Genome sequencing of the extremophile Thermus scotoductus SA-01 and expression of selected genes
Thermus scotoductus SA-01 is an extremophile that was isolated from groundwater samples at 3.2 km depth in a South African gold mine and has previously been shown to grow using nitrate, Fe(III), Mn(IV) or Sº as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III) and the quinone-containing compound anthraquinone-2,6-disulfonate. This study reports the sequencing of the T. scotoductus SA-01 genome using a strategy involving the GS20 and FLX pyrosequencing, which is a novel, rapid method of high-throughput sequencing, as well as Sanger technology. The GS20 and FLX pyrosequencing data was assembly into 35 contigs using the Newbler Assembly software. Mapping attempts using various software against the reference strain T. thermophilus, proved unsuccessful due to low levels of synteny and extensive rearrangement noticed between the two organisms. After using various strategies to close the gaps between the 35 contigs with Sanger sequencing, the complete chromosome sequence was obtained. The genome consists of a 2 346 803 bp chromosome and a plasmid, which could not be closed with all sequencing attempts. The draft plasmid sequence consists of 8 383 bp with about 65% in agreement with the chromosome sequence. Automatic annotation of the complete chromosome and draft plasmid sequence performed by TIGR (J. Craig Venter Institute formerly known as The Institute for Genome Research) revealed the presence of 2482 and 12 ORFs, respectively. ORF correction was performed using the Artemis software package. Manual annotation was performed using the ERGO Tool software on half of the genome using various public databases and criteria. BLAST results of the plasmid sequence against the chromosome show that four ORFs present on the draft plasmid are also present in an identical copy (one or more than one copy) on the T. scotoductus SA-01 chromosome, providing evidence of genetic exchange between the chromosome and the extrachromosomal element. Comparative genome analysis was done using strains that are related (3 genomes) to T. scotoductus, isolated from a South African goldmine (1 genome) and metal reducing organisms (2 genomes). Using this data, analysis of metabolism and thermophily of T. scotoductus SA-01 could be comparatively elucidated as well as determining the orthologous gene content. The complete chromosome and draft sequence of T. scotoductus SA-01 not only provides valuable basic data in terms of the organism’s lifestyle and capabilities but is also consists of many genes of potential interest for biotechnological applications. Due to its thermophilic nature, T. scotoductus SA-01 would contain many thermostable enzymes, which possess qualities that make them more robust and better suited for use in molecular applications. The DNA polymerase I and single stranded DNA binding (SSB) protein was chosen for expression studies for their potential use in a PCR reaction. A partially purified DNA polymerase I protein was obtained; however the protein was found to be non-functional in a PCR. Expression of the SSB was performed, but the protein could not be purified for further analysis. Obtaining expression at higher levels and complete protein characterization would be required in order to understand the capabilities of these proteins.