Draft Genome Sequence of Firmicutes Strain S0AB, a Heterotrophic Iron/Sulfur-Oxidizing Extreme Acidophile.

The draft whole-genome sequence of the extremely acidophilic and novel Firmicutes strain S0AB is reported. The genome comprises 3.3 Mbp and has a GC content of 43.72%. In total, 3,240 protein-coding genes, 56 tRNA genes, and 11 rRNA genes were predicted.

ANNOUNCEMENT

The novel Gram-positive, endospore-forming Firmicutes strain S0AB, which is mesophilic and extremely acidophilic, was isolated from laboratory-grade S0 (elemental) sulfur obtained from Peak Trading Company (Nottingham, UK) on a low-pH solid medium containing potassium tetrathionate and ferrous sulfate (1, 2). It requires an organic source of carbon for growth. It can oxidize and reduce iron and catalyzes the dissimilatory oxidation of S0 and tetrathionate. S0AB was cultured aerobically at 32°C in a liquid basal salt medium containing 20 mM ferrous sulfate and 0.02% (wt/vol) yeast extract, with an initial pH of 1.7 (3). DNA was extracted in two steps, as follows: (i) cells were lysed at 37°C with lysozyme and RNase A, and (ii) proteinase K and SDS were added for incubation at 65°C. DNA was purified using solid-phase reversible immobilization (SPRI) beads (Qiagen) and quantified with the Quant-iT double-stranded DNA (dsDNA) high-sensitivity (HS) kit (Thermo Fisher Scientific). The Illumina library was prepared using the Nextera XT library preparation kit (Illumina) with a Microlab STAR automated liquid-handling system (Hamilton Bonaduz AG, Switzerland) and was sequenced with the high-throughput Illumina NovaSeq 6000 system (producing 907,704 paired-end raw reads of 2 × 250 bp). The library for long-read DNA was prepared with the SQK-RBK004 kit (Oxford Nanopore Technologies [ONT], UK), loaded in a FLO-MIN106 (R.9.4.1) flow cell, and sequenced with a GridION system (ONT) (producing 6,814 raw reads with a mean length of 14,720 bp). Illumina reads were trimmed using Trimmomatic v0.30 with a sliding window quality cutoff of Q15 (4), and quality was assessed using SAMtools (5, 6), BEDTools (7), and BWA-MEM (bio-bwa.sourceforge.net), generating 889,712 raw reads. Hybrid assembly of the reads was performed using Unicycler v0.4.0 (8). Assembly metrics were calculated using QUAST (9). The genome size of S0AB is 3,342,730 bp, consisting of 150 contigs (size ≥200 bp; N50, 247,888 bp) with a GC content of 43.72% and mean coverage of 128×. CheckM (10) analysis showed 97.8% completeness with <3.5% contamination. The genome was annotated using Prokka 1.14.6 (11), which predicted 3,240 protein-coding genes, including genes for 1,519 hypothetical proteins and 1,721 proteins with predicted functional assignments, as well as 56 tRNA genes and 11 rRNA genes. Default parameters were used for all software unless otherwise noted.

The genome contains sor, sdoB, and soxB, which are involved in the dissimilatory oxidation of S0 and tetrathionate, and sqr, sat, and cysC, which are involved in the uptake of sulfate and its oxidation to sulfide, supporting previous observations of S0AB growth (1). It also contains a cupredoxin superfamily gene (sulfocyanin-like) clustered with cytochromes that have been shown to be involved in Fe2+ oxidation in Sulfobacillus thermosulfidooxidans, which may provide a favorable redox potential for chalcopyrite bioleaching (12). The genome is also predicted to have feoE, which is important for survival during anaerobic iron respiration (13), and a natural resistance-associated macrophage protein (NRAMP) family of proteins, which control metal ion transport and homeostasis (e.g., Mn2+, Cu2+, Fe2+, and Co2+) and are involved in cellular responses to fluctuating environmental concentrations of metal ions (14). The genome is predicted to contain a suite of genes encoding proteins for sporulation, including spoIIR and spoIIGA, and genes for acid resistance, such as the voltage-gated potassium channel kch and the high-affinity ATP-driven potassium transporter kdpABCDE, which are involved in generating a reverse membrane potential that repels the intrusion of protons in acidic environments (15).

Data availability.

The draft whole-genome shotgun project for Firmicutes strain S0AB has been deposited in DDBJ/ENA/GenBank under the accession number JALBUF000000000. The version described in this paper is version JALBUF010000000. The raw read data are available under the SRA accession numbers SRX15640773 and SRX15640772. Project data are available under the BioProject accession number PRJNA813843.