Draft Genome Sequence of the Symbiotically Competent Cyanobacterium Nostoc sp. Strain KVJ20

Nostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla. This cyanobacterium has been shown to have broad symbiotic competence, and bacterial extracts have inhibitory effects on cancer cell lines and microbes. An array of genes for the production of secondary metabolites is present.

N ostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla L., found as a weed in a plant school on Kvaløya Island in northern Norway (1). During the isolation process, the strain was unusually clean from the first steps of cultivation and did not require additional treatments to bring it into an axenic state. This was an indication that the organism produces antibiotic compounds. We had a similar experience with the genetically similar Nostoc sp. strain SKSF3, which originated from soil. Metabolic profiling by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed similarities between the secondary metabolite sets produced by these two isolates (1). In addition, the cell extracts of Nostoc sp. KVJ20 showed inhibitory effects on the A2068 metastatic human melanoma cell line and MRC5 fibroblasts (2). Insights into the metabolic capacities of the strain, based on full-genome sequencing, may help explain its antimicrobial and anticancer properties. Nostoc sp. KVJ20 has broad symbiotic competence and has successfully infected seedlings of the angiosperm Gunnera manicata Linden, where it existed as an intracellular symbiont (3). Thus, the genome will add valuable information on the core features underlying the symbiotic capacity of the genus.
Isolation and culturing of Nostoc sp. KVJ20 from the symbiotic organs of the liverwort Blasia pusilla were carried out as described earlier (1,4). The strain is maintained at the Department of Arctic and Marine Biology, Faculty of Biosciences, Fisheries and Economics, UiT The Arctic University of Norway. Photos of Nostoc sp. KVJ20 at different life stages and the phylogenetic placement are presented in Fig. 1.
Genomic DNA from Nostoc sp. KVJ20 was purified according to the method for bacterial genomic DNA isolation using CTAB, v3 (5). The only modification is two washes in 5 M NaCl preceding the DNA isolation. The DNA was quantified using a Qubit fluorometer. For the library, a Nextera DNA library prep kit (catalog number FC-121-1031) was used. The library was sequenced on a MiSeq machine (Illumina) at UiT The Artic University of Norway using the MiSeq reagent kit v3 (2 ϫ 300 bp; catalog number MS-102-3003). The reads were quality trimmed, adapter trimmed, and assembled using CLC Genomics Workbench 8.5 (Qiagen) with default settings. A total of 9,259,128 reads with an average read length of 208 bases were assembled. The NCBI Prokaryotic Genome Annotation Pipeline v3.1 was used for annotation, with GeneMarkSϩ used for gene identification (6, 7). The draft genome is composed of 425 contigs (332 scaffolds), with an N 50 value of 41,001 bp, a total size of 9.2 Mbp, an average read coverage of 200ϫ, and a GC content of 41.69%. In total, 7,676 genes were predicted, with 7,210 coding sequences (CDSs), 104 RNAs, and 362 pseudogenes.
The symbiotic competence, production of antibacterial and anticancer compounds, cellular differentiation mechanism, and how these properties are connected to the production of secondary metabolites from the identified gene clusters will be interesting to pursue further.
Data availability. The genome sequence is deposited in the NCBI under BioProject number PRJNA310825, SRA project number SRS4677145, and BioSample number SAMN04453661. The assembled genome is deposited under GenBank accession number LSSA00000000.

ACKNOWLEDGMENTS
This work was supported by the UiT The Arctic University of Norway and the NorZymeD project of the Research Council of Norway (grant number 221568).
Erik Hjerde (Department of Chemistry, UiT The Arctic University of Norway) is greatly acknowledged for help during assembly. Nodularia spumigena PCC73104, DQ185241. 16S rRNA genes, aligned using MUSCLE (10,11), were used as input to the Web server Phylogeny.fr (12). A total of 1,402 positions, not including gaps, were compared and used to reconstruct and analyze the phylogenetic relationships. Confidence index values are shown in red, and the bar represents the amount of nucleotide substitutions per site. Gloeobacter violaceus (GenBank accession number FR798924) was used as an outgroup.