Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont

Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales,... Antonie van Leeuwenhoek (2021) 114:2189–2203 https://doi.org/10.1007/s10482-021-01672-x(0123456789().,-volV)(0123456789().,-volV) ORIGINAL PAPER Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont . . . Guido Bonthond Sergei Shalygin Till Bayer Florian Weinberger Received: 28 May 2021 / Accepted: 7 October 2021 / Published online: 21 October 2021 The Author(s) 2021 Abstract This work introduces Waterburya agaro- numerous vitamins, W. agarophytonicola is poten- phytonicola Bonthond and Shalygin gen. nov., sp. nov, tially capable of producing cobalamin (vitamin B ), a baeocyte producing cyanobacterium that was iso- for which A. vermiculophyllum is an auxotroph. With a lated from the rhodophyte Agarophyton vermiculo- taxonomic description of the genus and species and a phyllum (Ohmi) Gurgel et al., an invasive seaweed that draft genome, this study provides as a basis for future has spread across the northern hemisphere. The new research, to uncover the nature of this geographically species genome reveals a diverse repertoire of chemo- independent association between seaweed and taxis and adhesion related genes, including genes cyanobiont. coding for type IV pili assembly proteins and a high number of genes coding for filamentous hemagglu- Keywords Gracilaria vermiculophylla tinin family (FHA) proteins. Among a genetic basis for Cobalamin  Holobiont  Pleurocapsales  Symbiosis the synthesis of siderophores, carotenoids and Vitamin B Guido Bonthond and Sergei Shalygin have contributed equally. Introduction Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ Since the inception of the holobiosis concept by s10482-021-01672-x. Meyer-Abich (1934) and the term ‘holobiont’ was G. Bonthond (&) coined by Margulis (1990), our view of multicellular Institute for Chemistry and Biology of the Marine organisms has changed. The notion that multicellular Environment (ICBM), Carl von Ossietzky University organisms are colonised by complex communities of Oldenburg, Schleusenstrasse 1, 26382 Wilhelmshaven, Germany microbes that affect their physiology and ecology, has e-mail: guidobonthond@gmail.com given rise to new questions. Using amplicon or metagenome sequencing, large amounts of data have G. Bonthond  T. Bayer  F. Weinberger been obtained to characterise substrate and host GEOMAR Helmholtz Centre for Ocean Research Kiel, Du¨sternbrooker Weg 20, 24105 Kiel, Germany associated microbial communities. While these tech- nologies have facilitated a revolution in the field of S. Shalygin microbial ecology, at the same time they have revealed Plant and Environmental Sciences Department, New that the extent of microbial diversity that has not been Mexico State University, 945 College Drive, Las Cruces, NM 88003, USA 123 2190 Antonie van Leeuwenhoek (2021) 114:2189–2203 described and/or cultured is far greater than previously species diversity residing in the order (Shalygin et al. thought (Amann and Rossello´-Mo´ra 2016). To study 2019a). host-microbe interactions and microbial communities The aim of the present work was to isolate, describe in general, it is important that more of these taxa are and sequence the genome of the pleurocapsalean characterised. More isolates, new taxonomic descrip- cyanobacterium that clustered in Bonthond et al. tions and epitypifications are needed to achieve this (2020) into the core OTU associated with A. vermicu- and to ultimately upgrade the available reference lophyllum, to gain insight to its putative functional records (e.g., SILVA; Quast et al. 2013, RefSeq; roles in the seaweed holobiont. Consequently, this O’Leary et al. 2016) on which amplicon and study introduces Waterburya agarophytonicola gen. metagenome sequencing approaches rely. However, and sp. nov. based on the type strain Waterburya while these culture-independent studies are on the one agarophytonicola KI4 . In addition, we highlight hand limited by the substantial number of unknown some genome characteristics that may be of relevance reads, they can at the same time help to point in which to the symbiosis with the host A. vermiculophyllum direction particularly relevant and undescribed species and based on this posit that the cyanobiont may may be found. represent an important source of cobalamin (vitamin In the course of a global study on the invasive B ) for its cobalamin auxotroph host. seaweed Agarophyton vermiculophyllum (Ohmi) Gur- gel et al. (synonym: Gracilaria vermiculophylla), using 16S rRNA gene amplicon sequencing, an Methods operational taxonomic unit (OTU) classified to the cyanobacteria was detected as core holobiont member, Collection i.e. in virtually every sampled host (Bonthond et al. 2020). Besides that the cyanobacterial OTU was During August/September 2017, algae of the Rhodo- associated with A. vermiculophyllum across its global phyte species Agarophyton vermiculophyllum distribution range, it was also the overall most (Fig. S1) were collected from several populations abundant OTU in the macroalgal holobiont and was across the northern hemisphere (see Bonthond et al. rarely detected in the surrounding seawater. Based on 2020 for location details). One of the visited popula- comparison with the SILVA 16S rRNA gene database tions is located at the Falckensteiner Strand near the 0 00 0 00 (Quast et al. 2013) the OTU was initially classified to city Kiel (Germany, 5423 55.3 N, 1011 27.6 E). the genus Pleurocapsa (Pleurocapsales). However, it Aiming to either obtain axenic cultures of the host A. only retrieved poor sequence hits using BLAST, vermiculophyllum itself or cultures from intimately which suggested it to be rather an undescribed related associated symbionts, we took samples of the young- species or a relative without available sequence data. est part of the algae (which are the apical tips) of Cultivation efforts made for the present study yielded approximately one millimetre length. To remove as a non-axenic culture of a slow growing baeocytous many microbes as possible without harming the host, cyanobacterium with some striking morphological the tips were thoroughly rinsed with sterile artificial differences to the genera Pleurocapsa, Chroococcid- seawater (ASW) and transferred to 20 mL test tubes iopsis and other genera in the Pleurocapsales. The with aluminium caps containing 10 mL fresh sterile Pleurocapsales is an ecologically diverse group, ASW and incubated at 15 degrees in near-darkness to including marine, freshwater, endolithic, epiphytic create conditions where microbial productivity and and sponge associated species (Al-Thukair and Gol- proliferation would be minimal but the host is still able ubic 1991; Anagnostidis and Pantazidou 1991; Rippka to grow. Over the period of approximately a year, the et al. 2015; Konstantinou et al. 2018) that counts 247 water was replaced a few times with fresh sterile ASW species in 25 genera (Shalygin et al. 2019b and and the apical fragments rinsed with fresh sterile ASW references therein). Many morphologically described as well. During a visual inspection in late 2018, one of species currently lack DNA sequence data of type the incubations contained high numbers of cyanobac- material and it is suspected that the currently described teria-like cells that appeared to express baeocytous taxa constitute only a minor portion of the actual growth. A small number of the cells was transferred to a new vial and this subculture (without host) was 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2191 incubated under the same conditions. While we were metaSPADES option (Nurk et al. 2017) and the successful maintaining this subculture (labelled KI4), default error correction tool and K-mer sizes of 21, 33 microscopic examination showed that it still contained and 55. Initial bin predictions were done with other (but much smaller) bacterial cells and attempts CONCOCT v1.1.0 (Alneberg et al. 2014), MaxBin2 to obtain completely axenic cyanobacterial isolates v2.2.6 (Wu et al. 2016) and metaBAT2 v2.12.1 (Kang from KI4 by further subculturing single cells failed as et al. 2019). Bins were refined and evaluated with the cells either died or were still non-axenic. An CheckM v1.0.12 (Parks et al. 2015), reassembled with herbarium specimen was prepared from a subsample SPAdes and classified with Megablast (Altschul et al. of KI4 by filtration of the medium and cells through a 1990). The reassembled bin corresponding with 0.2 lm membrane. The filter containing the cyanobac- Waterburya agarophytonicola was extracted and terial cells was submitted to the Algal herbarium of annotated automatically with the prokaryotic genome Natural History Museum of Denmark, Copenhagen. annotation pipeline (PGAP) from NCBI (Tatusova et al. 2016) and the integrated microbial genomes DNA extraction and genome sequencing annotations pipeline (IMGAP) v5.0.20 from the JGI- IMG portal (Chen et al. 2021). The sequence files were Due to the limited amount of cells and the slow deposited in Genbank (accession: PRJNA680001) and growth, a DNA extraction from strain KI4 did not IMG (accession: 246459). To identify secondary resolve detectable DNA concentrations. Therefore, a metabolite clusters we used ANTISMASH v5.2.0 PCR was conducted directly on cells from the culture. (Gruene et al. 2018) with default settings. This was done with the universal forward primer 27F; 0 0 5 -AGAGTTTGATCMTGGCTCAG-3 and a reverse Abundance of W. agarophytonicola on the host primer specific for the Pleurocapsales OTU from Bonthond et al. (2020, pleuro592R; 5 - To compare occurrence of the W. agarophytonicola CACTGCTTGCCAGAAGTTG-3 ). The product was core OTU from Bonthond et al. (2020) among sequenced in both directions at the Institute of Clinical seawater, algal surface and algal tissue, OTU counts Molecular Biology in Kiel using an Applied Biosys- from populations for which seawater, algal surface and tems 3730xl DNA Analyzer and the sequence was algal tissue were sampled, were extracted from the deposited in Genbank (accession: MW113706). 16S-V4 rRNA gene read count table of the respective To sequence the genome, we also transferred one to study. To compare read counts in proportions of the a few cells from the culture to two PCR tubes and used total count among sampled substrates (seawater, algal the EquiPhi29 DNA Polymerase (Thermo Fisher surface, algal tissue), a generalised linear mixed mo- Scientific) for whole genome amplification, following del was fitted, assuming a beta binomial distribution the protocol of the manufacturer with the incubation and a logit in the link function (Douma and Weedon step at 45 C for one hour. Both samples and a blank 2019) and with population and individual identity as were submitted to the Beijing Genomics Institute random intercepts, using the R package glmmTMB (BGI, Shenzhen, China) where library preparation was (Magnusson et al. 2017, sampling and population conducted and both samples were sequenced on the details in Bonthond et al. 2020). MGISEQ-2000 platform. Phylogenetic analyses Processing of sequence data, genome analysis A total of 9593 sequences (16S rRNA genes, with As the culture of Waterburya agarophytonicola KI4 average length about 1400 bp), including the majority was not axenic, the genome sequence data was treated of newly described cyanobacterial taxa and a full as a metagenome and the assembly was performed length 16S rRNA gene consensus sequence (1471 using the tools that are available in the software nucleotides) of W. agarophytonicola extracted from wrapper METAWRAP v1.3.2 (Uritskiy et al. 2018). In the initial genome assembly, were aligned in SINA brief, raw read files were trimmed using cutadapt (Pruesse et al. 2012) based on the secondary structure v1.18 (Martin 2011) and assembled with SPAdes of the 16S rRNA molecule. A maximum likelihood v3.13.0 (Bankevich et al. 2012) with the analysis was conducted with FastTreeMP (Price et al. 123 2192 Antonie van Leeuwenhoek (2021) 114:2189–2203 2010) using the GTR ? G substitution model with 0.39% average read count rare (Fig. 1). These occur- default settings and a 1000 bootstrap iterations, rence data show that W. agarophitonicola is specifi- running on XSEDE (Towns et al. 2014) of the cally found on A. vermiculophyllum and suggest it may CIPRES Gateway (Miller et al. 2015). occur as both endo- and epiphyte on its host. In addition, 95 cyanobacterial genomes and the genomes of four outgroup taxa were downloaded to Phylogenetic analyses construct an alignment, including 31 conserved pro- teins (see Wu and Eisen 2008) which have been used The 16S rRNA gene phylogeny showed a typical in studies on cyanobacterial phylogenetics (Shih et al. clustering of cyanobacterial clades at the order level 2013; Komarek et al. 2014;Osterholm et al. 2020). (Fig. 2a, Zimba et al. 2021). Only Nostocales and With a few exceptions, the genomes used in these Gloeobacterales were monophyletic. The order Pleu- previous studies were also included in our analysis, rocapsales with the new taxon Waterburya agarophy- which was, however, expanded with all currently tonicola was found within a large clade (2803 available Pleurocapsales genomes in RefSeq (O’Leary sequences) containing Chroococcales, Oscillatoriales et al. 2016) and the metagenome sequence of Pleuro- and Spirulinales. A well supported clade (87% on Fast capsa minor HA4230-MV1 , which was recently Maximum Likelihood) including 185 pleurocapsalean designated as epitype for P. minor (Shalygin et al. sequences was in the neighbouring position to the 2019b) and sequenced (Ward et al. 2021). Genomes paraphyletic Chroococcales (Fig. 2b). A large clade from Rhodobacter sphaeroides 2.4.1, Chlorobium including W. agarophytonicola and, surprisingly, the tepidum TLS, Chloroflexus aurantiacus J-10-fl and pseudofilamentous Hyella caespitosa PCC 7516 Heliobacterium modesticaldum Ice1 were acquired to together with the nanocyte producer Chamaecalyx serve as outgroups in the analysis. Using the 31 protein incrassatus PCC 7326 were in the sister position to the sequences from the Stanieria cyanosphaera PCC 7437 as query, a local tblastn (Camacho et al. 2009) was conducted against all downloaded genomes and the draft of W. agarophytonicola KI4 . Amino acid sequences of the best hits from each genome were aligned by protein using MAFFT v7.475 (Katoh et al. 2002). Alignments were then examined, trimmed and concatenated manually, resulting in a 9245 amino acid alignment. A Maximum-likelihood phylogenetic anal- ysis was conducted with RAxML-HPC2 v8.2.12 (Stamatakis 2014) on XSEDE (Towns et al. 2014) using the PROTGAMMA model and partitioned protein substitution models, selected based on the Akaike information criterion, and a 1000 bootstrap iterations. Results and discussion Relative abundance in the holobiont Fig. 1 Estimated abundances of the W. agarophytonicola core The most abundant OTU from Bonthond et al. 2020, OTU, across sampled substrates (i.e., seawater, algal surface and which closely related to W. agarophytonicola, consti- algal tissue) from 6 populations (Japan, China, Germany, France, Virginia, California, details in Bonthond et al. 2020). tutes on average 7.37% of all amplicon reads in tissue The 95% confidence intervals are indicated with shaded samples but also represents 4.68% of the reads in columns. Estimates and intervals were back-transformed from samples taken from the algal surface. However, in the the log scale and multiplied by a 100 to be presented in seawater surrounding the holobiont the OTU is with a percentages 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2193 BC Fig. 2 16S rRNA megaphylogeny with a total of 9593 taxa, Pleurocapsales, with several Chrooccocales clades as sister taxa. showing the position of the new genus and species Waterburya c Detailed view of the order Pleurocapsales, including W. agaro- agarophytonicola. a General view on the collapsed phylogeny phytonicola. A large asterisk indicates the maximum support with leaves depicting major cyanobacterial orders. Units near the value of the Maximum Likelihood, ‘‘hyphen’’ depicts sup- names of the orders show the number of sequences in the port \ 50. Note a, b and c are the same tree with different levels collapsed clades. High support values on the backbone are not of resolution (b and c are zoomed parts of the tree focused on shown (they were 89–99); the node connecting the Prochloro- Pleurocapsales and on Waterburya agarophytonicola). See thrix clade with the rest of the phylogeny did not show high Fig. S2 for an uncollapsed version of the order Pleurocapsales support. b Zoomed view on the clade containing the order fraction in panel c 123 2194 Antonie van Leeuwenhoek (2021) 114:2189–2203 newly described Odorella benthonica clade (Fig. 2c). the related Hyella patelloides LEGE 07179 counting The cryptic clade of Stanieria sensu stricto was 675 scaffolds, and may indicate a high proportion of relatively distant from W. agarophytonicola and the repetitive regions. With a predicted number new taxon W. agarophytonicola definitely forms an 5,168,141 bp in total and an estimated gene count of independent lineage. Whether or not the neighbouring 4902, the genome of W. agarophytonicola is the sequences are part of the new genus Waterburya is a smallest of all 9 genomes currently available in the question outside of this study, for which detailed Pleurocapsales, of which Pleurocapsa sp. CCALA p-distance analysis and analyses of 16S-23S ITS 161 has the next smallest genome (counting rRNA in all of the respective neighbouring strains 5,463,308 bp and 5033 genes) and Pleurocapsa sp. would be required. It can, however, already be stated PCC 7319 has the largest (7,386,997 bp, 6749 genes). that ‘‘Stanieria’’ PCC7103 and ‘‘Stanieria’’ UE7A The draft includes 36 tRNA genes, 3 noncoding have been previously misidentified as members of RNAs, a transfer-messenger RNA gene and 4842 Stanieria and are either members of Waterburya or protein coding genes. As the assembly is based on belong to separate genera (similarity values among short reads only, the number and order of ribosomal these taxa and W. agarophytonicola are around 97%). operon copies could not be resolved. The reassembled The clade distribution in the multiprotein phylogeny genome does not contain 16S rRNA gene copies and (Fig. 3) is similar to phylogenies from previous the full 16S rRNA gene sequence used for the 16S studies based on the same set of proteins (Shih et al. phylogeny (Genbank accession OK044280) was 2013; Koma´rek et al. 2014; Maresˇ 2018;Osterholm retrieved from the initial assembly and likely repre- et al. 2020). Furthermore, the phylogenetic position of sents a consensus sequences of the different copies. W. agarophytonicola in the multiprotein tree is in line Functions were predicted for 3375 genes, resulting in a with our 16S rRNA gene analysis (sister to Pleuro- total count of 1467 hypothetical proteins (Table 1). capsa sensu stricto). In the multiprotein tree, W. Besides W. agarophytonicola, the metagenome agarophytonicola grouped adjacent to the clade con- assembly of the culture contained 5 additional bins taining the recently epitypified Pleurocapsa minor that were predicted to be [ 70% complete, which HA4230-MV1 (Shalygin et al. 2019b). The same were classified to the Proteobacteria (i.e., Alteromon- study also selected a neotype for the generic type; adaceae, Methylophilaceae, Alphaproteobacteria, Pleurocapsa fuliginosa, which is closely related to P. Gammaproteobacteria) and Bacteriodetes (Flavobac- minor (both grouped in the 16S rRNA gene phylogeny teriaceae, see Table S1). In line with the absence of under Pleurocapsa sensu stricto, see Fig. 2. and cyanobacterial bins in the assembly, other cyanobac- Fig. S2). Besides the clear distinct morphology (e.g., teria were never observed microscopically from KI4, P. minor and P. fuliginosa both form pseudofila- supporting that the non-axenic culture is at least uni- ments), as a macroalgal symbiont, W. agarophytoni- cyanobacterial. cola is also ecologically unique and differs from the The W. agarophytonicola genome contains six Pleurocapsa species which were both isolated from KEGG-classified genes that are absent in all other rocky substrates (Shalygin et al. 2019b). However, currently available Pleurocapsales genomes future study is needed to elucidate whether W. (Table S2). These are p-hydroxybenzoate monooxy- agarophytonicola can be found on other substrates genase, hydroxymethyl cephem carbamoyltrans- and if the genus may accommodate more species with ferase, L-ectoine synthase, 7,8-dihydropterin-6-yl- similar symbiotic lifestyles. methyl-4-(beta-D-ribofuranosyl)aminobenzene 5 - phosphate synthase, a solute carrier family 10 General genome statistics (sodium/bile acid cotransporter) protein and an uncharacterised protein possibly involved in the The draft of Waterburya agarophytonicola KI4 biosynthesis of archaeosine. Altogether 73 COG- counts 5,107,674 bp and is predicted to be 98.83% classified genes that are present in the W. agarophy- complete, consisting of 149 scaffolds with an average tonicola genome are absent from other known 289 9 coverage and an N50 of 51,633. This is similar genomes of Pleurocapsales (Table S3). to a recent study from (Brito et al. 2020) who published a short-read based genome assembly of 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2195 123 2196 Antonie van Leeuwenhoek (2021) 114:2189–2203 b Fig. 3 Maximum-likelihood tree based on 31 conserved While the capacity to fix nitrogen is common in the proteins extracted from 96 cyanobacterial genomes. The Pleurocapsales (Rippka et al. 2015) and the nitroge- analysis was conducted with 4 outgroup taxa which have been nase proteins are present in several of the sequenced removed from the figure. Waterburya agarophytonicola is Pleurocapsales genomes (i.e., Myxosarcina sp. GI1, displayed in bold. Branches corresponding to nodes with [ 95 bootstrap support values are thickened and ex-type strains are Pleurocapsales sp. LEGE 06147, Pleurocapsales sp. labelled with an uppercase ‘T’ LEGE 10410 and Xenococcus sp. PCC 7305, Table S1), none of the NifD,-H or -K genes were found in W. agarophytonicola KI4 . Therefore, the genome does not support a nitrogen fixing role for W. Metabolic features agarophytonicola in the A. vermiculophyllum holo- biont, as was hypothesised in Bonthond et al. Besides genes encoding proteins involved in pathways for photosynthesis and carbon fixation, W. agarophy- (2020, 2021). However, the draft contains the ferre- doxin-nitrate and ferredoxin-nitrite reductase (NarA, tonicola’s draft contains a duplicated set of the cytochrome c oxidase subunits I-III and the genes NirA), and also reveals several transporter genes for for succinate dehydrogenase/fumarate reductase extracellular nitrate/nitrite (NrtA, -B, -C). Therefore, (sdhA, -B, -C), which may indicate the capacity for a the abundant occurrence of W. agarophytonicola may heterotrophic lifestyle as well. nonetheless affect nitrogen fluxes in the A. vermicu- lophyllum holobiont at the microscale, decreasing Table 1 Attributes of the Attribute bp Genes % of total Waterburya agarophytonicola KI4 Genome size 5,107,674 100.00% draft genome DNA coding 4,436,783 86.87% GC content 1,978,698 38.74% DNA scaffolds 149 100.00% Total gene count 4902 100.00% RNA genes 40 0.82% tRNA genes 36 0.73% Regulatory and miscellaneous features 20 0.41% Protein coding genes 4842 98.78% With function prediction 3375 68.85% With enzymes 922 18.81% Connected to KEGG pathways 1046 21.34% Connected to KEGG Orthology 1730 35.29% Connected to MetaCyc pathways 798 16.28% With COGs 3270 66.71% With Pfam 3551 72.44% With TIGRfam 1145 23.36% With SMART 1084 22.11% With SUPERFam 3757 76.64% With CATH FunFam 2966 60.51% In internal clusters 1156 23.58% Coding signal peptides 170 3.47% Coding transmembrane proteins 1163 23.73% COG clusters 1658 50.70% Pfam clusters 2030 57.17% TIGRfam clusters 893 77.99% 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2197 nitrate and nitrate concentrations in exchange for attachment and plant-microbe symbiosis, with COG ammonia. annotations for18 genes coding cheY-like chemotaxis Further, a genetic basis for assimilatory sulfate proteins, 3 genes coding signal transduction histidine reduction is present and this pathway is linked with kinase/cheY-like chemotaxis proteins and one gene genes catalysing sulfite production from Alkanesul- for a two-component sensor histidine kinase/cheY- fonate, 3’-phosphoadenosine-5’-phosphosulfate like chemotaxis protein. Moreover, the presence of 23 (PAPS), adenosine-5’-phosphosulfate (PAPS), genes coding for filamentous hemagglutinin family methanesulfonate and thiosulfate. Waterburya agaro- (FHA) proteins and 5 copies for the large exoprotein phytonicola KI4 has a high number of genes coding involved in heme utilization and adhesion suggest W. for carbonic anhydrase (5 KEGG and 8 COG anno- agarophytonicola is well equipped with cellular tations), which is more than any of the other 8 mechanisms related to adhesion (Locht et al. 1993; Pleurocapsales genomes in the IMG database. In Paulsrud and Lindblad 2002). Hemagglutinins such as addition, W. agarophytonicola contains the genes FHA proteins have been associated in adhesion, coding for phycobilisome proteins, i.e.; allophyco- adherence and virulence in plant pathogens (Gottig cyanin, phycocyanin, phycoerythrocyanin and phyco- et al. 2009) but are also utilised by endophytic plant erythrin. Also several carotenoid biosynthesis mutualists (Taghavi et al. 2010). pathways were found, suggesting W. agarophytoni- In addition, 8 genes coding for type IV pili (Tfp) assembly proteins (Pil) and 2 genes coding for cola is capable of synthesising b-carotene, zeaxanthin and canthaxathin. Similar with several other Pleuro- uncharacterised surface proteins with fasciclin capsales the W. agarophytonicola genome further (FAS1) repeats, may further support the presence of features both endoglucanase and b-glucosidase, as adhesion and/or infection mechanisms. Such pili are, well as an endo-1,4-beta-xylanase (KEGG annota- for example, found in plant endophytic Nostoc tions, Table S1). Besides these enzymes for cellulose cyanobacteria, where they are expressed abundantly and hemicellulose degradation the genome contains on the surface of hormogonia, and allow gliding four copies of catechol 2,3-dioxygenase-like lactoyl- motility towards the plant host (Duggan et al. 2007; glutathione lyase (COG annotation; Table S2) that Adams and Duggan 2012). Tfps are typical for a plant could enable W. agarophytonicola to degrade lignine. endophytic lifestyle (Frank 2018) and have also been However, despite its apparent close and, based on the found in unicellular and baeocytous cyanobacteria absence of records from other hosts or habitats, (Herdman and Rippka 1988) and even in bacteria from specific association with a red algal agarophyte, W. other phyla, where they may function in motility, agarophytonicola features no agarase. adhesion, DNA exchange and pathogenesis (Mattick 2002; Adams and Duggan 2012). Chemotaxis and adhesion Vitamins We found various chemotaxis and motility associated genes in Waterburya agarophytonicola’s draft gen- Similar to the other available Pleurocapsales genomes ome, some of which in high copy numbers, which W. agarophytonicola KI4 has the genetic basis for the suggest the cyanobacterium may be responsive to synthesis of various vitamins, including biotin (B ), different stimuli and be capable of directed movement. folate (B ), nicotinic acid (B ), panthothenate (B ), 11 3 5 Notably, many KEGG clusters associated with chemo- riboflavin (B ), thiamine (B ), a-tocopherol (E), 2 1 taxis are present, including genes for the methyl- phylloquinone (K ), menaquinone (K ) and pyridoxal 1 2 accepting chemotaxis protein (MCP), purine-binding 5’-phosphate (B ). Moreover, the genome contains the chemotaxis proteins (cheW) and the two-component pathway for the synthesis of cobalamin (vitamin B ), system (cheA, -B, and -R). The genome also contains including genes encoding cob(I)alamin adenosyltrans- multiple copies of the chemosensory pili system ferase and adenosylcobinamide-GDP ribazoletrans- protein ChpA and the twitching motility proteins ferase. As the production of vitamins is energetically (PilG, -H, -I, -J), as well as gene homologs required for expensive, these metabolites may represent potential positive phototactic motility (PixG, -H, -I, -J, -L). The benefits to W. agarophytonicola’s host. In particular, genome further yields proteins associated with surface vitamin B (cobalamin) is likely a valuable molecule 123 2198 Antonie van Leeuwenhoek (2021) 114:2189–2203 for the red algal host. Most Rhodophyta, including the 2020). The genome also contains a copy of the Florideophyceae –to which A. vermiculophyllum menaquinone-specific isochorismate synthase, a key belongs– express the cobalamin-dependent methion- enzyme in the synthesis of a siderophore group ine synthase (METH, Provasoli and Carlucci 1974) containing NRPSs (Walsh and Gary Marshall 2004). but are cobalamin auxotroph and depend on microbial Iron or other metal scavenging compounds are symbionts to acquire this vitamin (Croft et al. 2005). produced by most bacteria and operate extracellularly. Cobalamin auxotrophs may obtain the vitamin in Siderophores may increase the local iron availability exchange for fixed carbon (e.g., glycerol, Kazamia not only to their producer but at the same time to other et al. 2012). Also Gracilariopsis chorda, the closest microbes with the matching receptors for uptake. relative of A. vermiculophyllum of which a genome While their production can thus be favourable to sequence is currently available, encodes METH and is selective taxa, they may at the same time promote iron thus dependent on the acquisition of cobalamin from starvation in other taxa and therewith strongly influ- microbial sources. Given the core and dominant ence the taxonomic composition in microbial com- presence of W. agarophytonicola in the holobiont munities (Kramer et al. 2020). Mutualistic (Bonthond et al. 2020), this is an important observa- associations between terrestrial plants and siderophore tion and could imply that the cyanobacterium repre- producing endophytic bacteria have been documented sents a major or primary cobalamin source for A. for some time (Loaces et al. 2011; Frank 2018) and also for macroalgae it is thought that microbial vermiculophyllum. If this is true, and cobalamin from W. agarophytonicola is indeed functioning as a mutualists play an important role in the regulation of coenzyme in A. vermiculophyllum METH, another availability of iron and other trace metals (Wichard important question is what the cyanobacterium 2016). The genome further reveals three genes coding receives from the host in return. Rhodophytes produce for ferric uptake regulators (Fur), a common key various carbohydrates (Ito and Hori 1989) and as W. regulator in synthesis and activity of siderophores agarophytonicola has the genetic basis for aerobic (Kramer et al. 2020) and several other proteins related respiration, one hypothesis could be that the cyano- to siderophore transport, including 9 major facilitator biont is able to switch to a heterotrophic lifestyle, superfamily (MFS) permeases and TonB, ExbB, ExbD utilising both oxygen and a carbon source such as transporter proteins (Arstøl and Hohmann-Marriott glycerol from the host. This would also avoid the 2019, Tables S1, S2). Altogether, the presence of necessity to compete for light between the two these different genetic signatures associated with phototrophs. siderophore synthesis thus hint at a role for W. agarophytonicola in the cycling of iron and/or other Secondary metabolites and siderophores trace metals within the holobiont. In total 15 secondary metabolite clusters were Taxonomy detected, including 6 non-ribosomal peptide syn- thetases (NRPSs) or NRPS-like gene clusters, 3 The genus Waterburya and the species W. agarophy- terpenes, 2 bacteriocins, 1 lanthidin, 1 ectoine, a T3- tonicola are here described according to the Interna- polykethinde synthase (PKS) cluster and a hybrid tional Code of Nomenclature for algae, fungi, and NRPS-T1PKS cluster. Some of these clusters may plants (Turland et al. 2018). indicate the capacity for toxin production, including e.g., bacteriocins, which are toxins often produced to inhibit growth of other bacteria (Cotter et al. 2013) and Waterburya Bonthond and Shalygin gen. nov. NRPSs, PKSs or hybrids that may be involved in the synthesis of various toxins, including, Anatoxin-A, Diagnosis Cylindrospermopsin and microcystin (Kehr et al. 2011). Akin to Stanieria by morphology, different from NRPSs are also commonly at the basis for the which by marine habitat, epi-endophytic growth on the synthesis of siderophores and other metallophores rhodophyte A. vermiculophyllum, and based on 16S (Arstøl and Hohmann-Marriott 2019; Kramer et al. rRNA gene phylogeny. 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2199 Description Description In culture, small groups of cells forming blackish Macrocolonies slowly growing in liquid BG11 visible aggregates (macrocolonies). Microcolonies medium (20 PSU) under low light conditions in form consist of mostly individual, spherical cells; or of tight blackish clusters. Microcolonies small, con- oblate-spherical cells in groups growing together. sisting of released baeocytes (3.5–6 lm in diameter), Cells in the microcolony of different sizes, terracotta growing baeocytes (up to 8 lm in diameter), and or pale grey-brown by color. Cells surrounded by thin, large, mature cells ready to form baeocytes through colorless mucilaginous envelopes, never with com- multiple fission (7–20 lm in diameter). Mucilaginous mon slime. Baeocytes formation through multiple envelopes colorless, adherent to the cell walls, rarely fission of the mature cells, which are much larger than slightly widened and more robust especially in the stressed cultures. Released baeocytes growing to large baeocytes. Baeocytes are released by cell wall break- age. Reproduction exclusively by baeocyte sizes (up to 19–20 lm), after which multiple fission production. occurs. Sometimes baeocytes form in the smaller mother cells, 10–15 lm. Growing cells spherical or Etymology oblate-spherical, terracotta by color, released baeo- cytes pale grey-brown. Unreleased baeocytes abun- Named in honour of John B. Waterbury—an important dant, 64 or 128 per one mother cell; small, 2–2.5(3) in cyanobacterial researcher, who worked on morphol- diameter. ogy and physiology of many marine baeocyte producers. Etymology Type species Agarophyton- from the marine red alga Agarophyton vermiculophyllum (Ohmi) Gurgel et al., and -cola Waterburya agarophytonicola from L. n. incola; an inhabitant. Type locality Waterburya agarophytonicola Bonthond 0 00 and Shalygin sp. nov. (Fig. 4) Falckensteiner Strand Kiel (5423 55.3 N, 0 00 1011 27.6 E), within one meter from the surface Holotype (Baltic sea) on Agarophyton vermiculophyllum. Here designated: Specimen on filter, lyophilised. GenBank accession numbers Algal herbarium of Natural History Museum of Denmark, Copenhagen; accession number: C-A- MW113706 (16S rRNA gene partial sequence 99685. obtained with Sanger sequencing), OK044280 (16S rRNA gene complete, from initial genome assembly), Diagnosis PRJNA680001 (draft genome). Morphologically similar to Stanieria sublitoralis, but Taxonomic notes differing by phylogenetic position on the 16S rRNA gene tree and by being associated with Agarophyton The genus Stanieria is highly polyphyletic, even vermiculophyllum. Akin to Chroococcopsis gigantea, though the type species S. cyanosphaera PCC 7437 different by 16S rRNA gene phylogeny, smaller cells, was phylogenetically established. There are at least 5 lacking daughter cells and its distribution in marine to phylogenetically distant clades resembling members brackish habitats. of Stanieria outside of the Stanieria sensu stricto clade. One of them contains Stanieria sp. PCC 7302 isolated from a seawater tank at California Bay (Mexico) (Waterbury and Stanier 1978). This isolate 123 2200 Antonie van Leeuwenhoek (2021) 114:2189–2203 Fig. 4 Microphotographs showing the morphology of Water- mother cell (arrow); d Massive baeocyte formation adjacent to burya agarophytonicola. a Released baeocytes; b Growing the small group of growing cells; e Clear example of baeocyte cells, note that adjacent cells are not a product of binary fission; production; f: Unreleased, released and growing baeocytes. The c Aggregation of the cells and initial baeocyte formation within scale bar equals 10 lm is similar by morphology to Waterburya agarophy- (A. Lindstedt) Anagnostidis & Pantazidou, but this is tonicola, however different by habitat and 16S rRNA questionable, considering vast differences in ecology gene phylogeny. According to Anagnostidis and and geography between these two taxa (isolated in a Pantazidou (1991), Stanieria sp. PCC 7302 may Swedish sublittoral on various sea animals, valves and belong to the established taxon Stanieria sublitoralis algae versus isolated from a water tank in Mexico). To 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2201 Acknowledgements We are grateful to the Institute of confirm the true affiliation of Stanieria sublitoralis Clinical Molecular Biology in Kiel for conducting the Sanger DNA sequencing of type material from the type sequencing as supported in part by the DFG Cluster of locality is required. Excellence ‘‘Future Ocean’’ and we thank especially the technicians T. Naujoks and C. Noack for technical support. We also thank Dr. Stacy Krueger-Hadfield for providing and allowing us to use the photo she took of Agarophyton Conclusion vermiculophyllum which is displayed in Fig. S1. With this work we introduce Waterburya agarophy- Author contributions GB performed sampling, laboratory tonicola Bonthond and Shalygin gen. nov. sp. nov. and work and processed and analysed data. SS performed laboratory work, drafted the taxonomic description and present a draft of its genome. While the exact nature of processed and analysed data. FW performed sampling and this rhodophyte-cyanobacterium symbiosis remains to laboratory work and analysed data. TB performed sampling and be determined in future work, the genome reveals supported with bioinformatic data processing and analyses. GB, clues to its functional roles as a core member in the A. SS, TB and FW conceived the study and wrote the manuscript. vermiculophyllum holobiont (Bonthond et al. 2020). Funding Open Access funding enabled and organized by Altogether, a high number of chemotaxis, adhesion Projekt DEAL. This study was funded by the Deutsche and adherence related genes support a host-associated Forschungsgemeinschaft (DFG, project no. WE2700/5-1 lifestyle for W. agarophytonicola. Genes for adher- granted to FW and BA5508/2-1 granted to TB). ence and virulence (genes for Tfps and FHAs), Data availability The holotype Waterburya combined with occurrence data (Fig. 1) especially agarophytonicola KI4 was deposited in the Algal herbarium hint at an endophytic lifestyle. However, we have not of Natural History Museum of Denmark, Copenhagen; been able to microscopically confirm whether W. accession number: C-A-99685. The partial 16S rRNA gene agarophytonicola occurs endo- and/or epiphytically Sanger sequence and a full 16S rRNA gene consensus sequence obtained from the initial genome assembly were deposited in and whether it is associated intra- and/or extracellu- Genbank under the accessions MW113706 and OK044280, larly, which thus remains a question for feature respectively. The draft genome assembly and PGAP annotation research. This study does not support a diazotrophic are available in the Short Read Archive (SRA, accession role for the cyanobacterium in the holobiont, but PRJNA680001). Annotations from the IMGAP can be accessed on IMG (Taxon ID: 2913235824, submission ID: 246459). instead demonstrates it has the potential to function as a source of vitamins, in particular cobalamin, to the Declarations vitamin B -auxotrophic host. In addition, W. agaro- phytonicola may possibly facilitate uptake of iron and/ Conflicts of interest The authors declare that they have no or other trace metals by its host. To further investigate conflict of interest. the relationship between W. agarophytonicola and A. Open Access This article is licensed under a Creative Com- vermiculophyllum, advanced microscopic work is mons Attribution 4.0 International License, which permits use, needed to confirm the endo- and/or epiphytic occur- sharing, adaptation, distribution and reproduction in any med- rence of the cyanobiont and identify whether W. ium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative agarophytonicola produces chlorophyll in association Commons licence, and indicate if changes were made. The with the host, to decipher if it can adapt a heterotrophic images or other third party material in this article are included in lifestyle. Moreover, this may help to explore whether the article’s Creative Commons licence, unless indicated pili-like structures, such as those richly reflected in the otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your genome, are expressed by the cyanobiont. To shed intended use is not permitted by statutory regulation or exceeds light on how specific the relationship between A. the permitted use, you will need to obtain permission directly vermiculophyllum and W. agarophytonicola is, related from the copyright holder. To view a copy of this licence, visit hosts need to be studied as well. Finally, metabolic http://creativecommons.org/licenses/by/4.0/. assays should be carried out in an experimental context to quantify cobalamin production and to screen for host cell wall carbohydrates or other host molecules that are potentially metabolised by W. agarophytonicola. 123 2202 Antonie van Leeuwenhoek (2021) 114:2189–2203 References canker, is involved in bacterial virulence. PLoS ONE 4:e4358 Gruene T, Wennmacher JTC, Zaubitzer C et al (2018) Rapid Adams DG, Duggan PS (2012) Signalling in cyanobacteria– structure determination of microcrystalline molecular plant symbioses. Signaling and communication in plant compounds using electron diffraction. Angew Chemie Int symbiosis. Springer, New York, pp 93–121 Ed 57:16313–16317 Alneberg J, Bjarnason BS, De Bruijn I et al (2014) Binning Herdman M, Rippka R (1988) [22] Cellular differentiation: metagenomic contigs by coverage and composition. Nat hormogonia and baeocytes. Methods Enzymol Methods 11:1144–1146 167:232–242 Al-Thukair AA, Golubic S (1991) New endolithic cyanobacteria Ito K, Hori K (1989) Seaweed: chemical composition and from the arabian gulf. I. Hyella immanis sp. nov. 1. J Phy- potential food uses. Food Rev Int 5:101–144 col 27:766–780 Kang DD, Li F, Kirton E et al (2019) MetaBAT 2: an adaptive Altschul SF, Gish W, Miller W et al (1990) Basic local align- binning algorithm for robust and efficient genome recon- ment search tool. J Mol Biol 215:403–410 struction from metagenome assemblies. PeerJ 7:e7359 ´ ´ Amann R, Rossello-Mora R (2016) After all, only millions? Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel Mbio 74:e0999-e1016 method for rapid multiple sequence alignment based on Anagnostidis K, Pantazidou A (1991) Marine and aerophytic fast Fourier transform. Nucleic Acids Res 30:3059–3066 cyanosarcina, staniera and pseudocapsa (Chroococcales) Kazamia E, Czesnick H, Van NTT et al (2012) Mutualistic species from Hellas (Greece). Algol Stud fu¨r Hydrobiol interactions between vitamin B12-dependent algae and Suppl Vol 141–157 heterotrophic bacteria exhibit regulation. Environ Micro- Arstøl E, Hohmann-Marriott MF (2019) Cyanobacterial side- biol 14:1466–1476 rophores—physiology, structure, biosynthesis, and appli- Kehr J-C, Picchi DG, Dittmann E (2011) Natural product cations. Mar Drugs 17:281 biosyntheses in cyanobacteria: a treasure trove of unique Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: a new enzymes. Beilstein J Org Chem 7:1622–1635 genome assembly algorithm and its applications to single- ´ ˇ ´ ˇ Komarek J, Kastovsky J, Mares J, Johansen JR (2014) Taxo- cell sequencing. J Comput Biol 19:455–477 nomic classification of cyanoprokaryotes (cyanobacterial Bonthond G, Bayer T, Krueger-Hadfield SA et al (2020) How do genera) 2014, using a polyphasic approach. Preslia microbiota associated with an invasive seaweed vary 86:295–335 across scales? Mol Ecol 29:2094–2108. https://doi.org/10. Konstantinou D, Gerovasileiou V, Voultsiadou E, Gkelis S 1111/mec.15470 (2018) Sponges-cyanobacteria associations: global diver- Bonthond G, Bayer T, Krueger-Hadfield SA et al (2021) The sity overview and new data from the eastern mediter- role of host promiscuity in the invasion process of a sea- ranean. PLoS ONE 13:e0195001 weed holobiont. ISME J 15:1668–1679. https://doi.org/10. ¨ ¨ Kramer J, Ozkaya O,Ku¨mmerli R (2020) Bacterial siderophores 1038/s41396-020-00878-7 in community and host interactions. Nat Rev Microbiol Brito A, Vieira J, Vieira CP et al (2020) Comparative genomics 18:152–163 discloses the uniqueness and the biosynthetic potential of Loaces I, Ferrando L, Scavino AF (2011) Dynamics, diversity the marine cyanobacterium hyella patelloides. Front and function of endophytic siderophore-producing bacteria Microbiol 11:1527 in rice. Microb Ecol 61:606–618 Camacho C, Coulouris G, Avagyan V et al (2009) BLAST?: Locht C, Berlin P, Menozzi FD, Renauld G (1993) The fila- architecture and applications. BMC Bioinformatics 10:1–9 mentous haemagglutinin, a multifaceted adhesin produced Chen I-MA, Chu K, Palaniappan K et al (2021) The IMG/M data by virulent Bordetella spp. Mol Microbiol 9:653–660 management and analysis system v. 6.0: new tools and Magnusson A, Skaug H, Nielsen A, et al (2017) Package advanced capabilities. Nucleic Acids Res 49:D751–D763 ‘glmmTMB.’ R packag version 02 0 Cotter PD, Ross RP, Hill C (2013) Bacteriocins—a viable Maresˇ J (2018) Multilocus and SSU rRNA gene phylogenetic alternative to antibiotics? Nat Rev Microbiol 11:95–105 analyses of available cyanobacterial genomes, and their Croft MT, Lawrence AD, Raux-Deery E et al (2005) Algae relation to the current taxonomic system. Hydrobiologia acquire vitamin B 12 through a symbiotic relationship with 811:19–34 bacteria. Nature 438:90–93 Margulis L (1990) Words as battle cries–symbiogenesis and the Douma JC, Weedon JT (2019) Analysing continuous propor- new field of endocytobiology. Bioscience 40:673–677. tions in ecology and evolution: a practical introduction to https://doi.org/10.2307/1311435 beta and Dirichlet regression. Methods Ecol Evol Martin M (2011) Cutadapt removes adapter sequences from 10:1412–1430 high-throughput sequencing reads. Embnet J 17:10–12 Duggan PS, Gottardello P, Adams DG (2007) Molecular anal- Mattick JS (2002) Type IV pili and twitching motility. Annu ysis of genes in Nostoc punctiforme involved in pilus Rev Microbiol 56:289–314 biogenesis and plant infection. J Bacteriol 189:4547–4551 Meyer-Abich A (1934) Beitra¨ge zur theorie der evolution der Frank AC (2018) The genomes of endophytic bacteria. Endo- organismen. I. das typologische grundgesetz und seine fol- phytes of forest trees. Springer, New York, pp 141–176 gerungen fu¨r phylogenie und entwicklungsphysiologie. Acta Gottig N, Garavaglia BS, Garofalo CG et al (2009) A filamen- Biotheor 7:1–80. https://doi.org/10.1007/BF01603792 tous hemagglutinin-like protein of Xanthomonas axono- Miller MA, Schwartz T, Pickett BE et al (2015) A RESTful API podis pv. citri, the phytopathogen responsible for citrus for access to phylogenetic tools via the CIPRES science gateway. Evol Bioinform 11:EBO-S21501 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2203 Nurk S, Meleshko D, Korobeynikov A, Pevzner PA (2017) Stamatakis A (2014) RAxML version 8: a tool for phylogenetic metaSPAdes: a new versatile metagenomic assembler. analysis and post-analysis of large phylogenies. Bioinfor- Genome Res 27:824–834 matics 30:1312–1313 O’Leary NA, Wright MW, Brister JR et al (2016) Reference Taghavi S, Van Der Lelie D, Hoffman A et al (2010) Genome sequence (RefSeq) database at NCBI: current status, tax- sequence of the plant growth promoting endophytic bac- onomic expansion, and functional annotation. Nucleic terium Enterobacter sp 638. PLoS Genet 6:e1000943 Acids Res 44:D733–D745 Tatusova T, DiCuccio M, Badretdin A et al (2016) NCBI Osterholm J, Popin RV, Fewer DP, Sivonen K (2020) Phy- prokaryotic genome annotation pipeline. Nucleic Acids logenomic analysis of secondary metabolism in the toxic Res 44:6614–6624 cyanobacterial genera Anabaena. Dolichospermum Apha- Towns J, Cockerill T, Dahan M et al (2014) XSEDE: acceler- nizomenon Toxins (basel) 12:248 ating scientific discovery. Comput Sci Eng 16:62–74 Parks DH, Imelfort M, Skennerton CT et al (2015) CheckM: Turland NJ, Wiersema JH, Barrie FR, et al (2018) International assessing the quality of microbial genomes recovered from code of nomenclature for algae, fungi, and plants (Shen- isolates, single cells, and metagenomes. Genome Res zhen Code) adopted by the nineteenth international 25:1043–1055 botanical Congress shenzhen, China, July 2017. Koeltz Paulsrud P, Lindblad P (2002) Fasciclin domain proteins are Botanical Books present in Nostoc symbionts of lichens. Appl Environ Uritskiy GV, DiRuggiero J, Taylor J (2018) MetaWRAP—a Microbiol 68:2036–2039 flexible pipeline for genome-resolved metagenomic data Price MN, Dehal PS, Arkin AP (2010) FastTree 2–approxi- analysis. Microbiome 6:1–13 mately maximum-likelihood trees for large alignments. Walsh CT, Gary Marshall C (2004) Siderophore biosynthesis in PLoS ONE 5:e9490 bacteria. Iron Transp Bact 18–37 Provasoli L, Carlucci AF (1974) Vitamins and growth regula- Ward RD, Stajich JE, Johansen JR et al (2021) Metagenome tors. Bot Monogr sequencing to explore phylogenomics of terrestrial Pruesse E, Peplies J, Glockner FO (2012) SINA: accurate high- cyanobacteria. Microbiol Resour Announc 10:e00258- throughput multiple sequence alignment of ribosomal e321 RNA genes. Bioinformatics 28:1823–1829 Waterbury JB, Stanier RY (1978) Patterns of growth and Quast C, Pruesse E, Yilmaz P et al (2013) The SILVA ribosomal development in pleurocapsalean cyanobacteria. Microbiol RNA gene database project: improved data processing and Rev 42:2 web-based tools. Nucleic Acids Res 41:D590–D596. Wichard T (2016) Identification of metallophores and organic https://doi.org/10.1093/nar/gks1219 ligands in the chemosphere of the marine macroalga Ulva Rippka R, Waterbury JB, Herdman M, Castenholz RW (2015) (Chlorophyta) and at land-sea interfaces. Front Mar Sci Pleurocapsa-group. Bergey’s manual of systematics of 3:131 archaea and bacteria. Wiley, New Jersey, pp 1–9 Wu M, Eisen JA (2008) A simple, fast, and accurate method of Shalygin S, Huang I, Allen EH et al (2019a) Odorella ben- phylogenomic inference. Genome Biol 9:1–11 thonica gen. & sp. nov. (Pleurocapsales, Cyanobacteria): Wu Y-W, Simmons BA, Singer SW (2016) MaxBin 2.0: an an odor and prolific toxin producer isolated from a Cali- automated binning algorithm to recover genomes from fornia aqueduct. J Phycol 55:509–520 multiple metagenomic datasets. Bioinformatics 32:605–607 Shalygin S, Kavulic KJ, Pietrasiak N et al (2019b) Neotypifi- Zimba PV, Shalygin S, Huang I-S et al (2021) A new boring cation of Pleurocapsa fuliginosa and epitypification of P. toxin producer–perforafilum tunnelli gen. & sp. nov. (Os- minor (Pleurocapsales): resolving a polyphyletic cillatoriales, Cyanobacteria) isolated from Laguna Madre, cyanobacterial genus. Phytotaxa 392:245 Texas, USA. Phycologia 60:10–24 Shih PM, Wu D, Latifi A et al (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome Publisher’s Note Springer Nature remains neutral with sequencing. Proc Natl Acad Sci 110:1053–1058 regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antonie van Leeuwenhoek Springer Journals

Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont

Download PDF
15 pages

Loading next page...
 
/lp/springer-journals/draft-genome-and-description-of-waterburya-agarophytonicola-gen-nov-sp-O3sgn9FGDd

References (76)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2021
ISSN
0003-6072
eISSN
1572-9699
DOI
10.1007/s10482-021-01672-x
Publisher site
See Article on Publisher Site

Abstract

Antonie van Leeuwenhoek (2021) 114:2189–2203 https://doi.org/10.1007/s10482-021-01672-x(0123456789().,-volV)(0123456789().,-volV) ORIGINAL PAPER Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont . . . Guido Bonthond Sergei Shalygin Till Bayer Florian Weinberger Received: 28 May 2021 / Accepted: 7 October 2021 / Published online: 21 October 2021 The Author(s) 2021 Abstract This work introduces Waterburya agaro- numerous vitamins, W. agarophytonicola is poten- phytonicola Bonthond and Shalygin gen. nov., sp. nov, tially capable of producing cobalamin (vitamin B ), a baeocyte producing cyanobacterium that was iso- for which A. vermiculophyllum is an auxotroph. With a lated from the rhodophyte Agarophyton vermiculo- taxonomic description of the genus and species and a phyllum (Ohmi) Gurgel et al., an invasive seaweed that draft genome, this study provides as a basis for future has spread across the northern hemisphere. The new research, to uncover the nature of this geographically species genome reveals a diverse repertoire of chemo- independent association between seaweed and taxis and adhesion related genes, including genes cyanobiont. coding for type IV pili assembly proteins and a high number of genes coding for filamentous hemagglu- Keywords Gracilaria vermiculophylla tinin family (FHA) proteins. Among a genetic basis for Cobalamin  Holobiont  Pleurocapsales  Symbiosis the synthesis of siderophores, carotenoids and Vitamin B Guido Bonthond and Sergei Shalygin have contributed equally. Introduction Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ Since the inception of the holobiosis concept by s10482-021-01672-x. Meyer-Abich (1934) and the term ‘holobiont’ was G. Bonthond (&) coined by Margulis (1990), our view of multicellular Institute for Chemistry and Biology of the Marine organisms has changed. The notion that multicellular Environment (ICBM), Carl von Ossietzky University organisms are colonised by complex communities of Oldenburg, Schleusenstrasse 1, 26382 Wilhelmshaven, Germany microbes that affect their physiology and ecology, has e-mail: guidobonthond@gmail.com given rise to new questions. Using amplicon or metagenome sequencing, large amounts of data have G. Bonthond  T. Bayer  F. Weinberger been obtained to characterise substrate and host GEOMAR Helmholtz Centre for Ocean Research Kiel, Du¨sternbrooker Weg 20, 24105 Kiel, Germany associated microbial communities. While these tech- nologies have facilitated a revolution in the field of S. Shalygin microbial ecology, at the same time they have revealed Plant and Environmental Sciences Department, New that the extent of microbial diversity that has not been Mexico State University, 945 College Drive, Las Cruces, NM 88003, USA 123 2190 Antonie van Leeuwenhoek (2021) 114:2189–2203 described and/or cultured is far greater than previously species diversity residing in the order (Shalygin et al. thought (Amann and Rossello´-Mo´ra 2016). To study 2019a). host-microbe interactions and microbial communities The aim of the present work was to isolate, describe in general, it is important that more of these taxa are and sequence the genome of the pleurocapsalean characterised. More isolates, new taxonomic descrip- cyanobacterium that clustered in Bonthond et al. tions and epitypifications are needed to achieve this (2020) into the core OTU associated with A. vermicu- and to ultimately upgrade the available reference lophyllum, to gain insight to its putative functional records (e.g., SILVA; Quast et al. 2013, RefSeq; roles in the seaweed holobiont. Consequently, this O’Leary et al. 2016) on which amplicon and study introduces Waterburya agarophytonicola gen. metagenome sequencing approaches rely. However, and sp. nov. based on the type strain Waterburya while these culture-independent studies are on the one agarophytonicola KI4 . In addition, we highlight hand limited by the substantial number of unknown some genome characteristics that may be of relevance reads, they can at the same time help to point in which to the symbiosis with the host A. vermiculophyllum direction particularly relevant and undescribed species and based on this posit that the cyanobiont may may be found. represent an important source of cobalamin (vitamin In the course of a global study on the invasive B ) for its cobalamin auxotroph host. seaweed Agarophyton vermiculophyllum (Ohmi) Gur- gel et al. (synonym: Gracilaria vermiculophylla), using 16S rRNA gene amplicon sequencing, an Methods operational taxonomic unit (OTU) classified to the cyanobacteria was detected as core holobiont member, Collection i.e. in virtually every sampled host (Bonthond et al. 2020). Besides that the cyanobacterial OTU was During August/September 2017, algae of the Rhodo- associated with A. vermiculophyllum across its global phyte species Agarophyton vermiculophyllum distribution range, it was also the overall most (Fig. S1) were collected from several populations abundant OTU in the macroalgal holobiont and was across the northern hemisphere (see Bonthond et al. rarely detected in the surrounding seawater. Based on 2020 for location details). One of the visited popula- comparison with the SILVA 16S rRNA gene database tions is located at the Falckensteiner Strand near the 0 00 0 00 (Quast et al. 2013) the OTU was initially classified to city Kiel (Germany, 5423 55.3 N, 1011 27.6 E). the genus Pleurocapsa (Pleurocapsales). However, it Aiming to either obtain axenic cultures of the host A. only retrieved poor sequence hits using BLAST, vermiculophyllum itself or cultures from intimately which suggested it to be rather an undescribed related associated symbionts, we took samples of the young- species or a relative without available sequence data. est part of the algae (which are the apical tips) of Cultivation efforts made for the present study yielded approximately one millimetre length. To remove as a non-axenic culture of a slow growing baeocytous many microbes as possible without harming the host, cyanobacterium with some striking morphological the tips were thoroughly rinsed with sterile artificial differences to the genera Pleurocapsa, Chroococcid- seawater (ASW) and transferred to 20 mL test tubes iopsis and other genera in the Pleurocapsales. The with aluminium caps containing 10 mL fresh sterile Pleurocapsales is an ecologically diverse group, ASW and incubated at 15 degrees in near-darkness to including marine, freshwater, endolithic, epiphytic create conditions where microbial productivity and and sponge associated species (Al-Thukair and Gol- proliferation would be minimal but the host is still able ubic 1991; Anagnostidis and Pantazidou 1991; Rippka to grow. Over the period of approximately a year, the et al. 2015; Konstantinou et al. 2018) that counts 247 water was replaced a few times with fresh sterile ASW species in 25 genera (Shalygin et al. 2019b and and the apical fragments rinsed with fresh sterile ASW references therein). Many morphologically described as well. During a visual inspection in late 2018, one of species currently lack DNA sequence data of type the incubations contained high numbers of cyanobac- material and it is suspected that the currently described teria-like cells that appeared to express baeocytous taxa constitute only a minor portion of the actual growth. A small number of the cells was transferred to a new vial and this subculture (without host) was 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2191 incubated under the same conditions. While we were metaSPADES option (Nurk et al. 2017) and the successful maintaining this subculture (labelled KI4), default error correction tool and K-mer sizes of 21, 33 microscopic examination showed that it still contained and 55. Initial bin predictions were done with other (but much smaller) bacterial cells and attempts CONCOCT v1.1.0 (Alneberg et al. 2014), MaxBin2 to obtain completely axenic cyanobacterial isolates v2.2.6 (Wu et al. 2016) and metaBAT2 v2.12.1 (Kang from KI4 by further subculturing single cells failed as et al. 2019). Bins were refined and evaluated with the cells either died or were still non-axenic. An CheckM v1.0.12 (Parks et al. 2015), reassembled with herbarium specimen was prepared from a subsample SPAdes and classified with Megablast (Altschul et al. of KI4 by filtration of the medium and cells through a 1990). The reassembled bin corresponding with 0.2 lm membrane. The filter containing the cyanobac- Waterburya agarophytonicola was extracted and terial cells was submitted to the Algal herbarium of annotated automatically with the prokaryotic genome Natural History Museum of Denmark, Copenhagen. annotation pipeline (PGAP) from NCBI (Tatusova et al. 2016) and the integrated microbial genomes DNA extraction and genome sequencing annotations pipeline (IMGAP) v5.0.20 from the JGI- IMG portal (Chen et al. 2021). The sequence files were Due to the limited amount of cells and the slow deposited in Genbank (accession: PRJNA680001) and growth, a DNA extraction from strain KI4 did not IMG (accession: 246459). To identify secondary resolve detectable DNA concentrations. Therefore, a metabolite clusters we used ANTISMASH v5.2.0 PCR was conducted directly on cells from the culture. (Gruene et al. 2018) with default settings. This was done with the universal forward primer 27F; 0 0 5 -AGAGTTTGATCMTGGCTCAG-3 and a reverse Abundance of W. agarophytonicola on the host primer specific for the Pleurocapsales OTU from Bonthond et al. (2020, pleuro592R; 5 - To compare occurrence of the W. agarophytonicola CACTGCTTGCCAGAAGTTG-3 ). The product was core OTU from Bonthond et al. (2020) among sequenced in both directions at the Institute of Clinical seawater, algal surface and algal tissue, OTU counts Molecular Biology in Kiel using an Applied Biosys- from populations for which seawater, algal surface and tems 3730xl DNA Analyzer and the sequence was algal tissue were sampled, were extracted from the deposited in Genbank (accession: MW113706). 16S-V4 rRNA gene read count table of the respective To sequence the genome, we also transferred one to study. To compare read counts in proportions of the a few cells from the culture to two PCR tubes and used total count among sampled substrates (seawater, algal the EquiPhi29 DNA Polymerase (Thermo Fisher surface, algal tissue), a generalised linear mixed mo- Scientific) for whole genome amplification, following del was fitted, assuming a beta binomial distribution the protocol of the manufacturer with the incubation and a logit in the link function (Douma and Weedon step at 45 C for one hour. Both samples and a blank 2019) and with population and individual identity as were submitted to the Beijing Genomics Institute random intercepts, using the R package glmmTMB (BGI, Shenzhen, China) where library preparation was (Magnusson et al. 2017, sampling and population conducted and both samples were sequenced on the details in Bonthond et al. 2020). MGISEQ-2000 platform. Phylogenetic analyses Processing of sequence data, genome analysis A total of 9593 sequences (16S rRNA genes, with As the culture of Waterburya agarophytonicola KI4 average length about 1400 bp), including the majority was not axenic, the genome sequence data was treated of newly described cyanobacterial taxa and a full as a metagenome and the assembly was performed length 16S rRNA gene consensus sequence (1471 using the tools that are available in the software nucleotides) of W. agarophytonicola extracted from wrapper METAWRAP v1.3.2 (Uritskiy et al. 2018). In the initial genome assembly, were aligned in SINA brief, raw read files were trimmed using cutadapt (Pruesse et al. 2012) based on the secondary structure v1.18 (Martin 2011) and assembled with SPAdes of the 16S rRNA molecule. A maximum likelihood v3.13.0 (Bankevich et al. 2012) with the analysis was conducted with FastTreeMP (Price et al. 123 2192 Antonie van Leeuwenhoek (2021) 114:2189–2203 2010) using the GTR ? G substitution model with 0.39% average read count rare (Fig. 1). These occur- default settings and a 1000 bootstrap iterations, rence data show that W. agarophitonicola is specifi- running on XSEDE (Towns et al. 2014) of the cally found on A. vermiculophyllum and suggest it may CIPRES Gateway (Miller et al. 2015). occur as both endo- and epiphyte on its host. In addition, 95 cyanobacterial genomes and the genomes of four outgroup taxa were downloaded to Phylogenetic analyses construct an alignment, including 31 conserved pro- teins (see Wu and Eisen 2008) which have been used The 16S rRNA gene phylogeny showed a typical in studies on cyanobacterial phylogenetics (Shih et al. clustering of cyanobacterial clades at the order level 2013; Komarek et al. 2014;Osterholm et al. 2020). (Fig. 2a, Zimba et al. 2021). Only Nostocales and With a few exceptions, the genomes used in these Gloeobacterales were monophyletic. The order Pleu- previous studies were also included in our analysis, rocapsales with the new taxon Waterburya agarophy- which was, however, expanded with all currently tonicola was found within a large clade (2803 available Pleurocapsales genomes in RefSeq (O’Leary sequences) containing Chroococcales, Oscillatoriales et al. 2016) and the metagenome sequence of Pleuro- and Spirulinales. A well supported clade (87% on Fast capsa minor HA4230-MV1 , which was recently Maximum Likelihood) including 185 pleurocapsalean designated as epitype for P. minor (Shalygin et al. sequences was in the neighbouring position to the 2019b) and sequenced (Ward et al. 2021). Genomes paraphyletic Chroococcales (Fig. 2b). A large clade from Rhodobacter sphaeroides 2.4.1, Chlorobium including W. agarophytonicola and, surprisingly, the tepidum TLS, Chloroflexus aurantiacus J-10-fl and pseudofilamentous Hyella caespitosa PCC 7516 Heliobacterium modesticaldum Ice1 were acquired to together with the nanocyte producer Chamaecalyx serve as outgroups in the analysis. Using the 31 protein incrassatus PCC 7326 were in the sister position to the sequences from the Stanieria cyanosphaera PCC 7437 as query, a local tblastn (Camacho et al. 2009) was conducted against all downloaded genomes and the draft of W. agarophytonicola KI4 . Amino acid sequences of the best hits from each genome were aligned by protein using MAFFT v7.475 (Katoh et al. 2002). Alignments were then examined, trimmed and concatenated manually, resulting in a 9245 amino acid alignment. A Maximum-likelihood phylogenetic anal- ysis was conducted with RAxML-HPC2 v8.2.12 (Stamatakis 2014) on XSEDE (Towns et al. 2014) using the PROTGAMMA model and partitioned protein substitution models, selected based on the Akaike information criterion, and a 1000 bootstrap iterations. Results and discussion Relative abundance in the holobiont Fig. 1 Estimated abundances of the W. agarophytonicola core The most abundant OTU from Bonthond et al. 2020, OTU, across sampled substrates (i.e., seawater, algal surface and which closely related to W. agarophytonicola, consti- algal tissue) from 6 populations (Japan, China, Germany, France, Virginia, California, details in Bonthond et al. 2020). tutes on average 7.37% of all amplicon reads in tissue The 95% confidence intervals are indicated with shaded samples but also represents 4.68% of the reads in columns. Estimates and intervals were back-transformed from samples taken from the algal surface. However, in the the log scale and multiplied by a 100 to be presented in seawater surrounding the holobiont the OTU is with a percentages 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2193 BC Fig. 2 16S rRNA megaphylogeny with a total of 9593 taxa, Pleurocapsales, with several Chrooccocales clades as sister taxa. showing the position of the new genus and species Waterburya c Detailed view of the order Pleurocapsales, including W. agaro- agarophytonicola. a General view on the collapsed phylogeny phytonicola. A large asterisk indicates the maximum support with leaves depicting major cyanobacterial orders. Units near the value of the Maximum Likelihood, ‘‘hyphen’’ depicts sup- names of the orders show the number of sequences in the port \ 50. Note a, b and c are the same tree with different levels collapsed clades. High support values on the backbone are not of resolution (b and c are zoomed parts of the tree focused on shown (they were 89–99); the node connecting the Prochloro- Pleurocapsales and on Waterburya agarophytonicola). See thrix clade with the rest of the phylogeny did not show high Fig. S2 for an uncollapsed version of the order Pleurocapsales support. b Zoomed view on the clade containing the order fraction in panel c 123 2194 Antonie van Leeuwenhoek (2021) 114:2189–2203 newly described Odorella benthonica clade (Fig. 2c). the related Hyella patelloides LEGE 07179 counting The cryptic clade of Stanieria sensu stricto was 675 scaffolds, and may indicate a high proportion of relatively distant from W. agarophytonicola and the repetitive regions. With a predicted number new taxon W. agarophytonicola definitely forms an 5,168,141 bp in total and an estimated gene count of independent lineage. Whether or not the neighbouring 4902, the genome of W. agarophytonicola is the sequences are part of the new genus Waterburya is a smallest of all 9 genomes currently available in the question outside of this study, for which detailed Pleurocapsales, of which Pleurocapsa sp. CCALA p-distance analysis and analyses of 16S-23S ITS 161 has the next smallest genome (counting rRNA in all of the respective neighbouring strains 5,463,308 bp and 5033 genes) and Pleurocapsa sp. would be required. It can, however, already be stated PCC 7319 has the largest (7,386,997 bp, 6749 genes). that ‘‘Stanieria’’ PCC7103 and ‘‘Stanieria’’ UE7A The draft includes 36 tRNA genes, 3 noncoding have been previously misidentified as members of RNAs, a transfer-messenger RNA gene and 4842 Stanieria and are either members of Waterburya or protein coding genes. As the assembly is based on belong to separate genera (similarity values among short reads only, the number and order of ribosomal these taxa and W. agarophytonicola are around 97%). operon copies could not be resolved. The reassembled The clade distribution in the multiprotein phylogeny genome does not contain 16S rRNA gene copies and (Fig. 3) is similar to phylogenies from previous the full 16S rRNA gene sequence used for the 16S studies based on the same set of proteins (Shih et al. phylogeny (Genbank accession OK044280) was 2013; Koma´rek et al. 2014; Maresˇ 2018;Osterholm retrieved from the initial assembly and likely repre- et al. 2020). Furthermore, the phylogenetic position of sents a consensus sequences of the different copies. W. agarophytonicola in the multiprotein tree is in line Functions were predicted for 3375 genes, resulting in a with our 16S rRNA gene analysis (sister to Pleuro- total count of 1467 hypothetical proteins (Table 1). capsa sensu stricto). In the multiprotein tree, W. Besides W. agarophytonicola, the metagenome agarophytonicola grouped adjacent to the clade con- assembly of the culture contained 5 additional bins taining the recently epitypified Pleurocapsa minor that were predicted to be [ 70% complete, which HA4230-MV1 (Shalygin et al. 2019b). The same were classified to the Proteobacteria (i.e., Alteromon- study also selected a neotype for the generic type; adaceae, Methylophilaceae, Alphaproteobacteria, Pleurocapsa fuliginosa, which is closely related to P. Gammaproteobacteria) and Bacteriodetes (Flavobac- minor (both grouped in the 16S rRNA gene phylogeny teriaceae, see Table S1). In line with the absence of under Pleurocapsa sensu stricto, see Fig. 2. and cyanobacterial bins in the assembly, other cyanobac- Fig. S2). Besides the clear distinct morphology (e.g., teria were never observed microscopically from KI4, P. minor and P. fuliginosa both form pseudofila- supporting that the non-axenic culture is at least uni- ments), as a macroalgal symbiont, W. agarophytoni- cyanobacterial. cola is also ecologically unique and differs from the The W. agarophytonicola genome contains six Pleurocapsa species which were both isolated from KEGG-classified genes that are absent in all other rocky substrates (Shalygin et al. 2019b). However, currently available Pleurocapsales genomes future study is needed to elucidate whether W. (Table S2). These are p-hydroxybenzoate monooxy- agarophytonicola can be found on other substrates genase, hydroxymethyl cephem carbamoyltrans- and if the genus may accommodate more species with ferase, L-ectoine synthase, 7,8-dihydropterin-6-yl- similar symbiotic lifestyles. methyl-4-(beta-D-ribofuranosyl)aminobenzene 5 - phosphate synthase, a solute carrier family 10 General genome statistics (sodium/bile acid cotransporter) protein and an uncharacterised protein possibly involved in the The draft of Waterburya agarophytonicola KI4 biosynthesis of archaeosine. Altogether 73 COG- counts 5,107,674 bp and is predicted to be 98.83% classified genes that are present in the W. agarophy- complete, consisting of 149 scaffolds with an average tonicola genome are absent from other known 289 9 coverage and an N50 of 51,633. This is similar genomes of Pleurocapsales (Table S3). to a recent study from (Brito et al. 2020) who published a short-read based genome assembly of 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2195 123 2196 Antonie van Leeuwenhoek (2021) 114:2189–2203 b Fig. 3 Maximum-likelihood tree based on 31 conserved While the capacity to fix nitrogen is common in the proteins extracted from 96 cyanobacterial genomes. The Pleurocapsales (Rippka et al. 2015) and the nitroge- analysis was conducted with 4 outgroup taxa which have been nase proteins are present in several of the sequenced removed from the figure. Waterburya agarophytonicola is Pleurocapsales genomes (i.e., Myxosarcina sp. GI1, displayed in bold. Branches corresponding to nodes with [ 95 bootstrap support values are thickened and ex-type strains are Pleurocapsales sp. LEGE 06147, Pleurocapsales sp. labelled with an uppercase ‘T’ LEGE 10410 and Xenococcus sp. PCC 7305, Table S1), none of the NifD,-H or -K genes were found in W. agarophytonicola KI4 . Therefore, the genome does not support a nitrogen fixing role for W. Metabolic features agarophytonicola in the A. vermiculophyllum holo- biont, as was hypothesised in Bonthond et al. Besides genes encoding proteins involved in pathways for photosynthesis and carbon fixation, W. agarophy- (2020, 2021). However, the draft contains the ferre- doxin-nitrate and ferredoxin-nitrite reductase (NarA, tonicola’s draft contains a duplicated set of the cytochrome c oxidase subunits I-III and the genes NirA), and also reveals several transporter genes for for succinate dehydrogenase/fumarate reductase extracellular nitrate/nitrite (NrtA, -B, -C). Therefore, (sdhA, -B, -C), which may indicate the capacity for a the abundant occurrence of W. agarophytonicola may heterotrophic lifestyle as well. nonetheless affect nitrogen fluxes in the A. vermicu- lophyllum holobiont at the microscale, decreasing Table 1 Attributes of the Attribute bp Genes % of total Waterburya agarophytonicola KI4 Genome size 5,107,674 100.00% draft genome DNA coding 4,436,783 86.87% GC content 1,978,698 38.74% DNA scaffolds 149 100.00% Total gene count 4902 100.00% RNA genes 40 0.82% tRNA genes 36 0.73% Regulatory and miscellaneous features 20 0.41% Protein coding genes 4842 98.78% With function prediction 3375 68.85% With enzymes 922 18.81% Connected to KEGG pathways 1046 21.34% Connected to KEGG Orthology 1730 35.29% Connected to MetaCyc pathways 798 16.28% With COGs 3270 66.71% With Pfam 3551 72.44% With TIGRfam 1145 23.36% With SMART 1084 22.11% With SUPERFam 3757 76.64% With CATH FunFam 2966 60.51% In internal clusters 1156 23.58% Coding signal peptides 170 3.47% Coding transmembrane proteins 1163 23.73% COG clusters 1658 50.70% Pfam clusters 2030 57.17% TIGRfam clusters 893 77.99% 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2197 nitrate and nitrate concentrations in exchange for attachment and plant-microbe symbiosis, with COG ammonia. annotations for18 genes coding cheY-like chemotaxis Further, a genetic basis for assimilatory sulfate proteins, 3 genes coding signal transduction histidine reduction is present and this pathway is linked with kinase/cheY-like chemotaxis proteins and one gene genes catalysing sulfite production from Alkanesul- for a two-component sensor histidine kinase/cheY- fonate, 3’-phosphoadenosine-5’-phosphosulfate like chemotaxis protein. Moreover, the presence of 23 (PAPS), adenosine-5’-phosphosulfate (PAPS), genes coding for filamentous hemagglutinin family methanesulfonate and thiosulfate. Waterburya agaro- (FHA) proteins and 5 copies for the large exoprotein phytonicola KI4 has a high number of genes coding involved in heme utilization and adhesion suggest W. for carbonic anhydrase (5 KEGG and 8 COG anno- agarophytonicola is well equipped with cellular tations), which is more than any of the other 8 mechanisms related to adhesion (Locht et al. 1993; Pleurocapsales genomes in the IMG database. In Paulsrud and Lindblad 2002). Hemagglutinins such as addition, W. agarophytonicola contains the genes FHA proteins have been associated in adhesion, coding for phycobilisome proteins, i.e.; allophyco- adherence and virulence in plant pathogens (Gottig cyanin, phycocyanin, phycoerythrocyanin and phyco- et al. 2009) but are also utilised by endophytic plant erythrin. Also several carotenoid biosynthesis mutualists (Taghavi et al. 2010). pathways were found, suggesting W. agarophytoni- In addition, 8 genes coding for type IV pili (Tfp) assembly proteins (Pil) and 2 genes coding for cola is capable of synthesising b-carotene, zeaxanthin and canthaxathin. Similar with several other Pleuro- uncharacterised surface proteins with fasciclin capsales the W. agarophytonicola genome further (FAS1) repeats, may further support the presence of features both endoglucanase and b-glucosidase, as adhesion and/or infection mechanisms. Such pili are, well as an endo-1,4-beta-xylanase (KEGG annota- for example, found in plant endophytic Nostoc tions, Table S1). Besides these enzymes for cellulose cyanobacteria, where they are expressed abundantly and hemicellulose degradation the genome contains on the surface of hormogonia, and allow gliding four copies of catechol 2,3-dioxygenase-like lactoyl- motility towards the plant host (Duggan et al. 2007; glutathione lyase (COG annotation; Table S2) that Adams and Duggan 2012). Tfps are typical for a plant could enable W. agarophytonicola to degrade lignine. endophytic lifestyle (Frank 2018) and have also been However, despite its apparent close and, based on the found in unicellular and baeocytous cyanobacteria absence of records from other hosts or habitats, (Herdman and Rippka 1988) and even in bacteria from specific association with a red algal agarophyte, W. other phyla, where they may function in motility, agarophytonicola features no agarase. adhesion, DNA exchange and pathogenesis (Mattick 2002; Adams and Duggan 2012). Chemotaxis and adhesion Vitamins We found various chemotaxis and motility associated genes in Waterburya agarophytonicola’s draft gen- Similar to the other available Pleurocapsales genomes ome, some of which in high copy numbers, which W. agarophytonicola KI4 has the genetic basis for the suggest the cyanobacterium may be responsive to synthesis of various vitamins, including biotin (B ), different stimuli and be capable of directed movement. folate (B ), nicotinic acid (B ), panthothenate (B ), 11 3 5 Notably, many KEGG clusters associated with chemo- riboflavin (B ), thiamine (B ), a-tocopherol (E), 2 1 taxis are present, including genes for the methyl- phylloquinone (K ), menaquinone (K ) and pyridoxal 1 2 accepting chemotaxis protein (MCP), purine-binding 5’-phosphate (B ). Moreover, the genome contains the chemotaxis proteins (cheW) and the two-component pathway for the synthesis of cobalamin (vitamin B ), system (cheA, -B, and -R). The genome also contains including genes encoding cob(I)alamin adenosyltrans- multiple copies of the chemosensory pili system ferase and adenosylcobinamide-GDP ribazoletrans- protein ChpA and the twitching motility proteins ferase. As the production of vitamins is energetically (PilG, -H, -I, -J), as well as gene homologs required for expensive, these metabolites may represent potential positive phototactic motility (PixG, -H, -I, -J, -L). The benefits to W. agarophytonicola’s host. In particular, genome further yields proteins associated with surface vitamin B (cobalamin) is likely a valuable molecule 123 2198 Antonie van Leeuwenhoek (2021) 114:2189–2203 for the red algal host. Most Rhodophyta, including the 2020). The genome also contains a copy of the Florideophyceae –to which A. vermiculophyllum menaquinone-specific isochorismate synthase, a key belongs– express the cobalamin-dependent methion- enzyme in the synthesis of a siderophore group ine synthase (METH, Provasoli and Carlucci 1974) containing NRPSs (Walsh and Gary Marshall 2004). but are cobalamin auxotroph and depend on microbial Iron or other metal scavenging compounds are symbionts to acquire this vitamin (Croft et al. 2005). produced by most bacteria and operate extracellularly. Cobalamin auxotrophs may obtain the vitamin in Siderophores may increase the local iron availability exchange for fixed carbon (e.g., glycerol, Kazamia not only to their producer but at the same time to other et al. 2012). Also Gracilariopsis chorda, the closest microbes with the matching receptors for uptake. relative of A. vermiculophyllum of which a genome While their production can thus be favourable to sequence is currently available, encodes METH and is selective taxa, they may at the same time promote iron thus dependent on the acquisition of cobalamin from starvation in other taxa and therewith strongly influ- microbial sources. Given the core and dominant ence the taxonomic composition in microbial com- presence of W. agarophytonicola in the holobiont munities (Kramer et al. 2020). Mutualistic (Bonthond et al. 2020), this is an important observa- associations between terrestrial plants and siderophore tion and could imply that the cyanobacterium repre- producing endophytic bacteria have been documented sents a major or primary cobalamin source for A. for some time (Loaces et al. 2011; Frank 2018) and also for macroalgae it is thought that microbial vermiculophyllum. If this is true, and cobalamin from W. agarophytonicola is indeed functioning as a mutualists play an important role in the regulation of coenzyme in A. vermiculophyllum METH, another availability of iron and other trace metals (Wichard important question is what the cyanobacterium 2016). The genome further reveals three genes coding receives from the host in return. Rhodophytes produce for ferric uptake regulators (Fur), a common key various carbohydrates (Ito and Hori 1989) and as W. regulator in synthesis and activity of siderophores agarophytonicola has the genetic basis for aerobic (Kramer et al. 2020) and several other proteins related respiration, one hypothesis could be that the cyano- to siderophore transport, including 9 major facilitator biont is able to switch to a heterotrophic lifestyle, superfamily (MFS) permeases and TonB, ExbB, ExbD utilising both oxygen and a carbon source such as transporter proteins (Arstøl and Hohmann-Marriott glycerol from the host. This would also avoid the 2019, Tables S1, S2). Altogether, the presence of necessity to compete for light between the two these different genetic signatures associated with phototrophs. siderophore synthesis thus hint at a role for W. agarophytonicola in the cycling of iron and/or other Secondary metabolites and siderophores trace metals within the holobiont. In total 15 secondary metabolite clusters were Taxonomy detected, including 6 non-ribosomal peptide syn- thetases (NRPSs) or NRPS-like gene clusters, 3 The genus Waterburya and the species W. agarophy- terpenes, 2 bacteriocins, 1 lanthidin, 1 ectoine, a T3- tonicola are here described according to the Interna- polykethinde synthase (PKS) cluster and a hybrid tional Code of Nomenclature for algae, fungi, and NRPS-T1PKS cluster. Some of these clusters may plants (Turland et al. 2018). indicate the capacity for toxin production, including e.g., bacteriocins, which are toxins often produced to inhibit growth of other bacteria (Cotter et al. 2013) and Waterburya Bonthond and Shalygin gen. nov. NRPSs, PKSs or hybrids that may be involved in the synthesis of various toxins, including, Anatoxin-A, Diagnosis Cylindrospermopsin and microcystin (Kehr et al. 2011). Akin to Stanieria by morphology, different from NRPSs are also commonly at the basis for the which by marine habitat, epi-endophytic growth on the synthesis of siderophores and other metallophores rhodophyte A. vermiculophyllum, and based on 16S (Arstøl and Hohmann-Marriott 2019; Kramer et al. rRNA gene phylogeny. 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2199 Description Description In culture, small groups of cells forming blackish Macrocolonies slowly growing in liquid BG11 visible aggregates (macrocolonies). Microcolonies medium (20 PSU) under low light conditions in form consist of mostly individual, spherical cells; or of tight blackish clusters. Microcolonies small, con- oblate-spherical cells in groups growing together. sisting of released baeocytes (3.5–6 lm in diameter), Cells in the microcolony of different sizes, terracotta growing baeocytes (up to 8 lm in diameter), and or pale grey-brown by color. Cells surrounded by thin, large, mature cells ready to form baeocytes through colorless mucilaginous envelopes, never with com- multiple fission (7–20 lm in diameter). Mucilaginous mon slime. Baeocytes formation through multiple envelopes colorless, adherent to the cell walls, rarely fission of the mature cells, which are much larger than slightly widened and more robust especially in the stressed cultures. Released baeocytes growing to large baeocytes. Baeocytes are released by cell wall break- age. Reproduction exclusively by baeocyte sizes (up to 19–20 lm), after which multiple fission production. occurs. Sometimes baeocytes form in the smaller mother cells, 10–15 lm. Growing cells spherical or Etymology oblate-spherical, terracotta by color, released baeo- cytes pale grey-brown. Unreleased baeocytes abun- Named in honour of John B. Waterbury—an important dant, 64 or 128 per one mother cell; small, 2–2.5(3) in cyanobacterial researcher, who worked on morphol- diameter. ogy and physiology of many marine baeocyte producers. Etymology Type species Agarophyton- from the marine red alga Agarophyton vermiculophyllum (Ohmi) Gurgel et al., and -cola Waterburya agarophytonicola from L. n. incola; an inhabitant. Type locality Waterburya agarophytonicola Bonthond 0 00 and Shalygin sp. nov. (Fig. 4) Falckensteiner Strand Kiel (5423 55.3 N, 0 00 1011 27.6 E), within one meter from the surface Holotype (Baltic sea) on Agarophyton vermiculophyllum. Here designated: Specimen on filter, lyophilised. GenBank accession numbers Algal herbarium of Natural History Museum of Denmark, Copenhagen; accession number: C-A- MW113706 (16S rRNA gene partial sequence 99685. obtained with Sanger sequencing), OK044280 (16S rRNA gene complete, from initial genome assembly), Diagnosis PRJNA680001 (draft genome). Morphologically similar to Stanieria sublitoralis, but Taxonomic notes differing by phylogenetic position on the 16S rRNA gene tree and by being associated with Agarophyton The genus Stanieria is highly polyphyletic, even vermiculophyllum. Akin to Chroococcopsis gigantea, though the type species S. cyanosphaera PCC 7437 different by 16S rRNA gene phylogeny, smaller cells, was phylogenetically established. There are at least 5 lacking daughter cells and its distribution in marine to phylogenetically distant clades resembling members brackish habitats. of Stanieria outside of the Stanieria sensu stricto clade. One of them contains Stanieria sp. PCC 7302 isolated from a seawater tank at California Bay (Mexico) (Waterbury and Stanier 1978). This isolate 123 2200 Antonie van Leeuwenhoek (2021) 114:2189–2203 Fig. 4 Microphotographs showing the morphology of Water- mother cell (arrow); d Massive baeocyte formation adjacent to burya agarophytonicola. a Released baeocytes; b Growing the small group of growing cells; e Clear example of baeocyte cells, note that adjacent cells are not a product of binary fission; production; f: Unreleased, released and growing baeocytes. The c Aggregation of the cells and initial baeocyte formation within scale bar equals 10 lm is similar by morphology to Waterburya agarophy- (A. Lindstedt) Anagnostidis & Pantazidou, but this is tonicola, however different by habitat and 16S rRNA questionable, considering vast differences in ecology gene phylogeny. According to Anagnostidis and and geography between these two taxa (isolated in a Pantazidou (1991), Stanieria sp. PCC 7302 may Swedish sublittoral on various sea animals, valves and belong to the established taxon Stanieria sublitoralis algae versus isolated from a water tank in Mexico). To 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2201 Acknowledgements We are grateful to the Institute of confirm the true affiliation of Stanieria sublitoralis Clinical Molecular Biology in Kiel for conducting the Sanger DNA sequencing of type material from the type sequencing as supported in part by the DFG Cluster of locality is required. Excellence ‘‘Future Ocean’’ and we thank especially the technicians T. Naujoks and C. Noack for technical support. We also thank Dr. Stacy Krueger-Hadfield for providing and allowing us to use the photo she took of Agarophyton Conclusion vermiculophyllum which is displayed in Fig. S1. With this work we introduce Waterburya agarophy- Author contributions GB performed sampling, laboratory tonicola Bonthond and Shalygin gen. nov. sp. nov. and work and processed and analysed data. SS performed laboratory work, drafted the taxonomic description and present a draft of its genome. While the exact nature of processed and analysed data. FW performed sampling and this rhodophyte-cyanobacterium symbiosis remains to laboratory work and analysed data. TB performed sampling and be determined in future work, the genome reveals supported with bioinformatic data processing and analyses. GB, clues to its functional roles as a core member in the A. SS, TB and FW conceived the study and wrote the manuscript. vermiculophyllum holobiont (Bonthond et al. 2020). Funding Open Access funding enabled and organized by Altogether, a high number of chemotaxis, adhesion Projekt DEAL. This study was funded by the Deutsche and adherence related genes support a host-associated Forschungsgemeinschaft (DFG, project no. WE2700/5-1 lifestyle for W. agarophytonicola. Genes for adher- granted to FW and BA5508/2-1 granted to TB). ence and virulence (genes for Tfps and FHAs), Data availability The holotype Waterburya combined with occurrence data (Fig. 1) especially agarophytonicola KI4 was deposited in the Algal herbarium hint at an endophytic lifestyle. However, we have not of Natural History Museum of Denmark, Copenhagen; been able to microscopically confirm whether W. accession number: C-A-99685. The partial 16S rRNA gene agarophytonicola occurs endo- and/or epiphytically Sanger sequence and a full 16S rRNA gene consensus sequence obtained from the initial genome assembly were deposited in and whether it is associated intra- and/or extracellu- Genbank under the accessions MW113706 and OK044280, larly, which thus remains a question for feature respectively. The draft genome assembly and PGAP annotation research. This study does not support a diazotrophic are available in the Short Read Archive (SRA, accession role for the cyanobacterium in the holobiont, but PRJNA680001). Annotations from the IMGAP can be accessed on IMG (Taxon ID: 2913235824, submission ID: 246459). instead demonstrates it has the potential to function as a source of vitamins, in particular cobalamin, to the Declarations vitamin B -auxotrophic host. In addition, W. agaro- phytonicola may possibly facilitate uptake of iron and/ Conflicts of interest The authors declare that they have no or other trace metals by its host. To further investigate conflict of interest. the relationship between W. agarophytonicola and A. Open Access This article is licensed under a Creative Com- vermiculophyllum, advanced microscopic work is mons Attribution 4.0 International License, which permits use, needed to confirm the endo- and/or epiphytic occur- sharing, adaptation, distribution and reproduction in any med- rence of the cyanobiont and identify whether W. ium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative agarophytonicola produces chlorophyll in association Commons licence, and indicate if changes were made. The with the host, to decipher if it can adapt a heterotrophic images or other third party material in this article are included in lifestyle. Moreover, this may help to explore whether the article’s Creative Commons licence, unless indicated pili-like structures, such as those richly reflected in the otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your genome, are expressed by the cyanobiont. To shed intended use is not permitted by statutory regulation or exceeds light on how specific the relationship between A. the permitted use, you will need to obtain permission directly vermiculophyllum and W. agarophytonicola is, related from the copyright holder. To view a copy of this licence, visit hosts need to be studied as well. Finally, metabolic http://creativecommons.org/licenses/by/4.0/. assays should be carried out in an experimental context to quantify cobalamin production and to screen for host cell wall carbohydrates or other host molecules that are potentially metabolised by W. agarophytonicola. 123 2202 Antonie van Leeuwenhoek (2021) 114:2189–2203 References canker, is involved in bacterial virulence. PLoS ONE 4:e4358 Gruene T, Wennmacher JTC, Zaubitzer C et al (2018) Rapid Adams DG, Duggan PS (2012) Signalling in cyanobacteria– structure determination of microcrystalline molecular plant symbioses. Signaling and communication in plant compounds using electron diffraction. Angew Chemie Int symbiosis. Springer, New York, pp 93–121 Ed 57:16313–16317 Alneberg J, Bjarnason BS, De Bruijn I et al (2014) Binning Herdman M, Rippka R (1988) [22] Cellular differentiation: metagenomic contigs by coverage and composition. Nat hormogonia and baeocytes. Methods Enzymol Methods 11:1144–1146 167:232–242 Al-Thukair AA, Golubic S (1991) New endolithic cyanobacteria Ito K, Hori K (1989) Seaweed: chemical composition and from the arabian gulf. I. Hyella immanis sp. nov. 1. J Phy- potential food uses. Food Rev Int 5:101–144 col 27:766–780 Kang DD, Li F, Kirton E et al (2019) MetaBAT 2: an adaptive Altschul SF, Gish W, Miller W et al (1990) Basic local align- binning algorithm for robust and efficient genome recon- ment search tool. J Mol Biol 215:403–410 struction from metagenome assemblies. PeerJ 7:e7359 ´ ´ Amann R, Rossello-Mora R (2016) After all, only millions? Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel Mbio 74:e0999-e1016 method for rapid multiple sequence alignment based on Anagnostidis K, Pantazidou A (1991) Marine and aerophytic fast Fourier transform. Nucleic Acids Res 30:3059–3066 cyanosarcina, staniera and pseudocapsa (Chroococcales) Kazamia E, Czesnick H, Van NTT et al (2012) Mutualistic species from Hellas (Greece). Algol Stud fu¨r Hydrobiol interactions between vitamin B12-dependent algae and Suppl Vol 141–157 heterotrophic bacteria exhibit regulation. Environ Micro- Arstøl E, Hohmann-Marriott MF (2019) Cyanobacterial side- biol 14:1466–1476 rophores—physiology, structure, biosynthesis, and appli- Kehr J-C, Picchi DG, Dittmann E (2011) Natural product cations. Mar Drugs 17:281 biosyntheses in cyanobacteria: a treasure trove of unique Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: a new enzymes. Beilstein J Org Chem 7:1622–1635 genome assembly algorithm and its applications to single- ´ ˇ ´ ˇ Komarek J, Kastovsky J, Mares J, Johansen JR (2014) Taxo- cell sequencing. J Comput Biol 19:455–477 nomic classification of cyanoprokaryotes (cyanobacterial Bonthond G, Bayer T, Krueger-Hadfield SA et al (2020) How do genera) 2014, using a polyphasic approach. Preslia microbiota associated with an invasive seaweed vary 86:295–335 across scales? Mol Ecol 29:2094–2108. https://doi.org/10. Konstantinou D, Gerovasileiou V, Voultsiadou E, Gkelis S 1111/mec.15470 (2018) Sponges-cyanobacteria associations: global diver- Bonthond G, Bayer T, Krueger-Hadfield SA et al (2021) The sity overview and new data from the eastern mediter- role of host promiscuity in the invasion process of a sea- ranean. PLoS ONE 13:e0195001 weed holobiont. ISME J 15:1668–1679. https://doi.org/10. ¨ ¨ Kramer J, Ozkaya O,Ku¨mmerli R (2020) Bacterial siderophores 1038/s41396-020-00878-7 in community and host interactions. Nat Rev Microbiol Brito A, Vieira J, Vieira CP et al (2020) Comparative genomics 18:152–163 discloses the uniqueness and the biosynthetic potential of Loaces I, Ferrando L, Scavino AF (2011) Dynamics, diversity the marine cyanobacterium hyella patelloides. Front and function of endophytic siderophore-producing bacteria Microbiol 11:1527 in rice. Microb Ecol 61:606–618 Camacho C, Coulouris G, Avagyan V et al (2009) BLAST?: Locht C, Berlin P, Menozzi FD, Renauld G (1993) The fila- architecture and applications. BMC Bioinformatics 10:1–9 mentous haemagglutinin, a multifaceted adhesin produced Chen I-MA, Chu K, Palaniappan K et al (2021) The IMG/M data by virulent Bordetella spp. Mol Microbiol 9:653–660 management and analysis system v. 6.0: new tools and Magnusson A, Skaug H, Nielsen A, et al (2017) Package advanced capabilities. Nucleic Acids Res 49:D751–D763 ‘glmmTMB.’ R packag version 02 0 Cotter PD, Ross RP, Hill C (2013) Bacteriocins—a viable Maresˇ J (2018) Multilocus and SSU rRNA gene phylogenetic alternative to antibiotics? Nat Rev Microbiol 11:95–105 analyses of available cyanobacterial genomes, and their Croft MT, Lawrence AD, Raux-Deery E et al (2005) Algae relation to the current taxonomic system. Hydrobiologia acquire vitamin B 12 through a symbiotic relationship with 811:19–34 bacteria. Nature 438:90–93 Margulis L (1990) Words as battle cries–symbiogenesis and the Douma JC, Weedon JT (2019) Analysing continuous propor- new field of endocytobiology. Bioscience 40:673–677. tions in ecology and evolution: a practical introduction to https://doi.org/10.2307/1311435 beta and Dirichlet regression. Methods Ecol Evol Martin M (2011) Cutadapt removes adapter sequences from 10:1412–1430 high-throughput sequencing reads. Embnet J 17:10–12 Duggan PS, Gottardello P, Adams DG (2007) Molecular anal- Mattick JS (2002) Type IV pili and twitching motility. Annu ysis of genes in Nostoc punctiforme involved in pilus Rev Microbiol 56:289–314 biogenesis and plant infection. J Bacteriol 189:4547–4551 Meyer-Abich A (1934) Beitra¨ge zur theorie der evolution der Frank AC (2018) The genomes of endophytic bacteria. Endo- organismen. I. das typologische grundgesetz und seine fol- phytes of forest trees. Springer, New York, pp 141–176 gerungen fu¨r phylogenie und entwicklungsphysiologie. Acta Gottig N, Garavaglia BS, Garofalo CG et al (2009) A filamen- Biotheor 7:1–80. https://doi.org/10.1007/BF01603792 tous hemagglutinin-like protein of Xanthomonas axono- Miller MA, Schwartz T, Pickett BE et al (2015) A RESTful API podis pv. citri, the phytopathogen responsible for citrus for access to phylogenetic tools via the CIPRES science gateway. Evol Bioinform 11:EBO-S21501 123 Antonie van Leeuwenhoek (2021) 114:2189–2203 2203 Nurk S, Meleshko D, Korobeynikov A, Pevzner PA (2017) Stamatakis A (2014) RAxML version 8: a tool for phylogenetic metaSPAdes: a new versatile metagenomic assembler. analysis and post-analysis of large phylogenies. Bioinfor- Genome Res 27:824–834 matics 30:1312–1313 O’Leary NA, Wright MW, Brister JR et al (2016) Reference Taghavi S, Van Der Lelie D, Hoffman A et al (2010) Genome sequence (RefSeq) database at NCBI: current status, tax- sequence of the plant growth promoting endophytic bac- onomic expansion, and functional annotation. Nucleic terium Enterobacter sp 638. PLoS Genet 6:e1000943 Acids Res 44:D733–D745 Tatusova T, DiCuccio M, Badretdin A et al (2016) NCBI Osterholm J, Popin RV, Fewer DP, Sivonen K (2020) Phy- prokaryotic genome annotation pipeline. Nucleic Acids logenomic analysis of secondary metabolism in the toxic Res 44:6614–6624 cyanobacterial genera Anabaena. Dolichospermum Apha- Towns J, Cockerill T, Dahan M et al (2014) XSEDE: acceler- nizomenon Toxins (basel) 12:248 ating scientific discovery. Comput Sci Eng 16:62–74 Parks DH, Imelfort M, Skennerton CT et al (2015) CheckM: Turland NJ, Wiersema JH, Barrie FR, et al (2018) International assessing the quality of microbial genomes recovered from code of nomenclature for algae, fungi, and plants (Shen- isolates, single cells, and metagenomes. Genome Res zhen Code) adopted by the nineteenth international 25:1043–1055 botanical Congress shenzhen, China, July 2017. Koeltz Paulsrud P, Lindblad P (2002) Fasciclin domain proteins are Botanical Books present in Nostoc symbionts of lichens. Appl Environ Uritskiy GV, DiRuggiero J, Taylor J (2018) MetaWRAP—a Microbiol 68:2036–2039 flexible pipeline for genome-resolved metagenomic data Price MN, Dehal PS, Arkin AP (2010) FastTree 2–approxi- analysis. Microbiome 6:1–13 mately maximum-likelihood trees for large alignments. Walsh CT, Gary Marshall C (2004) Siderophore biosynthesis in PLoS ONE 5:e9490 bacteria. Iron Transp Bact 18–37 Provasoli L, Carlucci AF (1974) Vitamins and growth regula- Ward RD, Stajich JE, Johansen JR et al (2021) Metagenome tors. Bot Monogr sequencing to explore phylogenomics of terrestrial Pruesse E, Peplies J, Glockner FO (2012) SINA: accurate high- cyanobacteria. Microbiol Resour Announc 10:e00258- throughput multiple sequence alignment of ribosomal e321 RNA genes. Bioinformatics 28:1823–1829 Waterbury JB, Stanier RY (1978) Patterns of growth and Quast C, Pruesse E, Yilmaz P et al (2013) The SILVA ribosomal development in pleurocapsalean cyanobacteria. Microbiol RNA gene database project: improved data processing and Rev 42:2 web-based tools. Nucleic Acids Res 41:D590–D596. Wichard T (2016) Identification of metallophores and organic https://doi.org/10.1093/nar/gks1219 ligands in the chemosphere of the marine macroalga Ulva Rippka R, Waterbury JB, Herdman M, Castenholz RW (2015) (Chlorophyta) and at land-sea interfaces. Front Mar Sci Pleurocapsa-group. Bergey’s manual of systematics of 3:131 archaea and bacteria. Wiley, New Jersey, pp 1–9 Wu M, Eisen JA (2008) A simple, fast, and accurate method of Shalygin S, Huang I, Allen EH et al (2019a) Odorella ben- phylogenomic inference. Genome Biol 9:1–11 thonica gen. & sp. nov. (Pleurocapsales, Cyanobacteria): Wu Y-W, Simmons BA, Singer SW (2016) MaxBin 2.0: an an odor and prolific toxin producer isolated from a Cali- automated binning algorithm to recover genomes from fornia aqueduct. J Phycol 55:509–520 multiple metagenomic datasets. Bioinformatics 32:605–607 Shalygin S, Kavulic KJ, Pietrasiak N et al (2019b) Neotypifi- Zimba PV, Shalygin S, Huang I-S et al (2021) A new boring cation of Pleurocapsa fuliginosa and epitypification of P. toxin producer–perforafilum tunnelli gen. & sp. nov. (Os- minor (Pleurocapsales): resolving a polyphyletic cillatoriales, Cyanobacteria) isolated from Laguna Madre, cyanobacterial genus. Phytotaxa 392:245 Texas, USA. Phycologia 60:10–24 Shih PM, Wu D, Latifi A et al (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome Publisher’s Note Springer Nature remains neutral with sequencing. Proc Natl Acad Sci 110:1053–1058 regard to jurisdictional claims in published maps and institutional affiliations.

Journal

Antonie van Leeuwenhoek – Springer Journals

Published: Dec 1, 2021

Keywords: Gracilaria vermiculophylla; Cobalamin; Holobiont; Pleurocapsales; Symbiosis; Vitamin B12

There are no references for this article.