The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism

Abstract

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, approximately 2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.

FIG. 1.

Selected sections of a full multiple-sequence alignment of known and putative proteins involved in Fe oxidation performed by CLUSTAL W (1.83). Amino acids known or predicted to be involved with copper binding (4, 10, 25, 76) are shown in bold. Segments underlined and highlighted in gray and black show protein signatures for sulfocyanin and rusticyanin, respectively, with intermediate nonhighlighted amino acids failing to meet currently recognized positional consensus amino acids (25). Abbreviations: sso, S. solfataricus; sac, S. acidocaldarius; sto, S. tokodaii; msed, M. sedula; faci, F. acidarmanus; afe, A. ferrooxidans; soxE, sulfocyanin; hyp, hypothetical protein; bcp, blue copper protein; rus, rusticyanin.

FIG. 2.

Proposed organization of common elements in terminal oxidase complexes of M. sedula. Terminal oxidase complexes have been proposed to be hybrids of bacterial bc₁ complexes, containing Fe-S Rieske (SoxL or SoxF) and cytochrome b (SoxC, SoxG, or SoxN) components, and cytochrome oxidases, containing Cox subunits I and II (SoxB or SoxM and SoxA or SoxH, respectively). Also provided is the relative genome sequence location (ORF numbers) of these common elements; previous studies of these general subunit or cluster functions are listed at the far right. Sac, S. acidarmanus; Sme, S. metallicus; Sto, S. tokodaii; Aam, A. ambivalens.

FIG. 3.

The 3-hydroxypropionate cycle that is thought to operate in C. aurantiacus (adapted from references and 21). ORF numbers predicted to encode the required enzymes are as follows: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase; 3, propionyl-CoA synthase; 4, propionyl-CoA carboxylase; 5, methylmalonyl-CoA epimerase; 6, methylmalonyl-CoA mutase; 7, succinyl-CoA:l-malyl-CoA transferase; 8, succinate dehydrogenase; 9, fumarate dehydratase; 10, l-malyl-CoA lyase; 11, β-methylmalyl-CoA lyase; 12, unknown enzymes; 13, succinyl-CoA: citramalate-CoA transferase; 14, S/R-citramalyl-CoA lyase.

FIG. 4.

M. sedula attached to a FeS₂ substrate. Bar, 2 μm.

FIG. 5.

Volcano plot of M. sedula microarray dye-flip results (YE versus YE+FeSO₄). Negative x-axis values represent ORFs more highly transcribed on YE+FeSO₄ while positive x-axis values represent ORFs more highly transcribed on YE alone. As points of reference on the y axis, P values of 0.05, 5 × 10⁻⁵, and 5 × 10⁻¹⁰ correspond to significance values of 1.3, 4.3, and 9.3, respectively.

FIG. 6.

Schematic summarizing Fe oxidation, sulfur metabolism, and adhesion components believed to be important to M. sedula's biomining capacity. Elements of the schematic are linked to their annotations or putative functions by superscript numbers. Both genome sequence analysis and preliminary transcriptional profiling results (FeSO₄+YE versus YE) indicate that membrane-associated proteins have a prevalent role in biomining physiology.