QK54 Chamaesiphon polymorphus CCALA 037 Synechococcus sp. PCC 7003 A0A1J1LHZ9 A0YLI5 Q8RKB4 Leptolyngbya sp. PCC 7376 A0A2K8T8Q7 Chamaesiphon minutus ATCC 27169 / PCC 6605 A0A1J1KX57 A0A1Z4HA11 Limnothrix rosea IAM M-220 Cyanobacteria bacterium UBA11372 H1WK90 A0A2R5FKW6 Stanieria sp. NIES-3757 B5W1Z7 A0A2Z6D5E5 Cyanobacteria bacterium UBA11371

A0A0F5YL77 A0A1Z4FXV5 Cyanobacteria bacterium UBA11368 A0A1Z4K4L2 K1X9Q9 A0A1Z4JTS4 Cyanobacteria bacterium UBA11369 Synechococcus sp. ATCC 27264 / PCC 7002 / PR-6 Xenococcus sp. PCC 7305 D4ZXN0 A0A328V8I3 A0A251WH24 D8FV26 Nostoc sp. NIES-4103 Phormidesmis priestleyi A0A140K1C9 K9XPG1 Leptolyngbya sp. PCC 7375 Nostoc minutum NIES-26 A0A1U7HRA4 K9UGD8 K9T7B0 Leptolyngbya sp. 'hensonii' W0FKH9 K9SCE2 B8HRV7 Cyanobacteria bacterium M5B4 A0A1C0VSD9 K9RWU2

F5UNV7 A0A2D2Q3M8 Leptolyngbya sp. Phormidesmis priestleyiLeptolyngbya Ana foveolarum Q8DMS8 A7LBW5 Microcoleus sp. PCC 7113 A0A355CQY4 V5V6V6 B8HKI1 A0A2G4F1S4 K9VHX7 Nostoc sp. CENA543 A0A2T1C7L6 Stanieria cyanosphaera ATCC 29371 / PCC 7437 A0A1Z4RTD8 G9BWC5 Nostoc carneum NIES-2107 U5XHX4 A0A2A2TCU5 uncultured Oscillatoria sp. A0A2T1G0F2 A0A351KY97

U5XGZ2 A0A352YEM1 A0A2T1EVT9 Tolypothrix tenuis PCC 7101 Planktothrix rubescens A0A1U7HA71 K9T748 W0FL08 A0A1Q4RE36 Aulosira laxa NIES-50 Planktothrix sp. UBA10369 A0A1Z4SV04 A0A2H2XF21

L8LP00 W0FFW9 K9TDB4 A0A1Z4R9R1 Tolypothrix sp. PCC 7601 U7QIX6 A0A2A2TEC5 A0A068N5V0

L8AJF3 A0YTJ3 P73891 A0A2W7B8A5

B7K2A9

A3IV45

B1WZ86

G5JA75 T2II85 Fremyella diplosiphon NIES-3275

A0A2M8JQY0 Synechococcus sp. ATCC 29403 / PCC 7335 U9VZU3 Microchaete diplosiphon K9EQY9 K9PUV9 T2JNP4 T2J9Z8 Nostoc flagelliforme CCNUN1 Planktothrix agardhii T2IHT3 T2IWN3 Calothrix sp. NIES-2100

Q4BWI6 G5JA74 A3IV44 A0A0F5YES0

Planktothrix sp. PCC 11201 L8AI55 Nostoc commune NIES-4072

B1WZ85 P73890 B7K2A8

T2IX71

T2J9L4

T2JR58 Nostoc commune HK-02

K9SCZ5 Synechococcus sp. ATCC 29403 / PCC 7335 Calothrix sp. NIES-2098

Calothrix brevissima NIES-22 Planktothrix agardhii NIES-204 Calothrix brevissima NIES-22

Lyngbya aestuarii BL J A0A068N1X8 Alkalinema sp. CACIAM 70d

Planktothrix paucivesiculata PCC 9631 A0A2W7BQB2 Stanieria sp. NIES-3757

Lyngbya sp. PCC 8106 Stanieria cyanosphaera ATCC 29371 / PCC 7437 Planktothrix tepida PCC 9214 Planktothrix serta PCC 8927 Hydrococcus rivularis NIES-593

Pleurocapsa sp. PCC 7327

Geitlerinema sp. PCC 7407 Oscillatoriales cyanobacterium CG2_30_40_61 Cyanothece sp. PCC 7425 / ATCC 29141

Planktothrix agardhii NIVA-CYA 126/8 Arthrospira sp. PCC 8005 Synechococcus sp. ATCC 27167 / PCC 6312 Arthrospira sp. O9.13F Synechococcus lividus PCC 6715 Limnoraphis robusta CS-951 Thermosynechococcus elongatus BP-1

Thermosynechococcus vulcanus str. Copeland Arthrospira maxima CS-328 Arthrospira platensis C1 Thermosynechococcus sp. NK55a Cyanothece sp. PCC 7425 / ATCC 29141

Merismopedia glauca CCAP 1448/3

Chondrocystis sp. NIES-4102

Calothrix elsteri CCALA 953

Cyanobacteria bacterium UBA11371

Cyanobacteria bacterium UBA11368

Hydrococcus rivularis NIES-593 [Oscillatoria] sp. PCC 6506 Pleurocapsa sp. PCC 7327 Calothrix sp. HK-06

Calothrix sp. NIES-4105

Calothrix sp. NIES-4071

Gloeocapsa sp. PCC 73106

Calothrix sp. NIES-4101

Calothrix elsteri CCALA 953

Synechocystis sp. PCC 6714

Synechocystis sp. PCC6803 chimera

Synechocystis sp. PCC 6803 / Kazusa

Snowella sp.

Cyanothece sp. PCC 8801

Cyanothece sp. CCY0110

Cyanothece sp. ATCC 51142

Crocosphaera watsonii WH 0003 uncultured Phormidium sp. Crocosphaera watsonii WH 8502

"Flavobacterium sp." Microcoleus vaginatus FGP-2

Arthrospira platensis NIES-39 / IAM M-135 Oscillatoriales cyanobacterium USR001 Planktothrix phage PaV-LD

Tychonema bourrellyi FEM_GT703

Microcoleaceae bacterium UBA10368 Lyngbya aestuarii BL J Snowella sp.

uncultured Phormidium sp. Lyngbya sp. PCC 8106

Phormidium phage MIS-PhV1B uncultured Phormidium sp. Oscillatoria nigro-viridis PCC 7112 filamentous cyanobacterium Phorm 6 Phormidium phage MIS-PhV1A

filamentous cyanobacterium Phorm 46

Chamaesiphon minutus ATCC 27169 / PCC 6605 Limnoraphis robusta CS-951

Leptolyngbya sp. PCC 7375

Oscillatoria acuminata PCC 6304 Leptolyngbya sp. PCC 7376

Cyanothece sp. CCY0110 Leptolyngbya sp. Heron Island J

Cyanothece sp. PCC 8801

Cyanothece sp.ATCC 51142

Geitlerinema sp. PCC 7407 Crocosphaera watsonii WH 0402

Crocosphaera watsonii WH 0401

Synechocystis sp. PCC 6714 Crocosphaera watsonii WH 0005

Crocosphaera watsonii WH 8502

Crocosphaera watsonii WH 8501

Crocosphaera watsonii WH 0003

Crocosphaera watsonii WH 0402

Crocosphaera watsonii WH 0401

Crocosphaera watsonii WH 0005

Synechocystis sp. PCC6803 chimera

Synechocystis sp. PCC 6803 / Kazusa

Supplemen tary Figure S4 . Phylogen etic tree of NblA , Ycf18 and NblA -like sequ ences. Bra nches with bootstrap support valu es of at least 80% are indi cated with circl es. G1-NblA and G1b-Nb lA are indicated with asterisks. Interactive repre sentation of the tree is ava ilable at https://itol.embl.de/ tree/1326 810816 9281151544 017427. The taxon omy assign ed to the entries is le ft unchanged except in the follow ing cases: all no n-cyano bacteria l metagen omic assem blies ar e group ed into one category; L8A I55 and L8A JF3 are derived from a Syne chocystis-B acillus chi mera; A0A2 R7TRJ3 is appar ently from a sho rt fragme nt of a Synechococcu s genome mis-cla ssified as Pseu domona s sp. HMWF031 .