Role of an Archaeal PitA Transporter in the Copper and Arsenic Resistance of Metallosphaera sedula, an Extreme Thermoacidophile

Abstract

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.

The Confluence of Heavy Metal Biooxidation and Heavy Metal Resistance: Implications for Bioleaching by Extreme Thermoacidophiles

“…Specifically, for E. coli, this involves use of the Pst system as opposed to the less specific Pit system [156]. A loss of function for the Pit system in M. sedula increased resistance compared to a spontaneous mutant harboring a restored version, but found to reduce resistance to copper [157]. The result is consistent with mutations of low-affinity, high velocity transporters pit and corA being more tolerant to arsenate and cobalt, respectively [158][159][160][161].…”

“…Through the action of PPX, the microorganism can generate organic phosphate for metal chelation, or the reversible reaction catalyzed by PPK can generate additional ATP for heavy-metal efflux systems or other cellular metabolism associated with metal challenge [142]. The importance of phosphate metabolism, specifically import via an archaeal Pit system, to enhanced copper resistance for an M. sedula mutant has been established and indicates phosphate plays a key role in supernormal copper resistance [157].…”

Secretion and fusion of biogeochemically active archaeal membrane vesicles

“…All environmental samples were enriched in BS (Zinder & Brock, ) supplemented with 0.2% (w/v) tryptone at both pH 2.0 and 3.0 Brock salts supplemented with 0.2% Tryptone (BST). Metallosphaera sedula DSM5348 was cultivated as described (McCarthy et al., ) in BS at pH 2.0 supplemented with 0.2% (w/v) tryptone. Cultures (50 ml) were grown with aeration by agitation at 75°C to mid‐exponential phase and subcultured into Applikon 3L bioreactors containing 2.0 L BST medium.…”

Microbial copper resistance: importance in biohydrometallurgy

“…As suggested by McCarthy et al . () perhaps PitA and Pho84 comprise dual phosphate transporters that are inherent to these thermoacidophilic archaeons and are critical for their extreme metal resistance.…”