Draft genome of Myxosarcina sp. strain GI1, a baeocytous cyanobacterium associated with the marine sponge Terpios hoshinota

To date, genome sequences (complete or in draft form) from only six baeocytous cyanobacteria in four genera have been reported: Xenococcus, Chroococcidiopsis, Pleurocapsa, and Stanieria. To expand our knowledge on the diversity of baeocytous cyanobacteria, this study sequenced the genome of GI1, which is a Myxosarcina-like baeocytous cyanobacterium. GI1 is of interest not only because of its phylogenetic niche, but also because it is a cyanobiont isolated from the marine cyanobacteriosponge Terpios hoshinota, which has been shown to cause the death of corals. The ~7 Mb draft GI1 genome contains 6,891 protein-coding genes and 62 RNA genes. A comparison of genomes among the sequenced baeocytous cyanobacterial strains revealed the existence or absence of numerous discrete genes involved in nitrogen metabolism. It will be interesting to determine whether these genes are important for cyanobacterial adaptations and interactions between cyanobionts and their marine sponge hosts.


Introduction
In the latest (second) edition of Bergey's Manual of Systematic Bacteriology, cyanobacteria are classified into five subsections ("orders") [1]. All members in Subsection II (order Pleurocapsales) reproduce (exclusively or partially) via multiple fission, which produces small reproductive cells called baeocytes [2]; these species are thus described as "baeocytous". Baeocytous species are further divided into seven genera according to developmental characteristics, such as: the contribution of baeocyte formation to reproduction, the morphology of cell aggregates associated with successive binary fission in vegetative cells, and the presence of fibrous cell walls at the onset of baeocyte formation. The seven genera are Cyanocystis, Dermocarpella, Stanieria, Xenococcus, Chroococcidiopsis, Pleurocapsa, and Myxosarcina [2]. The taxa in Subsection II present considerable diversity in terms of physiology and ecology. Most baeocytous species are solitary (free-living) entities, which can be found in water or on land. Intertidal zones show a particularly rich diversity of baeocytous species, most of which are epilithic or endolithic [2]. A number of species associate with lichen [3] or sporadically occur as extracellular symbionts of marine sponges [4]. Terpios hoshinota is a marine cyanobacteriosponge that infests coral reefs in west Indo-Pacific regions [5]. T. hoshinota infestations have been named "black disease" because these sponges tend to overgrow live corals, resulting in the formation of black encrustations, which can spread within a few days and shut down photosynthesis. This causes the death of the coral, with none of the coral pulps able to regenerate following encrustation. In 2006, an unprecedented outbreak of black disease occurred in the waters of Green Island, located southeast of Taiwan. In that outbreak, more than 30% of coral were overgrown by sponge [6]. Little is known about the nature of coral black disease, although Montipora aequituberculae corals appear to be particularly susceptible. T. hoshinota is associated with a substantial quantity of cyanobacteria, mainly Aphanocaps type, which lives intercellularly within the sponge [5]. The cyanobacteria associated with T. hoshinota are able to perform photosynthesis; therefore, it has been suggested that cyanobionts provide nutrients to support the spread of their host [6]. In this work, we succeeded in purifying a cyanobacterium associated with T. hoshinota from Green Island, called GI1. We then cultivated the organisms in the laboratory to study their taxonomy and physiology. Specifically, we describe the morphological, biochemical, and genomic properties of GI1, which resemble those of a Myxosarcina species [2]. The genome sequence of GI1 may also provide insight into symbiotic interactions between cyanobionts and their marine sponge hosts.

Classification and features
A coral sample (Montipora sp.) overgrown by T. hoshinota was collected from the sub-tidal zone of Green Island in 2007. Black scrapings from the surface of the sample were suspended in sterile seawater and then streaked onto plates prepared by supplementing ASN-III medium [7] with 0.8% agar (ASN-III agar plates). After two months, only one type of cyanobacterium, characterized by a punctiform shape and blackish color, was found on the plate. This cyanobacterium was purified by successively transferring and streaking onto the same type of plates at two month intervals. An axenic culture was then established and added to our collection as strain GI1. This strain produced coccoid and motile baeocytes, which reacted photactically and lost mobility as they enlarged into spherical vegetative cells. Most of the vegetative cells performed successive binary fission in three planes, which resulted in the formation of cubic or irregular cell aggregates and eventually produced baeocytes ( Figure 1). Baeocyte diameters (2.3 ± 0.2 μm) differed little from those of parental (mature) vegetative cells (3.7 ± 0.7 μm) that were preparing to release baeocytes. The vegetative cells in GI1 had an average volume only 4.2 times larger than that of a newly released baeocytes; thus, each vegetative cell could produce no more than 4 baeocytes. These characteristics suggest that GI1 belongs to the Myxosarcina [2]. Phylogenetic analysis of 16S rRNA gene sequences led to the segregation of Subsection II cyanobacteria into two groups in the tree ( Figure 2). The first group contained Chroococcidiopsis cluster 1, which is similar to heterocyst-forming cyanobacteria; the second group contained the bulk of Subsection II cyanobacteria, including GI1. Note that with high bootstrap support, GI1 did not form a sister clade with Myxosarcina PCC 7325, which was located in the same clade containing Pleurocapsa, Dermocarpella, and Stanieria cluster 2. Stanieria cluster 2 also failed to form a sister clade with Stanieria cluster 1. These observations suggest that the phylogeny of the 16S rRNA gene sequence is not consistent with the taxonomic relationships among baeocytous cyanobacteria. GI1 is a facultative photoheterotroph. Supplementing the ASN-III medium with yeast extract and glucose in 1 and 2 g/L −1 stimulated the growth of GI1 but inhibited the growth of Myxosarcina strain PCC 7312, indicating that the ability of GI1 to use organic resources exceeds that of PCC 7312. The classification and general features of Myxosarcina sp. strain GI1 are summarized in Table 1.

Genome project history
The project information and its association with MIGS version 2.0 compliance [8] are summarized in Table 2. The genome was first sequenced in 2010 and this work provides a high-quality draft of genome. The assembled contigs have been deposited in NCBI.

Growth conditions and genomic DNA preparation
A single colony of GI1 was selected from the ASN-III agar plate and transferred into a 1 L serum bottle with 200 mL of ASN-III medium. The culture was shaken (90 rpm), aerated (0.2 volume per volume per minute, VVM), and illuminated laterally at 27.0 μmol photons · m −2 · s −1 , as measured at the surface of the bottle. Cells were then cultivated in a 12:12 light-dark cycle until the late exponential phase of growth. The cells The 16S rRNA gene sequences of GI1 and type strains belonging to different cyanobacterial subsections were subjected to phylogenetic analysis using MEGA5 software [31] in conjunction with the multiple alignment program CLUSTAL W to construct a maximum-likelihood tree, using bootstrap values of 1000 replicates. The GenBank accession numbers for each stain are shown in parenthesis.
in each culture were harvested by centrifugation at 5,000 × g for 15 min, rinsed twice using 10 mL deionized water, and extracted using Tri-Total Nucleic Acid Isolation Reagent (Geneaid, New Taipei City, Taiwan) to obtain genomic DNA. Extraction was performed according to manufacturer guidelines. Genomic DNA of GI1 was quantified using the Quant-iT dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA, USA) and quality checked on 0.6% agarose gel. Twenty micrograms of DNA was sheared using a Bioruptor ultrasonicator (Diagenode, Liège, Belgium) set at power on for 30 sec and power off for 30 sec. The fragmented DNA was then separated using either 1.5% or 0.6% agarose gel electrophoresis to obtain DNA strands of various lengths.

Genome sequencing and assembly
One  (1) it contained an unknown base "N", (2) the lowest quality score was less than 30, or (3) more than 95% of the bases were identical. The reads of the 3 kb MP library were further trimmed to a length of 60 bp. The 5 kb and 9 kb MP libraries were prepared using Illumina's Nextera kit. Reads Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [30].
containing the Nextera adaptor sequence were retained and the adaptor parts were removed. Resulting reads shorter than 40 bp or containing an unknown base "N" were further discarded. The processed reads of all four libraries accounted for an 1186X coverage based on a 7 Mb genome. These reads were assembled using ALLPATHS-LG (v47833) [9,10] with all parameters set to default.

Genome properties
The draft genome of GI1 contained 7.06 M bp in 76 contigs (or 21 scaffolds); the N50 length of the contigs was 195,043 bp ( Table 3). The GC content was 40.1%. Gene annotation revealed 6891 protein coding genes, 6 rRNA genes, and 56 tRNA genes. COG annotations of protein coding genes are presented in Table 4. Figure 3 presents the genome atlas of GI1.

Insights from the genome sequence
The seven baeocytous cyanobacterial genomes (including GI1) are compared in Table 5. By comparing COG annotations, we identified 13 genes that existed in all baeocytous cyanobacteria except GI1 (Table 6) and 36 genes that only appeared in GI1 (Table 7). Many products of these genes (e.g. UreE, SpeA, and GltD in Table 6 and ArgR, COG2070, HutG, COG4262, and NtrB in Table 7) are related to nitrogen metabolism. It can therefore be surmised that these genes participate in nitrogen cycles between cyanobionts and their hosts. Moreover, many GI1-specific genes are involved in processing a wide range of organic compounds as carbon, nitrogen, or energy sources. The putative products encoded by these genes include COG 2070 (dioxygenases related to 2-nitropropane dioxygenase) [25], HutG (N-formylglutamate amidohydrolase) [26], CelA (cellobiohydrolase A) [27], and Hdrc (heterodisulfide reductase, subunit C) [28]. These enzymes are rarely found in cyanobacteria but are common among heterotrophic bacteria and fungi. Exploring the origins and functions of these genes in GI1 will no doubt produce interesting results.

Conclusions
The assembly and analysis of GI1 genome revealed distinctive genes involved in nitrogen metabolism and  utilization of a large array of organic compounds. The GI1 genome is thus valuable for studying interactions between GI1 and its marine sponge host.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions YC and TL designed and carried out the experiments. CY, CL, HS, TC, and CH performed the data analysis and drafted the manuscript. All authors read and approved the final manuscript. HdrC C Heterodisulfide reductase, subunit C * C: energy production and conversion; D: cell division and chromosome partitioning; E: amino acid transport and metabolism; G: carbohydrate transport and metabolism; I: lipid metabolism; K: transcription; L: DNA replication, recombination, and repair; M: cell envelope biogenesis, outer membrane; P: inorganic ion transport and metabolism; R: general function prediction only; S: function unknown; T: signal transduction mechanisms; U: intracellular trafficking and secretion; V: defense mechanisms.