Background: Cyanobacteria of the genus Nostoc are capable of forming symbioses with a wide range of organism, including a diverse assemblage of cyanolichens. Only certain lineages of Nostoc appear to be able to form a close, stable symbiosis, raising the question whether symbiotic competence is determined by specific sets of genes and functionalities. Results: We present the complete genome sequencing, annotation and analysis of two lichen Nostoc strains. Comparison with other Nostoc genomes allowed identification of genes potentially involved in symbioses with a broad range of partners including lichen mycobionts. The presence of additional genes necessary for symbiotic competence is likely reflected in larger genome sizes of symbiotic Nostoc strains. Some of the identified genes are presumably involved in the initial recognition and establishment of the symbiotic association, while others may confer advantage to cyanobionts during cohabitation with a mycobiont in the lichen symbiosis. Conclusions: Our study presents the first genome sequencing and genome-scale analysis of lichen-associated Nostoc strains. These data provide insight into the molecular nature of the cyanolichen symbiosis and pinpoint candidate genes for further studies aimed at deciphering the genetic mechanisms behind the symbiotic competence of Nostoc. Since many phylogenetic studies have shown that Nostoc is a polyphyletic group that includes several lineages, this work also provides an improved molecular basis for demarcation of a Nostoc clade with symbiotic competence. Keywords: Cyanobacteria, Nostoc, Lichen, Symbiosis, Symbiotic competence Background In lichen symbioses cyanobacteria provide mycobionts Lichens are symbiotic associations between a fungus with photosynthate and/or fixed nitrogen. At the same (mycobiont) and a photosynthetic partner (photobiont) time, the fungal partners provide the cyanobacteria with that can be an eukaryotic alga (phycobiont), a cyanobac- moisture, carbon dioxide and inorganic ions, as well as terium (cyanobiont), or both . While the vast majority a relatively stable habitat, protected from environmental of lichen fungi (> 13,500 species), mainly from Ascomy- extremes and predation . cota, associate with green algae (Chlorophyta), over 1500 The association of fungal mycobiont partner and the species of lichen-forming fungi form so called “cyano- photobiont partner (e.g. Nostoc) can either be by codisper- lichens” that have cyanobacteria as primary photobionts sal, e.g. in the lecanoromycete lichen Lobaria pulmonaria (forming “bipartite” lichens) or accessory photobionts , or de novo by the pairing of a germinating spore (forming “tripartite” lichens) . Cyanobacterial sym- and a free-living photobiont, as generally found in the bioses have evolved repeatedly in different lineages lecanoromycete genus Peltigera [7, 8]. of lichen-forming fungi [3–5], resulting in conver- Most lichen symbioses are thought to be obligate as gently similar thallus morphology in distantly related the majority of mycobionts are refractory to propagation cyanolichens . in vitro and do not survive without their photosyn- thetic partners [9, 10]. And, although many cyanobacte- rial symbionts can be readily isolated and maintained in *Correspondence: email@example.com pure culture , they often appear unable to establish Faculty of Life and Environmental Sciences, University of Iceland, Sturlugata 7, aposymbiotic populations outside lichen thalli in nature . 101 Reykjavík, Iceland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 2 of 18 Nostoc is common in cyanolichens, especially in the Genome sequencing of N. punctiforme PCC 73102 temperate and cold regions of the world. All Nostoc , a model strain for cyanobacterial symbiosis with species are filamentous and have complex life cycles plants, together with transposon mutagenesis [32, 33] involving cellular differentiation. Their non-branching fil- and insertion of antibiotic resistance cassettes have aments consist of cylindrical or spherical vegetative cells identified a number of genes involved in the symbiosis with intercalary heterocysts, large specialized nitrogen- . Here we present the complete sequence and anal- fixing cells developing in mature trichomes . The ysis of genomes from lichen-symbiotic Nostoc strains - filaments of Nostoc strains are usually covered with a one from the bipartite lichen Peltigera membranacea and sheath of mucilage and many free-living Nostoc can form one from the tripartite lichen Lobaria pulmonaria,- gelatinous macroscopic colonies in nature. The ability together with a discussion of genes which appear distinc- to produce mucilage and to form hormogonia, slender tive for symbiotic Nostoc. In addition we make use of draft motile filaments, is generally used to distinguish Nostoc genome data from three more Nostoc strains derived from strains from the closely related genus Anabaena [13, 14], P. membranacea and metagenome data from the lichens but they can be more reliably differentiated by akinete P. membranacea and P. malacea, as well as currently avail- size and shape, together with other morphological char- able whole genome data from members of the Nostocales. acters . However, some strains of Nostoc only pro- duce hormogonia erratically or do not produce them at Results and discussion all [14, 16, 17]. Genome properties The taxonomy of the family Nostocaceae is still rather We have shotgun-sequenced DNA from five lichen- poorly resolved, as exemplified by the placement of associated Nostoc strains and two lichens (Table 1). Calothrix and Tolypothrix spp. in several phylogenetic Draft genome assemblies were generated for three of clades and the separation of Nostoc spp. into different the strains. The genomes of two strains, namely of clades . Symbiotic Nostoc strains of many cyano- the nosperin producer Nostoc sp. N6 and the lichens, including both bi- and tripartite species of Peltig- cyanobiont of L. pulmonaria, were completely assembled era, have traditionally been called Nostoc punctiforme,but and annotated. The genome of Nostoc sp. N6 (8.9 Mb) cyanobacterial strains resembling Nostoc muscorum, Nos- is similarinsizetothatofsymbiotic N. punctiforme toc sphaericum and Nostoc linckia have also been cultured PCC 73102 but it is larger than genomes of free-living from Peltigera species [19, 20]. Nostoc and Anabaena strains (Table 1 and Additional In the lichen symbiosis cyanobacteria tend to undergo file 1). It consists of one circular chromosome (8.21 Mb) several morphological and structural changes [19–23], (Fig. 1) and 10 extrachromosomal replicons – 7 circu- confounding phenotypic identification. Thus, molecular lar (pNPM1, 213,966 bp; pNPM2, 167,441 bp; pNPM3, techniques offer a powerful addition for studying the 44,778 bp; pNPM4, 44,777 bp; pNPM5, 41,255 bp; diversity of these organisms, and for comparing lichen pNPM6, 30,992 bp; pNPM7, 29,551 bp) (Fig. 1)and 3 symbiotic strains as well as free-living cyanobacteria. Dur- linear (pNPM8, 66,996 bp; pNPM9, 22,270 bp; pNPM10, ing the past fifteen years the cyanobacterial symbionts of 21,916 bp) (Fig. 2). Based on the sequence coverage lichens have been the subject of many molecular investi- obtained, the linear replicons are present in higher copy gations which have greatly increased our understanding of numbers than the circular ones. pNPM9 and pNPM10 symbiont diversity. However, most of these studies have are characterized by a lower GC content (36.6% and been based on a limited number of marker genes (e.g. 16S 37.7%, respectively) than the rest of the genome (Table 2). rDNA, rbcLX, trnL) and have mainly been focused on The ends of pNPM8, pNPM9 and pNPM10 are in each phylogenetic relationships of different strains [8, 24–30]. case composed of identical or nearly identical inverted So far little is known about what defines symbiotic com- repeats 2.73, 0.16 and 1.06 kb long, respectively, with petence of cyanobacteria on the genome level. a conserved AAATTAACRGAC sequence at each end Table 1 List of lichen-associated Nostoc strains sequenced in this study Strain Genome status Genome size DNA source Nostoc sp. N6 ‘Peltigera membranacea cyanobiont’ Complete 8.9 Mb Pure culture Nostoc sp. 210A ‘P. membranacea cyanobiont’ Draft 8.33 Mb Pure culture Nostoc sp. 213 ‘P. membranacea cyanobiont’ Draft 8.33 Mb Pure culture Nostoc sp. 232 ‘P. membranacea cyanobiont’ Draft 9.16 Mb Pure culture Nostoc sp. ‘Lobaria pulmonaria cyanobiont’ Complete 7.34 Mb Pure culture Nostoc sp. ‘P. malacea cyanobiont’ Draft 8.52 Mb Metagenomic Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 3 of 18 Fig. 1 Chromosome and seven circular plasmids of the Nostoc sp. N6 genome. The outermost and second circles indicate genes in forward and reverse orientation color-coded by their COG categories. The third circles show pseudogenes. The fourth circle of the chromosome shows the rRNA genes (brown) and tRNA genes (green). The two innermost circles show GC content in gray and black and the GC skew in green (+) and purple (–) Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 4 of 18 Fig. 2 Linear replicons of Nostoc sp. N6. The lowermost and second lines indicate genes in forward and reverse orientation color-coded by their COG categories (see Figure 1). The third lines show pseudogenes. The two uppermost lines show GC content in gray and black and the GC skew in green (+) and purple (–). Blue arrows represent terminal inverted repeats (IR) (Additional file 2: Figure S1). The ends of the linear plas- ends . Linear replicons are rarely found in Cyanobac- mids form covalently closed hairpins, in which one DNA teria, the only known examples being a 429.7 kb lin- strand loops around and becomes the complementary ear chromosome of Cyanothece sp. 51142 (accession strand. This was inferred from two observations: (i) pres- NC_010547)  and a 37.15 kb incision element of ence of reads with palindrome sequences in the Nextera Anabaena variabilis ATCC 29413 (accession NC_014000) XT library and (ii) drop in coverage close to the ends of . An interesting feature of the largest linear plas- the plasmids and absence of read pairs spanning puta- mid pNPM8 is that it carries 24 tRNA genes out of the tive palindromes in the Nextera mate pair library. These minimum of 32 tRNAs required for translation accord- methods use different DNA inputs for adapter addition: ing to Crick’s wobble hypothesis . Genes for tRNAs Nextera XT uses PCR to add adapters to denatured DNA carrying isoleucine and histidine are not present, while whereas Nextera Mate Pair ligates adapters to blunt ends there are 3 different tRNA genes for arginine and 2 for of double-stranded DNA. lysine, serine, glutamine and glutamic acid. tRNA genes Terminal inverted repeats and hairpins are common have frequently been found in phages where they facili- in linear DNA molecules enabling replication of genome tate expression of phage genes with codons that are rare Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 5 of 18 Table 2 Summary of the Nostoc sp. N6 and Nostoc sp. ‘Lobaria pulmonaria cyanobiont’ genomes Replicon GenBank accession Topology Size, bp GC, % CDS Pseudogenes Total rRNA operons tRNA genes CRISPR arrays Nostoc sp. N6 chromosome CP026681 circular 8,214,648 41.4 6205 518 6723 4 103 13 pNPM1 CP026682 circular 213,966 41.1 180 20 200 0 0 2 pNPM2 CP026684 circular 167,441 40.4 122 10 132 0 0 0 pNPM3 CP026685 circular 44,778 41.4 30 5 35 0 0 0 pNPM4 CP026686 circular 44,777 40.9 38 4 42 0 0 0 pNPM5 CP026687 circular 41,225 40.9 32 1 33 0 0 0 pNPM6 CP026688 circular 30,992 41.0 27 3 30 0 0 0 pNPM7 CP026689 circular 29,551 42.4 30 4 34 0 0 0 pNPM8 CP026690 linear 66,996 41.3 69 4 73 0 24 0 pNPM9 CP026691 linear 22,270 36.6 14 2 16 0 0 0 pNPM10 CP026683 linear 21,916 37.7 20 0 20 0 0 1 Nostoc sp. ‘L. pulmonaria cyanobiont’ chromosome CP026692 circular 7,061,466 41.6 5311 737 6048 3 74 7 pNLP1 CP026693 circular 121,770 40.3 82 9 91 0 0 0 pNLP2 CP026694 circular 63,064 42.2 36 10 46 0 0 0 pNLP3 CP026695 circular 58,727 42.0 60 4 64 0 0 0 pNLP4 CP026696 circular 34,881 42.4 33 2 35 0 0 0 Other genome features Nostoc sp. N6 Nostoc sp. ‘L. pulmonaria cyanobiont’ fMet Intron in tRNA gene Yes No Number of genes with inteins 5 0 Number of excision elements in nif cluster 3 1 in the host genome [41, 42]. However, codon frequen- to Nostoc sp. N6, the L. pulmonaria cyanobiont genome cies in the Nostoc sp. N6 chromosome vs. pNPM8 did contains asmallernumberofcodingregions,alarger not provide support for this (Additional file 2:FigureS2). number of pseudogenes and 3 ribosomal DNA (rDNA) The Nostoc sp. N6 linear elements might be phage rem- operons instead of the 4 copies generally found in Nosto- nants that have lost their structural proteins but retain cales . These features along with much slower growth the ability for self-replication in the host cells. Linear observed in pure culture (3–4 times slower than Nostoc plasmid prophages are uncommon in nature, e.g. N15 in sp. N6) suggest genome shrinkage, gene loss and a pos- E. coli , PY54 in Yersenia enterocolitica and ϕKO2 sible semi-obligate nature of the cyanobiont. Interest- in Klebsiella oxytoca . Genes involved in chromo- ingly, cbiM transporter genes, involved in the uptake of some partitioning and segregation, such as parAB and cobalt for cobalamin (vitamin B12) biosynthesis (locus parM , typical of many low copy number plasmids tags NLP_0266 and NLP_2774), were found to be pseudo and bacterial chromosomes, were not found on the linear in the L. pulmonaria cyanobiont. Several other essential replicons, except for pNPM8 which carries a presumptive genes had disabling mutations but had intact functional parA gene (NPM_80015). Interestingly, a gene encoding a homologues. typical phage protein, terminase, involved in DNA pack- Inversion of the GC skew ((G−C)/(G+C)) from posi- aging into empty phage capsids, was found in pNPM9 tive to negative, typically seen at the replication origin of (NPM_90012) disrupted by an insertion sequence. bacterial chromosomes, cannot be applied to predict the The genome of the L. pulmonaria cyanobiont is nearly location of oriC in Cyanobacteria since their DNA asym- 1.5Mbsmallerthanthatof Nostoc sp. N6 and has a metry is greatly disturbed by mutational pressure  total size of 7.34 Mb. It consists of one circular chromo- and extensive chromosome rearrangements (see below). some (7.06 Mb) and 4 circular extrachromosomal repli- Aputative oriC for the chromosomes was identified in cons – pNLP1 (121,770 bp), pNLP2 (63,064 bp), pNLP3 both strains downstream of dnaA, encoding a chromoso- (58,727 bp) and pNLP4 (34,881 bp) (Fig. 3). Compared mal replication initiation protein (locus tags NPM_0001 Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 6 of 18 Fig. 3 Chromosome and four plasmids of the Nostoc sp. ‘L. pulmonaria cyanobiont’ genome. The outermost and second circles indicate genes in forward and reverse orientation color-coded by their COG categories. The third circles show pseudogenes. The fourth circle of the chromosome shows the rRNA genes (brown) and tRNA genes (green). The two innermost circles show GC content in gray and black and the GC skew in green (+) and purple (–) Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 7 of 18 and NLP_0001) (Additional file 2: Figure S3). Both oriC the majority of ribosomal proteins (Clusters of Ortholo- regions contain 6 DnaA boxes, most with TTTTCCACA, gous Groups (COG) category J; locally collinear block 7) the DnaA box motif specific for Cyanobacteria . Loca- (Additional file 2: Figure S5, Additional file 3), which tion of oriC adjacent to the dnaN gene encoding the are known to be syntenic across species [54, 55], as β subunit of DNA polymerase III has been claimed to well as many genes involved in carbohydrate transport be universal among Cyanobacteria [50, 51]. In free-living and metabolism (G) and cell wall/membrane/envelope Nostoc strains and in Anabaena variabilis, oriC is located biogenesis (M). in the intergenic region between the dnaA and dnaN Although symbiotic Nostoc strains N6 and N. punc- genes. However, in lichen-associated Nostoc strains and in tiforme show a higher number of encoded proteins in N. punctiforme, dnaA and dnaN genes are not adjacent most COG categories than the free-living strains (mean (∼ 52 kb apart in N. punctiforme). Interestingly, no appar- total 4344 vs. 3587; Additional file 2:Table S5), thefrac- ent DnaA boxes were found adjacent to either the dnaA tion assigned to COG categories was similar (63-68%) gene (Npun_F0001) or the dnaN gene (Npun_F0034) in and the distribution among categories was similar for N. punctiforme whereas a putative oriC with a cluster of all analyzed Nostoc and Anabaena strains (Fig. 5). On DnaA boxes lies within the Npun_F0036–Npun_F0037 the average, clade II of symbiotic Nostoc strains has a intergenic region. higher proportion of genes devoted to carbohydrate trans- port and metabolism (G), lipid transport and metabolism Genome and proteome comparison (I) and secondary metabolite biosynthesis, transport and Phylogenetic analysis of 31 conserved single copy protein catabolism (Q) compared to clade I Nostoc and Anabaena genes from Nostocales strains available in GenBank and strains. Interestingly, clade I, comprised of free-living Nos- JGI-IMG showed that lichen-associated Nostoc strains toc and Anabaena strains (Additional file 1), exhibits the and N. punctiforme group together in a clade (Nostoc II; highest number of genes for inorganic ion transport and Fig. 4) suggesting a monophyletic origin of these sym- metabolism (P) (Fig. 5). In contrast, the Nostoc strains in biotic Nostoc strains. The recently sequenced symbiotic symbiosis may benefit from host (plant or fungus) pro- strains Nostoc sp. KVJ20 and Nostoc sp. Moss 2 vision of inorganic ions, e.g. by the action of mycobiont  also associate with this clade, whereas two other siderophores . moss derived isolates together with terrestrial soil isolates The genome of Nostoc sp. N6 was found to encode of Nostoc calcicola and Nostoc linckia form a subclade the highest number of COG category L proteins (DNA within the Nostoc II clade. The free-living aquatic Nostoc replication, recombination and repair) (Additional file 2: Table S3). One possible explanation for this is that ter- strains group together with some Anabaena strains (clade Nostoc I; Fig. 4) while other Anabaena strains group with restrial cyanobacteria are generally subject to ultraviolet members of the genera Aphanizomenon and Dolichos- (UV) irradiation, and therefore are expected to pos- permum (clade Anabaena/Aphanizomenon). These major sess efficient mechanisms for repair of UV-induced DNA phylogenetic relationships are in accord with what has damage . Nevertheless, one of the genes involved in been found by O’Brien and coworkers and by biosynthesis of the cyanobacterial sunscreen scytonemin Warshan . Although O’Brien’s clade Nostoc II con- (tyrosinase, tyrP)[58–60] is missing in the Nostoc sp. N6 tains three free-living terrestrial isolates – N. punctiforme genome, and another one (DSBA oxidoreductase, frnE) SAG 71.79, N. commune 02011101 and N. musco- was found to be a pseudogene due to an in-frame stop rum SAG 57.79 (currently known as Desmonostoc codon (Additional file 2: Table S6). Both genes are thought muscorum) – none of them have been tested for symbiotic to participate in oxidative dimerization of precursors to competence and two of the strains have P. membranacea form scytonemin . The cyanobionts of L. pulmonaria cyanobionts as their closest phylogenetic relatives. Nostoc and P. malacea appear to have all genes necessary for strains with specificity for symbiosis with Gunnera also scytonemin biosynthesis (Additional file 2:Table S6). It fall within cladeII but their genome sequences are is possible that Nostoc sp. N6 compensates for the lack currently not available. of scytonemin with a larger repertoire of enzymes for Large scale genome comparisons of lichen-associated DNA repair. strains with N. punctiforme PCC 73102 reveal a low level Nostoc sp. N6 has a high number of transposable of synteny between them (Additional file 2:FigureS4) elements and inteins (Additional file 2:TablesS7-S9). indicating high genome plasticity and genome shuffling The best studied case of inteins in Cyanobacteria in these strains (Additional file 2: Figure S5). The ten is in DnaE (the α subunit of DNA polymerase III) most prominent regions of synteny include the whole encoded by two different ORFs and assembled by trans-splicing . More information on trans- set of genes involved in nitrogen fixation (the nif gene cluster; locally collinear block 1), some photosynthetic posons and inteins can be found in Supporting genes (locally collinear block 3), and genes encoding Information. Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 8 of 18 Fig. 4 Maximum liklelihood phylogenomic tree of Nostocales strains based on 31 single-copy core bacterial phylogenetic markers . Arthrospira platensis NIES-39, Lyngbya sp. PCC 8106 and Planktothrix agardhii NIVA-CYA 126/8 from the order Oscillatoriales were used as the outgroup. Numbers at branch nodes are bootstrap percentages based on 100 replicates (only values >50 are shown). Scale bar indicates 5% sequence divergence. Selected clades are named according to . Predominantly symbiotic clade is highlighted with green, paraphyletic group is highlighted with blue. Lichen-associated strains are shown in bold Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 9 of 18 Fig. 5 COG category distribution of the proteins encoded in the genomes of selected Nostoc and Anabaena strains. The ordinate axes indicate the percentage of genes in each COG functional category relative to the genes of all COG categories (left) and percentage COG category distribution among different clades (right) The majority of lichen associated Nostoc strains stud- gene clusters (vnf ) is provided in Supporting Information ied appears to have an alternative vanadium-based nitro- (Additional file 2: Figures S9 and S10). genase in addition to the standard molybdenum-based nitrogenase. This includes three of the strains studied Comparison to minimal bacterial and cyanobacterial gene sets here, Nostoc spp. 210A, 213 and 232, as well as the P. In order to see what pathways might differ, be incom- malacea lichen cyanobiont . The reason for the com- plete or deteriorating in lichen cyanobionts, we performed mon occurrence of this alternative nitrogenase in lichen- comparative analyses with the minimal bacterial and associated cyanobacteria is not clear, but may relate to thecyanobacterial“core”and “shell”genesets, rep- low availability of molybdenum in cyanolichens and/or resented by 206 and 682 genes respectively (Additional a functional advantage at relatively low growth temper- files 4 and 5). The most prominent differences were atures . A novel finding is that these lichen Nostoc observed for pyrimidine metabolism, in split ribonu- strains carry a near complete duplication of VnfD, with cleotide reductase enzymes, carbohydrate catabolism and a cyanobacterial aminoacyl-tRNA synthetase domain potassium transport, as described in the Supporting (CAAD; pfam14159) inserted at the carboxy end, simi- Information. lar to peptide insertions found in GluRS, ValRS, LeuRS and IleRS amino acid tRNA synthetases in a variety of Identification of genes specific to symbiotic Nostoc strains cyanobacteria, where this domain is thought to direct To identify functions enriched in symbiotic Nostoc the proteins to thylakoid membranes, a key source of genomes (present in over 80% of group), we per- reducing power and ATP . Further information on formed an all-by-all BLASTP search of all the proteomes the molybdenum-based (nif ) and the vanadium-based from the Nostoc I and II clades plus the sister clade Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 10 of 18 (Fig. 4) and assigned identified hits into orthologous of hormogonia formation and return to the vegetative groups. For the lichen-associated Nostoc strains 152 pro- state . tein orthologs satisfied the criteria set (see Methods), In N. punctiforme the hormogonium regulating locus is 189 orthologs for the predominantly symbiotic clade, linked to genes involved in sugar transport (Fig. 6). 399 for the Nostoc II clade and 385 for the com- It has been hypothesized that these genes are involved in bined Nostoc II clade and sister clade (see Additional HRF-induced synthesis of a metabolite inhibitor of hor- file 6 for listing). A few of the most prominent mogonium differentiation, rather than a carbon catabolic gene collectives associated with symbiotic Nostoc are function . This metabolite, probably similar to galac- discussed below. turonate , binding to the HrmR protein, may act in a positive feedback loop alleviating repression of the hrm locus, leading to increased production of the metabo- Hormogonium regulating locus. Hormogonia are rela- lite and at the same time facilitating increased import tively short motile filaments, lacking heterocysts, formed of sugars such as glucose, fructose and sucrose. Since by cyanobacteria from the orders Nostocales and Stigone- PfkA (6-phosphofructokinase) appears to be nonessen- matales. A hormogonium-inducing factor (HIF) secreted tial in symbiotic Nostoc, these sugars must be channeled by plant hosts induces symbiotic cyanobacteria to differ- through the oxidative pentose phosphate (OPP) pathway entiate hormogonia and they then dedifferentiate back or the Entner-Doudoroff (ED) pathway, both produc- into nitrogen-fixing filaments after about 48 h . The ing NADPH reducing equivalents facilitating biosynthesis capacity of Nostoc strains to form hormogonia has been and decreasing dependence on the non-oxidative pen- found to be necessary, but not singularly sufficient, for tose phosphate reactions (Calvin cycle). This catabolic symbiotic competence [69, 70]. An aqueous extract of the shift may simultaneously induce development from hor- hosting hornwort Anthoceros punctatus appears to con- mogonia to vegetative cells and heterocysts. The shift tain a hormogonium repressing factor (HRF) because it from vegetative cells to heterocysts is accompanied by suppresses HIF-induced hormogonia formation. Analy- an increase of the OPP-specific Gnd and an even greater sis ofN. punctiforme mutants led to proposal of the fol- increase in Zwf , indicating increased carbon flow via lowing model of HRF-dependent modulation of HrmR the ED pathway. The hrm locus is restricted to the Nostoc transcriptional regulation : HRF enters the Nostoc cell II clade and its sister clade. and it, or a derivative similar to galacturonate, binds to the repressor protein HrmR, decreasing affinity for the hrmR and hrmE promoter regions. This derepresses tran- D-alanine-D-alanine ligase operon. In addition to a scription of these genes, somehow leading to inhibition conventional cell-wall specific D-Ala-D-Ala ligase (DdlA), Fig. 6 Hormogonium regulating and sugar transporter loci in symbiotic Nostoc strains. Pseudogenes are denoted with an asterisk. orpB, carbohydrate-selective porin; mviM, inositol-2-dehydrogenase; glpC, glucose permease; frtA1A2BC, ABC-type fructose transporters; hrmE,inositol oxygenase; hrmK, gluconate kinase; hrmR, LacI family transcriptional regulator; hrmI, glucuronate isomerase; hrmU, D-mannonate oxidoreductase; hrmA and unk, unknown. A broken genome line indicates 2 separate loci Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 11 of 18 the lichen associated Nostoc strains uniquely harbour The additional peptidoglycan and phosphonate lipid another D-Ala-D-Ala ligase, of type 3, thought to be functions may lead to cell wall modifications that are involved in modification of peptide moieties in pep- well tailored to the intrathalline environment, as well tidoglycans as described in Supporting Information as being recognized as compatible by a mycobiont dur- (Additional file 2: Figures S14 and S15). ing establishment of symbiosis. Despite being sheltered by a mycobiont, lichen cyanobionts are subjected to Phosphonate biosynthetic genes. Phosphonates are extracellular enzymes and metabolites produced by both organophosphorus compounds containing direct carbon- the mycobiont and intrathalline bacteria. Therefore, a phosphorus bonds, e.g. in phosphonolipids where they specific ability to withstand some unfavorable aspects can not be cleaved by regular phospholipases. The bio- of this cohabitation is expected from lichen associated chemical pathways and gene clusters for phosphonolipid Nostoc strains. synthesis are well studied , facilitating recognition in new settings as in the case of the lichen-associated Nostoc Chloramphenicol phosphotransferase. Chloramphenicol strains in this study (Fig. 7). This cluster is characteristic is an antibiotic produced by Streptomyces venezuelae of the Nostoc II clade. Extended information is provided in ATCC 10712 and several other actinomycetes . The the Supporting Information. bacteriostatic activity of chloramphenicol results from Fig. 7 Phosphonate biosynthetic gene clusters of lichen cyanobionts (a) and proposed encoded biosynthetic pathway (b) (adapted from ). A homologous gene cluster from Burkholderia is shown for comparison. CTP-APT, CDP-alcohol phosphatidyltransferase; OG-Fe(II), 2-oxoglutarate non-heme Fe(II) dependent oxidase; unk, conserved hypothetical proteins; NTPT, NTP transferase; pepM, phosphoenolpyruvate phosphomutase; ppd, phosphonopyruvate decarboxylase; AEPT, 2-aminoethylphosphonate aminotransferase; hpnL, putative membrane protein; higBA, toxin-antitoxin module. A broken genome line indicates separate loci Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 12 of 18 its binding to the 50S subunit of the bacterial ribosome Similarly, the single Blasia-habitat Nostoc strain show- blocking peptidyl transferase . S. venezuelae escapes ing nosperin does not exhibit any of the other metabo- the toxicity of its own lethal secondary metabolite by lites under study . Remnants of the nsp gene cluster expressing a chloramphenicol phosphotransferase (CPT) were found on the chromosome of the L. pulmonaria that phosphorylates the primary (C-3) hydroxyl of chlo- cyanobiont (Additional file 2: Figure S19), where almost ramphenicol (Additional file 2: Figure S16) . Genes the entire cluster has been deleted, probably due to the encoding CPT were found almost exclusively in the Nostoc absenceofselectivepressure. II clade. Whole genome sequencing of the nosperin producer Nostoc sp. N6 revealed that the nsp gene cluster is located on the chromosome. The abundance of inser- Gas vesicles, sulfur metabolism. Genes encoding gas vesicle proteins have been shown to be involved in hor- tion sequences surrounding the cluster and the apparent mogonium function and establishment of the N. puncti- mixed gene origin suggests that it has been acquired as forme symbiosis [79, 80]aswellasinthe symbiosisof a genomic island through horizontal transfer and under- Nostoc with feathermoss . Gas vesicle proteins GvpC, gone several intragenomic recombination events . The GvpV and GvpW appear to be characteristic for the Nostoc genome of Nostoc sp.N6was also foundtoencodepath- II clade and its sister clade. Several genes associated with ways for the biosynthesis of nostopeptolide- and assimilation of alkane sulfonates in the moss-Nostoc asso- banyaside/suomilide-like  compounds as well as nos- ciation  were also found to be enriched in the Nostoc tocyclopeptide  (Additional file 2: Table S14). Nos- II clade. topeptolide in Nostoc punctiforme has been found to be a major hormogonium-repressing factor and is there- Sensory mechanisms. All the comparison groups were fore considered responsible for cellular differentiation of found to have differences related to sensory mecha- Nostoc . nisms and motility, including signal transduction histidine Nostocyclopeptides are cyclic heptapeptides with a kinases, methyl-accepting proteins as well as diguanylate unique imino linkage in the macrocyclic ring, isolated cyclases, thought to be involved in regulating motility in from the lichen cyanobiont Nostoc sp. ATCC 53789 . cyanobacteria . The diversity and rapid divergence of Two homologous NRPS functions (locus tags NPM_1843 sensory mechanisms underlines the great variety of eco- and NPM_1844) were found in the genome of Nostoc sp. types found in the genus Nostoc, especially in strains with N6. A nostoclide-like compound with a very similar struc- symbiotic capacity . Differences in genes involved in ture, cyanobacterin, produced by the cyanobacterium Tolypothrix sp. PCC 9009 (Scytonema hofmanni UTEX sensory mechanisms were also found in the comparison 2349) [93, 94], was found to inhibit the growth of many made by Warshan et al. . cyanobacteria, as well as green algae and angiosperms [95, 96]. Based on the homology with Tolypothrix sp. Secondary metabolites PCC 9009, we identified putative gene clusters for biosyn- Cyanobacteria produce a multitude of secondary metabo- thesis of nostoclide-like compounds in the genomes of lites, many of them toxic [85, 86]. In a recent study, Nostoc spp. 210A and 232 (Additional file 2: Figure S20b). Liaimer et al. found that Nostoc symbionts of the liv- erwort Blasia pusilla more frequently produce nodularin More extensive information on secondary products can be and microcystin type compounds antagonistic to other found in the Supporting Information. Nostoc strains than free living Nostoc from the same local- ity. Most types of secondary compounds were detected in Conclusions only 1 to 4 out of the 20 strains examined. The occurrence The complete genome sequences and comparative of the main secondary metabolite pathways in Nostoc genomic analyses of two lichen-associated Nostoc strains punctiforme,inthe Nostoc strains from the Blasia habitats are presented here. The finished genomes, manually  and in the lichen-derived strains of the present study curated, are appropriate for all types of detailed anal- shows little overlap. One of the secondary compounds yses and act as high-quality references for comparative detected by Liaimer et al.  is the polyketide synthase purposes . Comparative genome analysis of symbiotic plus non-ribosomal peptide synthase (PKS-NRPS) prod- and free-living cyanobacteria allowed the identification uct nosperin . We previously suggested that nosperin of several pathways that may contribute to symbiotic might have cytotoxic properties analogous to cyanobiont competence of Nostoc strains. One pathway, encoded microcystins [87, 88] which can serve as protective com- by the hormogonium regulating (hrm)locus,was pre- pounds in cyanolichens, e.g. against grazers. Interestingly, viously identified in symbiotically competent N. puncti- Nostoc sp. 232 was found to be devoid of nsp genes encod- forme and plays a central role in abrogating hormogonia formation. This pathway is similar to pathways of sugar ing nosperin, but it has a putative microcystin gene cluster uronate metabolism in heterotrophic non-cyanobacterial not found in the nsp containing Nostoc sp. N6 strain. Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 13 of 18 prokaryotes [71, 72]. Although the hrm locus has been cyanobionts [20, 105, 106]. Isolation and genome showntobeimportant in the Nostoc-plant symbiosis, sequencing of these lichen-associated strains can add its presence in all of the lichen-associated Nostoc strains more support and knowledge to our current understand- from this study suggests it is also relevant to establish- ing of what determines symbiotic competence in Nostoc ing Nostoc-mycobiont symbioses. Pathways that may be and other cyanobacteria. involved in cell wall biogenesis of lichen cyanobionts were also identified, including novel gene clusters encoding Methods synthesis of phosphonate lipids and an MXAN_4097-like Isolation and culture of Nostoc strains amidoligase (D-Ala-D-Ala ligase). Peltigera membranacea thalli for cyanobiont isola- It is apparent that the ability to form and maintain sym- tion were collected from a moss carpet (Hylocomium biosis is a complex trait governed by many factors and splendens and Pleurozium schreiberi) at Keldur, Reykjavik, different combinations of these factors may result in dif- Iceland, and Lobaria pulmonaria thallus was collected ferent symbiotic associations – from loose to the most from a maple tree trunk (Acer macrophyllum)atCedar intimate. The study presented here is the first attempt to Road, Vancouver Island, British Columbia, Canada. determine, on a whole genome level, what genes and fea- Nostoc strains were isolated on BG-11 agar medium as tures may contribute to symbiotic competence of Nostoc previously described , purified by repeated streak- cyanobionts in lichens. Although we have pinpointed can- ing on the same medium and maintained at room didate symbiotic genes in the lichen-associated Nostoc temperature. genomes, a more thorough analysis, e.g. with targeted mutationsandresynthesisofsymbiosis,isrequiredtover- DNA extraction, library construction and sequencing ify the importance and involvement of individual genes Genomic DNA was prepared from Nostoc cultures grown and pathways. Some progress has been achieved in study- in liquid BG-11 medium at an illumination of 50 μmol −2 −1 ing plant-cyanobacterial symbioses using the readily cul- photons m s as described in . Sequencing tured hornwort Anthoceros and the liverwort Blasia as libraries were prepared using Nextera XT and, for some model organisms. However, there are substantial differ- strains, Nextera Mate Pair Sample Preparation Kits (Illu- ences between plant- and mycobiont-cyanobacterial sym- mina) according to the manufacturer’s protocols and bioses, e.g. due to the heterotrophic nature of fungi. In sequenced using MiSeq Reagent Kits v2 with 2×250 and contrast to many lichens with green algal photobionts, the 2×150 cycles, respectively (Additional file 2:Table S1). bionts of cyanolichens are difficult to culture and synthe- Roche 454 reads of P. membranacea and P. malacea size in the laboratory. Problems include slow growth or metagenomes generated previously werealsoused unculturability of most mycobionts, difficulties in obtain- in this study to increase the number of lichen-associated ing axenic cultures of photobionts, and in maintaining Nostoc strains. resynthesized biont cultures for long periods of time. Few attempts have been documented of cyanolichen resynthe- Genome assembly sis under laboratory conditions [98–103]and currently Draft assemblies of Nostoc spp. N6 and ‘Lobaria there are no available models to study mycobiont-Nostoc pulmonaria cyanobiont’ genomes were constructed using symbiosis. Theuseof theglomeromycete Geosiphon MIRA v3.2.1 (www.chevreux.org/projects_mira.html) pyriforme, which is easily culturable and capable of form- and further processed and verified using GAP5 (Staden ing symbiosis with Nostoc strains, can help to overcome package)  (Additional file 2: Table S1). Remaining some of these limitations . gaps were closed by PCR and Sanger sequencing. Draft Recent studies of ten genomes and proteomes from genomes of Nostoc spp. 210A, 213, 232 and the P. malacea moss-associated Nostoc strains compared to the non- metagenome were assembled using SPAdes v3.10.1  symbiotic Nostoc sp. CALU 996, identified a number of with default parameters. Prior to assembly Illumina gene families present in the symbiotic strains but not reads were processed with Trimmomatic v0.36 with in the comparison strain , . Several of these, “LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 including the hrm locus, genes encoding gas vesicle pro- MINLEN:20” parameters. SPAdes contigs >1 kb were teins, genes connected with sulfur metabolism and genes binned using MaxBin v2.2.4  and those belonging linked to sensory mechanisms were identical or simi- to Cyanobacteria were scaffolded using BESST v2.2.6 lar to symbiotic-specific gene clusters identified in the [114, 115]. The resulting assemblies were improved with lichen-associated Nostoc. FinishM v0.0.9 (https://github.com/wwood/finishm)and In addition to Nostoc, several other nostocean Pilon v2.11.6 . Scaffolds were taxonomically classified cyanobacteria have been reported in lichen symbioses. using Kaiju (http://kaiju.binf.ku.dk/)and PhyloPy- Members of the genera Scytonema, Calothrix, Dichothrix, thiaS+ (http://phylopythias.bifo.helmholtz-hzi.de/) and Tolypothrix have also been found in lichens as web servers. Those not assigned to Cyanobacteria were Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 14 of 18 manually checked using a BLAST search , and rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, contaminating scaffolds were removed. Completeness rpsK, rpsM, rpsS, smpB and tsf ) were extracted from and contamination of the assemblies were assessed with the genomes using the AMPHORA2 pipeline and CheckM v1.0.7  (Additional file 2:Table S2). aligned with MUSCLE . An alignment mask was generated using Zorro . The marker alignments were Genome annotation further concatenated into a single partitioned alignment and the best protein substitution model for each of the Draft genome assemblies were annotated using the NCBI Prokaryotic Genome Annotation Pipeline . For com- markers was predicted using the concat_align.pl script plete genomes ORFs were predicted with Prodigal , of phylogenomics-tools (https://github.com/kbseah/ followed by manual correction in Artemis using phylogenomics-tools; https://doi.org/10.5281/zenodo. the gene prediction improvement pipeline GenePRIMP 46122). A maximum-likelihood phylogeny was derived . All encoded proteins were assigned functions by using the PROTCATWAG model for tree search in combining results from InterProScan , CDD  RAxML v8.2.4 automated by the tree_calculations.pl and BLAST searches  against the NCBI nonredun- script of phylogenomics-tools. Branch support was dant (nr) database. Transfer RNA genes were identified assessed using the approximate likelihood ratio test for with tRNAScan-SE-1.23  and ribosomal RNA genes branches (SH-like aLRT)  with 100 replicates. (5S, 16S, 23S) were predicted using RNAmmer . Other non-coding RNAs were identified with Infernal Genome and proteome comparison (v.1.1)  using RFAM convariance models (http://ftp. Whole genome comparisons were performed using ebi.ac.uk/pub/databases/Rfam). Identification of CRISPR PROmer (MUMmer 3.0 package; ) and Mauve . elements was performed using CRISPRfinder and To identify orthologous groups specific to different clades PILER-CR . Pseudogenes were annotated using the (Fig. 4) an all-by-all BLASTP search was performed on GenePRIMP pipeline and rechecked manually in Artemis. proteomes of 56 strains belonging to a) Nostoc Iclade Single in-frame stop codons and frameshifts were con- (16 strains), b) Nostoc II clade (27 strains), c) sister firmed in the original assemblies. Ribosomal slippage clade to Nostoc II clade (13 strains) with soft mask- −10 was annotated according to standard operating proce- ing and thresholds: E-value < 10 , percentage iden- dures (SOP) at the GenePRIMP website (http://studylib. tity 50% and percentage match 50%. The result- net/doc/7260119). Finally, short ORFs (encoding < 100 ing hits were clustered into orthologous groups using −2 aa) without any significant homology (E-value> 10 ) OrthoMCL [142, 143]. Orthologous groups specific to to the nr database, and ORFs represented solely by low- different clades were extracted as shown in Additional complexity sequences (e.g. spanning micro- and min- file 2: Figure S21. For COG category distribution compar- isatellite regions) were removed from the annotation. ison proteins encoded in the genomes of selected Nostoc Intein-containing proteins were identified by the presence and Anabaena strains were classified into COG func- of an intein/homing endonuclease domain (COG1372). tional categories  using RPS-BLAST against PSSMs Excision of nifD and fdxN excision elements in Nostoc (Position-Specific Scoring Matrices) from the updated −2 sp. N6 was confirmed by previously generated RNA-Seq COG database  with an E-value < 10 and the top data mappedwithBowtie2. Origins of repli- hit retained. cation (oriC) were identified by locating DnaA boxes (TT / TNCACA) . Thelocationofaclusterof Additional files DnaA boxes, especially adjacent to dnaA and/or dnaN genes, is considered an indicator for the location of Additional file 1: Nostocales strains used in this study. (XLS 44 kb) oriC. Transposases were classified into IS families using Additional file 2: Supporting Information (SI) Appendix. (PDF 8156 kb) ISfinder (https://www-is.biotoul.fr/;). Additional file 3: Proteins encoded in 10 most prominent locally collinear blocks in Nostoc punctiforme PCC 73102, Nostoc sp. N6 and Nostoc sp. ‘Lobaria pulmonaria cyanobiont’, identified by Mauve. (XLS 84 kb) Phylogenomic analysis Additional file 4: Comparison of the minimal bacterial gene set to Nostoc Available genomes of Nostocales strains along with sp. N6 and Nostoc sp. ‘Lobaria pulmonaria cyanobiont’. (XLS 61 kb) Arthrospira platensis NIES-39, Lyngbya sp. PCC 8106 Additional file 5: Comparison of the cyanobacterial core and shell gene and Planktothrix agardhii NIVA-CYA 126/8 (order Oscil- set to Nostoc sp. N6 and Nostoc sp. ‘Lobaria pulmonaria cyanobiont’. latoriales) as an outgroup were retrieved from GenBank (XLS 172 kb) and the Joint Genome Institute’s Integrated Microbial Additional file 6: Orthologous groups enriched in different clades. (XLS 756 kb) Genomes database (JGI-IMG) in January 2018. Thirty- one marker proteins that are universally conserved across Abbreviations the bacterial domain (dnaG, frr, infC, nusA, pgk, pyrG, AAG: Alkyl-acylglycerol; ATP: Adenosine triphosphate; CAAD: Cyanobacterial rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, Aminoacyl-tRNA synthetase domain; CDP: Cytidine Diphosphate; CDP-APT: Gagunashvili and Andrésson BMC Genomics (2018) 19:434 Page 15 of 18 CDP-alcohol Phosphatidyltransferase; CMP: Cytidine monophosphate; COG: symbiosis. Molecular Ecology. 2012;21(13):3159–72. https://doi.org/10. Clusters of Orthologous group; CPT: Chloramphenicol Phosphotransferase; 1111/j.1365-294X.2012.05482. CTP: Cytidine triphosphate; DAG: Ddiacylglycerol; dCMP: Deoxycytidine 7. O’Brien HE, Miadlikowska J, Lutzoni F. Assessing reproductive isolation monophosphate; dCTP: Deoxycytidine triphosphate; dNTP: Nucleoside in highly diverse communities of the lichen-forming fungal genus triphosphate; dTTP: Thymidine triphosphate; dUMP: Deoxyuridine Peltigera. Evolution. 2009;63(8):2076–86. monophosphate; dUTP: Uridine diphosphate; ED: Entner–Doudoroff; FAD: 8. O’Brien HE, Miadlikowska J, Lutzoni F. Assessing population structure Flavin adenine dinucleotide; Gnd: 6-Gluconophosphate dehydrogenase; and host specialization in lichenized cyanobacteria. New Phytologist. HAEP: 1-hydroxy-2-aminoethylphosphonate; HIF: Hormogonium inducing 2013;198(2):557–66. factor; HRF: Hormogonium repressing factor; IS: Insertion sequence; KEGG: 9. Crittenden P, David J, Hawksworth D, Campbell F. Attempted isolation Kyoto encyclopedia of genes and genomes; LAM: Lysine 2,3-Aminomutase; and success in the culturing of a broad spectrum of lichen-forming and NADP: Nicotinamide adenine dinucleotide phosphate; NNI: Nearest neighbor lichenicolous fungi. New Phytologist. 1995;130:267–97. interchange; NRPS: Non-ribosomal peptide synthetase; OPP: Oxidative 10. Stocker-Wörgötter E, Hager A. Culture methods for lichens and lichen pentose pathway; ORF: Open reading frame; PEP: Phosphoenolpyruvate; symbionts. In: Nash TH, editor. Lichen Biology. Cambridge: Cambridge PepM: Phosphoenolpyruvate mutase; PKS: Polyketide synthase; Ppd: University Press; 2008. p. 353–63. Phosphonopyruvate decarboxylase; PSSM: Position-specific scoring matrix; 11. Ahmadjian V. The Lichen Symbiosis. Hoboken: Wiley; 1993. RNR: Ribonucleoside reductase; SET: Lysine Methyltransferase; UDP: Uridine 12. Rikkinen J. 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