Genome sequence of the copper resistant and acid-tolerant Desulfosporosinus sp. BG isolated from the tailings of a molybdenum-tungsten mine in the Transbaikal area.

Here, we report on the draft genome of a copper-resistant and acidophilic Desulfosporosinus sp. BG, isolated from the tailings of a molybdenum-tungsten mine in Transbaikal area. The draft genome has a size of 4.52 Mb and encodes transporters of heavy metals. The phylogenetic analysis based on concatenated ribosomal proteins revealed that strain BG clusters together with the other acidophilic copper-resistant strains Desulfosporosinus sp. OT and Desulfosporosinus sp. I2. The K+-ATPase, Na+/H+ antiporter and amino acid decarboxylases may participate in enabling growth at low pH. The draft genome sequence and annotation have been deposited at GenBank under the accession number NZ_MASS00000000.

Direct link to deposited data

https://www.ncbi.nlm.nih.gov/nuccore/NZ_MASS00000000.1

Introduction

Members of the genus Desulfosporosinus are often found in acidic mining environments [6], [8], [11], [13], [17], [18], [20], [21], [22], [23]. These bacteria may participate in metal detoxification by precipitating them in the form of sulfides [5]. Desulfosporosinus acidiphilus [2] and Desulfosporosinus acididurans [19], both isolated from mining environments, represent the only two validly described, moderately acidophilic sulfate-reducing bacteria. Two other acidophilic Desulfosporosinus have been isolated and their genomes are available [1], [15]. Desulfosporosinus spp. are also known for their tolerance to metals, in particular to copper [1], [14], [15]. Recently, a novel acidophilic, copper-resistant Desulfosporosinus sp. BG was isolated from the tailings pond sediment of the Bom-Gorkhon molybdenum-tungsten mine in the Transbaikal area [10]. The acidophilic isolate could tolerate up to 6 g Cu2 +/L and grew at the pH range from 1.0 to 6.5. The purpose of this study was to sequence genome of Desulfosporosinus sp. BG to verify its phylogenetic relationship with the known acidophiles belonging to this genus and compare the putative mechanisms, which enable the bacteria to withstand acid and metal stress.

Experimental design, materials and methods

Sequencing and assembly of Desulfosporosinus sp. BG genome

Genomic DNA was extracted from Desulfosporosinus sp. BG biomass using the SDS-CTAB method [16]. Genomic DNA was sequenced with a Roche Genome Sequencer FLX (GS FLX), using the Titanium XL + protocol for a shotgun library. About 189 Mb of sequences with an average read length of 495 nt were generated. The reads were de novo assembled into contigs using the Newbler Assembler version 2.9 (454 Life Sciences, Branford, CT). The resulting draft genome sequence of Desulfosporosinus sp. BG consists of 156 contigs longer than 500 bp, with a total length of 4,536,051 bp. The total length of all 345 obtained contigs is 4,579,156 bp. The N50 contig size of the genome is 70,527 bp. Gene search and annotation were performed for all contigs longer than 500 bp using the RAST server [3] following manual curation.

Features of the Desulfosporosinus sp. BG genome

The draft genome of Desulfosporosinus sp. BG was around 4.52 Mb, which is a relatively small size, compared to Desulfosporosinus orientis – 5.86 Mb (NC_016584), Desulfosporosinus youngiae – 5.66 Mb (NZ_CM001441), Desulfosporosinus meridei – 4.87 Mb (NZ_CM001441.1), and Desulfosporosinus acidiphilus – 4.93 Mb (NC_018068). However, the acidophilic Desulfosporosinus acididurans has approximately the same size genome, 4.64 Mb (NZ_LDZY00000000.1), as strain BG. The GC content of the genome is 42.4%. The genome includes 4516 protein-coding genes, 67 tRNA genes, and 9 rRNA genes.

The phylogenetic analysis based on concatenation of 32 ribosomal proteins showed that strain BG clustered with the acidophilic, copper-resistant Desulfosporosinus sp. I2, and two uncultivated strains, Desulfosporosinus BRH_c37 and Desulfosporosinus BIKA1-9. Their composite genomes were obtained from the metagenomic data from groundwater (Fig. 1). The closest relative of strain BG was Desulfosporosinus sp. OT isolated previously from the Norilsk mining area [9].

Fig. 1

Phylogenetic analysis of representatives of the genus Desulfosporosinus based on concatenated amino acid sequences of 32 ribosomal proteins. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [7]. The tree with the highest log likelihood (− 27,426.3925) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 12 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 4259 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [12].

Several major mechanisms notable for the acid- and metal-tolerance were detected in the Desulfosporosinus sp. BG genome. The acid-tolerance determinants included the K+-transporting ATPase KdpABC (DSBG_RS17700-KdpA, DSBG_RS17705-KdpB, DSBG_RS17710-KdpC), which participates in the generation of internal positive membrane potential preventing proton influx to the cytoplasm. Strain BG along with Desulfosporosinus sp. OT has an Na+/H+ antiporter (DSBG_RS05820), known as the key transporter in maintaining the pH of actively metabolizing cells. The Na+/H+ antiporter did not occur in any other available Desulfosporosinus genomes. Phylogenetic analysis shows that the antiporter was likely acquired from Bacillus via lateral gene transfer. Additionally, strain BG has proton-consuming decarboxylases – the arginine decarboxylase (DSBG_RS13090) and the lysine decarboxylase (DSBG_RS07860), which are involved in the mechanism of coping with low pH environment enterobacteria [4]. Orthologous decarboxylases are present in all available Desulfosporosinus genomes.

A copper ATPase occurs in the Desulfosporosinus sp. BG genome. It comprises an operon with transcriptional regulator CsoR (DSBG_RS14110) and copper chaperon CopZ (DSBG_RS14120). Orthologous genes for the Cu-ATPase are present in all available Desulfosporosinus genomes. Consistent with the phylogenetic position derived from ribosomal proteins, the closest relatives of the Cu-ATPase of strain BG were proteins from Desulfosporosinus sp. OT (sequence similarity 97%) and Desulfosporoinus sp. I2 (80%).

Several other heavy metal-transporting ATPases occur in the genome of strain BG. Two IB P-type ATPases primarily responsible for translocating Cd2 + and other closely-related divalent Co, Hg, Pb, and Zn ions, were found in strain BG genome. One of the ATPases (DSBG_RS14085) is present in all available Desulfosporosinus genomes and located close to the Cu-ATPase. Interestingly, another cadmium ATPase (DSBG_RS14615) has orthologous genes only in Desulfosporosinus sp. OT (95% similarity), Desulfosorosinus sp. I2 (88%), Desulfosporosinus acidiphilus (85%), and uncultivated Desulfosporosinus sp. BRHc37 (90%), but not in any other available Desulfosporosinus genomes. The phylogenetic analysis shows that the cadmium ATPase was likely transferred laterally from Paenibacillus. Desulfosporosinus sp. OT encodes two ATPases of this type (WP_009618081, WP_009624106). Physiological studies did not reveal outstanding tolerance to cobalt, nickel, and cadmium in strain BG [10].

In conclusion, we have revealed additional transporters that are not available genomes of other members of this genus. These may enable Desulfosporosinus sp. BG to withstand low pH or high metal concentrations. The strain has heavy metal transporters that can enable it to tolerate high concentrations of lead, mercury, or zinc. The latter is a subject to be studied in the future research.

Conflict of interest

The authors declare no conflicts of interest in this study.

Specifications
Organism/cell line/tissue	Desulfosporosinus BG
Sex	N/A
Sequencer or array type	GS FLX (Roche)
Data format	Analyzed
Experimental factors	Bacterial strain
Experimental features	Assembled and annotated draft genome of Desulfosporosinus strain BG
Consent	N/A
Sample source location	Sediments of a tailings pond at a molybdenum-tungsten mine in Bom-Gorkhon, Transbaikal Area, Russia (51° 28′25″; 109° 49′15″).