Acidimicrobium ferrooxidans strain ICP^T (DSM 10331 = NBRC 103882 = JCM 15462) is the type strain of the species, and was first isolated by Clark and Norris from hot springs in the Krísuvík geothermal area, Iceland [1,2]. For over fifteen years A. ferrooxidans ICP^T remained extremely isolated phylogenetically as the sole type strain in the actinobacterial subclass Acidimicrobidae [3] (Figure 1). Only at the time this manuscript was written, Kurahashi et al. [8] and Johnson et al. [9] described three novel type strains representing one novel family, Iamiaceae [8], and two novel genera within the Acidimicrobiales [9]: Iamia majanohamensis (isolated from sea cucumber [8]), Ferromicrobium acidiphilum (from a mine site in North Wales, UK [9]) and Ferrithrix thermotolerans (from Yellowstone National Park, Wyoming, USA [9]). With the exception of I. majanohamensis, all these strains live in acidic environments. Here we present a summary classification and a set of features for A. ferrooxidans ICP^T (Table 1), together with the description of the complete genomic sequencing and annotation.

Figure 1 Phylogenetic tree highlighting the position of A. ferrooxidans strain ICPT relative to all other type strains within the Acidimicrobiales and the type strains of all other orders within the Actinobacteria. The tree was inferred from 1,306 aligned characters [4,5] of the 16S rRNA gene under the maximum likelihood criterion [6] and rooted with Rubrobacteriales. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [7] are shown in blue, published genomes in bold.

Members of the species A. ferrooxidans have been isolated or identified molecularly from warm, acidic, iron-, sulphur- or mineral-sulfide-rich environments. Strain TH3, isolated from a copper leaching dump [1], shares 100% 16S rRNA gene sequence identity with strain ICP^T, whose genome sequence is reported here. The moderately thermophilic bacterium N1-45-02 (EF199986) from a spent Canadian copper sulfide heap, and strain Y00168 from a geothermal site in Yellowstone National Park [14] are the only other pure cultivated members of the species. Acidimicrobium species in mixed cultures used for bioleaching were frequently reported [15]. Uncultured clone sequences with significant sequence similarity (>98%) were observed by Inskeep and colleagues from several hot springs in Yellowstone National Park (e.g. AY882832, DQ179032 and others), and from hydrothermally modified volcanic soil at Mount Hood (EU419128). Screening of environmental genomic samples and surveys reported at the NCBI BLAST server indicated no closely related phylotypes (the highest observed sequence identity was 91%) that can be linked to the species or genus. Several DGGE analyses indicated the presence of members of the genus Acidimicrobium in metal-rich mine waters and geothermal fields around the world.

Figure 1 shows the phylogenetic neighborhood of A. ferrooxidans strain ICP^T in a 16S rRNA based tree. The sequences of the two identical copies of the 16S rRNA genes in the genome differ in 16 positions (1.1%) from the previously published 16S rRNA sequence generated from of A. ferrooxidans DSM 10331 (U75647). The higher sequence coverage and overall improved level of sequence quality in whole-genome sequences, as compared to ordinary gene sequences, implies that the significant difference between the genome data and the previously reported 16S rRNA gene sequence might be due to sequencing errors in the previously reported sequence data.

Cells of strain ICP^T are rather small (0.4 µm × 1-1.5 µm) Gram-positive rods [1]. Optimal growth occurs at 45-50°C, pH 2, with a maximal doubling time of six hours at 48°C [1]. Cells are motile during heterotrophic growth on yeast extract. ICP^T forms small colonies when grown autotrophically on ferrous iron containing solid medium under air [1]. The closely related strain TH3 differs from the type strain ICP^T only by its tendency to grow in filaments, which has not been observed for strain ICP^T [1]. Strain ICP^T can be distinguished from members of the genus Sulfobacillus by its lower requirement of CO₂ for autotrophic growth [1]. Iron oxidation by ICP^T cells was not influenced by supplementation of either glucose nor by increased CO₂ concentration [1]. Thin section electron micrographs of A. ferrooxidans strains indicate intracellular vesicles when cells were grown on ferrous iron and yeast extract [1] (Figure 2).

Figure 2 Scanning electron micrograph of A. ferrooxidans ICPT (Manfred Rohde, Helmholtz Centre for Infection Research, Braunschweig)

The genome is 2,158,157 bp long and comprises one main circular chromosome with a 68.3% GC content (Table 3 and Figure 3). Of the 2,092 genes predicted, 2038 were protein coding genes, and 54 RNAs. Seventy four pseudogenes were also identified. A total of 75.7% of the genes were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 3 Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.