Draft genome sequence of chloride-tolerant Leptospirillum ferriphilum Sp-Cl from industrial bioleaching operations in northern Chile

Leptospirillum ferriphilum Sp-Cl is a Gram negative, thermotolerant, curved, rod-shaped bacterium, isolated from an industrial bioleaching operation in northern Chile, where chalcocite is the major copper mineral and copper hydroxychloride atacamite is present in variable proportions in the ore. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of chloride, sulfate and metals. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 2,475,669 bp draft genome is arranged into 74 scaffolds of 74 contigs. A total of 48 RNA genes and 2,834 protein coding genes were predicted from its annotation; 55 % of these were assigned a putative function. Release of the genome sequence of this strain will provide further understanding of the mechanisms used by acidophilic bacteria to endure high osmotic stress and high chloride levels and of the role of chloride-tolerant iron-oxidizers in industrial bioleaching operations.


Introduction
Extremely acidophilic leptospirilli exhibit considerable physiological and genetic variation [1] and have been classified into four species groups according to 16S rRNA phylogeny [2][3][4]. Group I is represented by Leptospirillum ferrooxidans, Group II by L. ferriphilum and Group III by "L. ferrodiazotrophum" [5,6]. Recently, metagenomic evidence has supported the recognition of a new species ascribed to Group IV [7].
As all leptospirilli, Group II members are aerobic and obligatly chemolithotrophic, ferrous iron oxidizing bacteria. However, they differ from the other groups in their G + C molar ratios, the number of copies of rrn genes and the size of 16S-23S rRNA gene spacers, as well as in their capacity to grow at 45°C [5].
L. ferriphilum has been shown to be the dominant microorganism in commercial biooxidation tanks in South Africa [5] and in PLS from heap bioleaching processes in Chile [8][9][10]. L. ferriphilum Sp-Cl is a key biological member in industrial biomining applications, becoming the most abundant or even the exclusive microorganism in certain stages of processes involving ferrous iron oxidation [11,12]. Competitive growth of L. ferriphilum Sp-Cl has been explained by the elevated temperature, particular electrochemical conditions and certain metal concentrations that develop during mineral leaching. Leptospirillum group II spp. have also been documented to act as the dominant primary producers on floating biofilms obtained from the Richmond Mine at Iron Mountain in USA [13,14].
The genomes of three isolates of L. ferriphilum are available: the draft genome of the type strain DSM 14647 obtained from an acid mine drainage in Peru [15], the complete genome of strain ML04 isolated from acidic water near a hot spring in China [16] and the complete genome of strain YSK [NCBI NZ_CP007243] isolated from an acid mine drainage in China. In addition, draft genomes for other three Group II members, 'C75' [13], '5-way CG' [17,18] and 'L. rubarum' [19] have been derived from metagenomic studies of acid mine drainages in the USA, together with several genomic variants emerging on short time evolutionary scales [13].
This work reports the microbiological and genomic properties of the first industrial isolate of L. ferriphilum. Strain Sp-Cl (DSM 22399) was isolated from the leaching solutions draining from bioleaching heaps at the Spence mine located in the Atacama Desert (northern Chile), where chalcocite is the major copper mineral and copper hydroxychloride atacamite [Cu 2 Cl(OH) 3 ] is present in variable proportions in the ore. The dissolution of atacamite is the main source of chloride in the PLS of the leaching process at Spence mine, which ranges between 1.5 and 12.5 g L −1 . The isolation of this industrially important, chloride tolerant, iron oxidizing acidophile is highly significant for both basic and applied reasons, being a relevant model for chloride leaching studies.

Classification and features
Phylogenetic analysis of the 16S rRNA gene sequence of the isolate Sp-Cl, and other 17 isolates and/or clones representing currently recognized leptospirilli groups and species, revealed its close relation to L. ferriphilum (Fig. 1). L. ferriphilum Sp-Cl cells are morphologically very similar to other L. ferriphilum strains described previously [5,15]. Sp-Cl cell are small sized (0.3 to 0.9 μm), curved rods (Fig. 2), depending on the culture state. The Gram stain for the Sp-Cl is consistently negative and a single polar flagellum enables its motility.
Like other known strains of the species, the Sp-Cl isolate utilizes ferrous iron as an energy source, but neither sulfur nor RISCs can be oxidized with energy conservation. It is also able to fix inorganic carbon (CO 2 ) and nitrogen (N 2 ) [20,21]. The pH for growth ranges from 1.3 to 2.0 and the registered highest tolerated temperature is 45°C, with an optimum between 30 and 37° (Table 1).
Previous work on related L. ferriphilum strains has confirmed the greater tolerance to copper, silver and sulfate by this species as compared to L. ferrooxidans and 'L. ferrodiazotrophum' members [10,16,22]. In addition, L. ferriphilum Sp-Cl has shown notable resistance to chloride (Cl − ) and iron concentrations being able to oxidize ferrous iron (3 g/L) in the presence of Cl − (12 g/L), making it a candidate for bioleaching with proportions of seawater  [11,12], which is an attractive opportunity in arid areas such as northern Chile and parts of Australia, or for chalcopyrite chloride leaching [23].

Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to members of the genus Leptospirillum. This Whole Genome Shotgun project has been deposited at GenBank under the accession LGSH00000000 [24]. The version described in this paper is the first version, LGSH01000000. Table 2 presents the project information and its association with MIGS version 2.0 compliance [25].

Growth conditions and genomic DNA preparation
Leptospirillum ferriphilum strain Sp-Cl (DSM 22399), was isolated from the PLS draining from a bioleaching  Phylum "Nitrospirae" TAS [38] Class "Nitrospira" TAS [38] Order "Nitrospirales" TAS [38] Family "Nitrospiraceae" TAS [1,38] Genus Leptospirillum TAS [39] Species Leptospirillum ferriphilum TAS [5] Strain Sp-Cl TAS [5] Gram stain Negative TAS [5] Cell 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 [40] heap at Spence mine, in the Antofagasta Region, Chile. The enrichment and isolation was performed at the Biotechnology Center (CBAR-UCN). Enrichment was performed using a PLS sample as inoculum followed by sequential dilutions and finally the culture was streaked on ABS solid media [26]. After repeated streaking of individual colonies growing on solid media an individual colony, designated Sp-Cl, was transferred to liquid medium.
The Sp-Cl strain was grown at 37°C in liquid ABS medium (pH 1.5) containing 50 mM Fe2+ on an orbital shaker at 150 rpm. The DNA was isolated from cells collected on a nitrocellulose filter (0.22 μm pore), using a High Pure PCR Template Preparation kit according to the manufacturer's instructions (Roche, Germany). The total amount of DNA was 10.4 μg (measured by Pico green assay). The quality of the DNA was assessed by agarose gel electrophoresis (0.8 % w/v).

Genome sequencing and assembly
The genome of L. ferriphilum strain Sp-Cl was sequenced at Beckman Coulter Genomics using 454 sequencing technology and mate pair libraries with insert sizes of 500 bp [27]. Pyrosequencing reads were assembled de novo using Newbler (v2.0.00.22). The final draft assembly contained 74 contigs in 74 scaffolds. The total size of the genome is~2,5 Mbp and the final assembly is based on 61 Mbp of 454 data, which provides an average 20 × coverage of the genome.

Genome annotation
Genes were identified using Glimmer 3.02 [28] as part of the RAST annotation pipeline [29]. The tRNA and tmRNA identification was achieved using ARAGORN v1.2.36 [30] and the rRNA prediction was carried out via HMMER3 [31]. Additional gene prediction analysis and functional annotation was performed at the Center for Bioinformatics and Genome Biology and at the Center for Biotechnology. The predicted CDSs were used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG and InterPro databases. Protein  Table 4. W: Extracellular structures coding genes were analyzed for signal peptides using SignalP v4.1 [32] and transmembrane helices using TMHMM v2.0 [33].

Genome properties
The draft genome size is 2,475,669 nucleotides, with an average G + C content of 54.41 % (Table 3). From a total of 2,882 genes, 2,834 were protein coding genes and 48 are RNA genes. A total of 41.83 % of the genes were assigned a putative function while the remaining ones were annotated as hypotheticals. The distribution of genes into COGs functional categories for L. ferriphilum Sp-Cl is presented in Table 4 and its comparison against the other sequenced L. ferriphilum genomes is presented in Fig. 3.

Insights from the genome sequence
Genomic analysis of L. ferriphilum strains Sp-Cl allowed several genes involved in the three known trehalose biosynthetic pathways in bacteria to be identified (Table 5): GalU-OtsA-OtsB (I); TreY-TreZ-TreX (V) and TreS (IV) [34,35]. Genes of IV and V synthetic pathways, considered as less-prominent routes for trehalose synthesis [36], were found in the genomes of L. ferriphilum DSM 14647 T , and strains Sp-Cl and LF-ML04 in similar genomic contexts as well as in A. ferrooxidans. Similar organization has previously found in Achromobacter xylosoxidans and Ralstonia eutropha H16 (NCBI accession numbers NC_023061.1 and NC_008313.1, respectively), suggesting co-regulation between both pathways. The enzyme encoded by TreS can also produce maltose from either glycogen or maltooligosaccharides and therefore TreS could also have glycogen debranching enzyme activity [36] and possibly maintain trehalose in equilibrium depending upon the osmotic requirement. In addition, another gene for a trehalose synthetase (Ble/Pep2) protein was located in the same genomic context in L. ferriphilum and strains Sp-Cl and LF-ML04 (Table 5) next to a gene for a maltosyltransferase (GlgE) in a similar configuration shown previously [34].
Recently, genes for both trehalose and ectoine biosynthetic pathways were identified in the draft genome of the L. ferriphilum type strain DSM 14647 [15]. Transcriptomic studies of L. ferrooxidans strain L3.2 (isolated from the Rio Tinto, Spain) have pinpointed genes involved in the synthesis of trehalose, ectoine and systems for the transport of potassium in response to the increase of sulfate [37]. In addition, all of the components involved in trehalose and ectoine synthetic pathways have been identified in proteomic analysis performed in biofilms populated by L. ferriphilum and 'L. ferrodiazotrophum' [19].

Conclusions
The 2.4 Mbp draft genome sequence of L. ferriphilum strain Sp-Cl is arranged in 74 high quality scaffolds, resembling in size the type strain DSM 14647 and the Chinese strain ML-04. It encodes 2,834 protein-coding genes, 42 % of which were assigned putative functions, exceeding the predicted gene content of the type strain, the ML-04 strain and the YSK strain, and suggesting recent acquisition of additional functions. A total of 48 RNA genes partitioned into 44 tRNAs, 1 tmRNA and 1 rRNA operon. The most abundant COG functional category in L. ferriphilum strain Sp-Cl and all sequenced strains of the species were translation, ribosomal structure and biogenesis (J), amino acid and transport metabolism (E) and cell wall and cell membrane biogenesis (M). Release of the genome sequence of this strain will provide further understanding of the mechanisms used by acidophilic bacteria to endure high osmotic stress and high chloride levels and of the role of chloride-tolerant ironoxidizers in industrial bioleaching operations.