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The genome of the metal sulfide-oxidizing, thermoacidophilic strain Metallosphaera cuprina Ar-4 has been completely sequenced and annotated. Originally isolated from a sulfuric hot spring, strain Ar-4 grows optimally at 65°C and a pH of 3.5. The M. cuprina genome has a 1,840,348-bp circular chromosome (2,029 open reading frames [ORFs]) and is 16% smaller than the previously sequenced Metallosphaera sedula genome. Compared to the M. sedula genome, there are no counterpart genes in the M. cuprina genome for about 480 ORFs in the M. sedula genome, of which 243 ORFs are annotated as hypothetical protein genes. Still, there are 233 ORFs uniquely occurring in M. cuprina. Genome annotation supports that M. cuprina lives a facultative life on CO2 and organics and obtains energy from oxidation of sulfidic ores and reduced inorganic sulfuric compounds.
Extremely thermoacidophilic archaea play important roles in mobilizing metal sulfide deposits in natural bioleaching environments (5, 9). Due to the ability to oxidize reduced inorganic sulfur compounds (RISCs) under high-temperature conditions, Metallosphaera has attracted increasing interest from the biomining industry (5, 6, 10–13). The bioleaching Metallosphaera sedula was explored at the genomic level (2). Here, we present the complete genome of a newly isolated, bioleaching, and thermoacidophilic Metallosphaera cuprina strain (8).
Genomic DNA of M. cuprina Ar-4 was purified from cells grown in modified Allen medium (3). The whole genome was sequenced by a Roche 454 genome sequencer FLX instrument. A total of 295,139 shotgun reads were produced and assembled into 55 contigs, providing 67-fold coverage. Gaps were closed by multiplex PCR and primer-walking methods. The gap-spanning PCR products were sequenced with an ABI 3730 DNA analyzer, and the resulting sequences were assembled using Phred/Phrap/Consed software. The final consensus quality level of each base was above 64. Protein-coding genes were identified with the Glimmer 3.02 program (4). Protein function was predicted by either homology searches in the GenBank and UniProt protein databases, function assignment searches in the CDD (COG) database, or domain/motif searches in the Pfam databases. The KEGG tool was used to reconstruct metabolic pathways. Membrane proteins were predicted by the LipoP, SignalP, and ConPred II programs. The tRNA genes were identified by using the tRNAScan-SE tool, and the rRNA genes were identified by using the RNAmmer 1.2 and BLASTn programs.
M. cuprina Ar-4 grew chemolithotrophically on CO2 with metal sulfide and RISCs as energy sources or chemoheterotrophically on various organics (8). Its genome consisted of a 1,840,348-bp circular chromosome. The genome carried 2,029 open reading frames (ORFs) in total. Genome annotation and metabolic reconstruction supported the idea that M. cuprina lived a facultative life. The M. cuprina strain fixed CO2 via the 3-hydroxypropionate/4-hydroxybutyrate cycle, and this strain assimilated carbohydrates via the nonphosphorylated Entner-Doudoroff (ED) pathway. It had a complete tricarboxylic acid (TCA) cycle and an incomplete phosphate pentose pathway. Oxidation of RISCs by the heterodisulfide reductase complex, sulfide:quinone oxidoreductase, thiosulfate:quinone oxidoreductase, tetrathionate hydrolase, and sulfite:acceptor oxidoreductase in M. cuprina was proposed. The terminal oxidase complexes of M. cuprina that channel electrons from RISC oxidation to oxygen were similar to those of “Metallosphaera yellowstonensis” (7) and M. sedula (1).
The M. cuprina genome was 16% smaller than the M. sedula genome. Analysis indicated that the counterpart genes of about 480 ORFs in the M. sedula genome were not found in the M. cuprina genome. Still, there were 233 ORFs uniquely occurring in M. cuprina. Most of those ORFs were annotated as hypothetical protein genes. Gene redundancy in M. cuprina was apparently kept low. For example, there was only one copy of the 4-hydroxybutyryl–coenzyme A (CoA) dehydratase gene in M. cuprina, but duplication of this function was observed in the M. sedula genome (2). The information provided in the M. cuprina genome sequence will facilitate additional researches on this organism, as well as defining the core genome and key physiological features of the genus Metallosphaera.
The M. cuprina genome sequence is available at GenBank under accession no. CP002656.
The work was supported by the Ministry of Science and Technology (973 Program; grant 2010CB630903) and the National Science Foundation of China (grants 31070042 and 30921065).
Published ahead of print on 6 May 2011.