High-Quality Draft Genome Sequence of the Siderophilic and Thermophilic Leptolyngbyaceae Cyanobacterium JSC-12

Despite the extreme oxidative stress induced by high Fe(II) concentrations (>100 μM) (1), contemporary circumneutral iron-depositing hot springs are densely populated with cyanobacteria (2–4). Here, we report the high-quality draft genome sequence of the cyanobacterium JSC-12, isolated from a phototrophic mat formed on iron deposits in Chocolate Pots Hot Springs, Yellowstone National Park, WY, USA (5). A sample was aseptically collected from a surface mat located ∼1 m downstream from the main mound (5), where the water was 40°C, pH 6.48, and contained 2.8 μM ferrous and 4.2 μM ferric iron. From this, we obtained a unialgal isolate of JSC-12 following references 2 and 4, using DH growth medium (6) supplemented with 0.04 mM FeCl₃·6H₂O. JSC-12 is a filamentous cyanobacterium with narrow trichomes (0.8 to 1.8 μm wide), resembling the morphological features of Leptolyngbya (7). Strain JSC-12 showed thermophilic and siderophilic behavior with an optimal growth temperature of 45°C and an Fe(III) concentration of 0.4 to 0.8 mM (3).

For genomic DNA isolation, JSC-12 was cultivated at 40°C under permanent white light (17 μE/m²/s) in 1-liter flasks containing 0.25 liter of DH medium supplemented with 0.04 mM FeCl₃·6H₂O on a rotating shaker (18 rpm). The genomic DNA was purified using the UltraClean microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA). The genome was sequenced from an Illumina regular fragment library using the KAPA-Illumina library creation kit (Kapa Biosystems) (96,333,258 reads and 7,321.3 Mb) and a paired-end 454 library (220,108 reads and 58.1 Mb) according to Peng et al. (8). Prior to assembly, the reads were filtered to remove low-quality reads, Escherichia coli strains, 454 adapters, and known contaminants using tools from the 454 assembler suite (Roche), including runMapping and runAssembly. The remaining data were assembled using a combination of Newbler v. 2.3-PreRelease-6/30/2009, VELVET v. 1.0.13 (9), and parallel Phrap v. 1.080812. The final high-quality draft assembly contained 20 contigs in 1 scaffold.

Annotation was performed by Oak Ridge National Laboratory using Prodigal v. 1.4 (10), followed by manual curation using the U.S. Department of Energy Joint Genome Institute’s GenePRIMP pipeline (11). For all computational steps, default parameters were used except where otherwise noted. The JSC-12 genome has a 5,530,491-bp genome with a GC content of 47.5; it contains 5,080 genes, 2 rRNA operons, and 44 tRNA genes. Phylogenetic analyses showed that JSC-12 is associated with Leptolyngbya, but the similarity in 16S rRNA (∼92%) and genomic amino acid identity (62 to 65%), along with genomic taxonomy from GTDB (12), suggest that it belongs to a potential new genus in the Leptolyngbyaceae family (13, 14).

Prediction of biosynthetic gene clusters revealed that the JSC-12 genome lacks the pathway for synthesizing bacterioferritin, the classical iron storage protein. However, the gene rbr, encoding rubrerythrin (15), was present. A putative feoABC operon (16) may be involved in the acquisition of Fe(II) through the cytoplasmic membrane for sequestration and detoxification within ferritin (ftn), which is likely cotranscribed with feoABC.

I.B. and S.S. thank Dan Garrison, Chief Scientist and Deputy Department Director at Jacobs Engineering, for patronage of our studies in 2006 to 2010. The study carried out at Jacobs was partially supported by the NASA JSC director discretionary fund in 2008 to 2009. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under contract number DE-AC02-05CH11231. J.A. was supported by the Chilean ANID doctoral fellowship number 21191763.

We thank D. A. Bryant and G. Shen (Penn State) for their cooperation in the additional purification of the isolated DNA. We thank A. Copeland for participation in the discussion of this manuscript.