Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile and of great relevance in bioleaching. However, the impacts of extreme thermoacidophiles propagated with different energy substrates on subsequent bioleaching of refractory chalcopyrite remain unknown. Transcriptional responses underlying their different bioleaching potentials are still elusive. Here, it was first showed that M. sedula inocula propagated with typical energy substrates have different chalcopyrite bioleaching capabilities. Inoculum propagated heterotrophically with yeast extract was deficient in bioleaching; however, inoculum propagated mixotrophically with chalcopyrite, pyrite or sulfur recovered 79%, 78% and 62% copper, respectively, in 12 days. Compared with heterotrophically propagated inoculum, 937, 859 and 683 differentially expressed genes (DEGs) were identified in inoculum cultured with chalcopyrite, pyrite or sulfur, respectively, including upregulation of genes involved in bioleaching-associated metabolism, e.g., Fe2+ and sulfur oxidation, CO2 fixation. Inoculum propagated with pyrite or sulfur, respectively, shared 480 and 411 DEGs with chalcopyrite-cultured inoculum. Discrepancies on repertories of DEGs that involved in Fe2+ and sulfur oxidation in inocula greatly affected subsequent chalcopyrite bioleaching rates. Novel genes (e.g., Msed_1156, Msed_0549) probably involved in sulfur oxidation were first identified. This study highlights that mixotrophically propagated extreme thermoacidophiles especially with chalcopyrite should be inoculated into chalcopyrite heaps at industrial scale.

Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78 % more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.