Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal "Shock" Reveal Generic and Specific Metal Responses

Abstract

The extremely thermoacidophilic archaeon Metallosphaera sedula mobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by "shocking" M. sedula with representative metals (Co(2+), Cu(2+), Ni(2+), UO2 (2+), Zn(2+)) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu(2+) (259 ORFs, 106 Cu(2+)-specific ORFs) and Zn(2+) (262 ORFs, 131 Zn(2+)-specific ORFs) triggered the largest responses, followed by UO2 (2+) (187 ORFs, 91 UO2 (2+)-specific ORFs), Ni(2+) (93 ORFs, 25 Ni(2+)-specific ORFs), and Co(2+) (61 ORFs, 1 Co(2+)-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu(2+) (6-fold) but also in response to UO2 (2+) (4-fold) and Zn(2+) (9-fold). Cu(2+) challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu(2+) resistance or issues in sulfur metabolism. The results indicate that M. sedula employs a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions.

Importance: The mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.

FIG 1

M. sedula growth in response to heavy metal shock. The specific growth rates with LD shock were comparable to those of the control (no metal added). The growth rates with HD shock were ∼30 to 60% of those of the control. On the basis of this finding, LD concentrations were set at 0.01 mM Co²⁺, 4 mM Cu²⁺, 1 mM Ni²⁺, 0.2 mM UO₂²⁺, and 15 mM Zn²⁺ and HD concentrations were set at 0.6 mM Co²⁺, 8 mM Cu²⁺, 8 mM Ni²⁺, 0.6 mM UO₂²⁺, and 30 mM Zn²⁺.

FIG 2

Overview of metal shock transcriptomic experiment. (A) Experimental strategy. (B) Metal dosage information and observed transcriptomic response (compared with that of the control), with the number of unique (Unq.) ORFs responding to each metal being indicated. The totals 131 and 554 represent the cumulative number of ORFs responding, while the totals 104 and 354 are the sum of all ORFs responsive to a single metal. (C) Heat plots displaying the least-squares means of the number of ORFs with significant differential transcription for at least one metal versus the control at the low and/or high dosage. If an ORF was not significantly differentially transcribed, the field was left blank. –, control; Co, cobalt; Cu, copper; Ni, nickel; U, uranium; Zn, zinc. The scale bar correlates to the LSM value. Violet, white, and green represent above average, average, and below average transcription levels, respectively.

FIG 3

Venn diagram of metal-responsive (compared with the response of the control) ORFs. (A) Low dose; (B) high dose. Red, the number of ORFs responsive to a single metal; black, the number of ORFs responsive to two metals; green, the number of ORFs responsive to three metals; blue, the number of ORFs responsive to four metals; violet, the number of ORFs responsive to five metals.

FIG 4

Comparison of the metal resistance mechanisms in M. sedula and extreme thermoacidophiles. (A) Copper resistance (cop) operon employing a P-type ATPase (copA), copper binding protein (copT), and regulator (copR). (B) Polyphosphate-based metal sequestration using exopolyphosphatase (ppX) and the arsenite resistance mechanism only represented by the arsenite transporter (arsB). (C) The mercury resistance (mer) operon comprised of mercury reductase (merA), mercury-trafficking protein (merH), and a regulator (merR). An ORF encoding a radical SAM domain could function in assembly/maturation. L, low dose; H, high dose; N, no metal added (control). The scale bar correlates to the LSM value. Violet, white, and green represent above average, average, and below average transcription levels, respectively.

FIG 5

Cu-responsive elements of M. sedula assimilatory sulfur metabolism. Enzymes were as follows: Msed_0963, sulfate adenylyltransferase (CysN); Msed_0962, phosphoadenylylsulfate reductase (thioredoxin) (CysH); Msed_0961, sulfite reductase (CysI); Msed_1616, thioredoxin; Msed_1210, rhodanese (thiosulfate sulfurtransferase); Msed_1675, phosphoglycerate dehydrogenase (PGDH; SerA); Msed_1674, class V aspartate aminotransferase (AspAT; SerC); Msed_1607, O-phosphoserine sulfhydrylase (OPSS); Msed_0602, l-glutamine synthetase (GS); Msed_0164, N-acetyl-gamma-glutamyl-phosphate reductase (LysY/ArgC). Substrates were as follows: adenosine 5′-phosphosulfate (APS), AMP, ATP, 3-phospho-hydroxypyruvate (PHP), O-phospho-l-serine (Sep), l-cysteine (l-Cys), l-glutamate (l-Glu), l-arginine (l-Arg), l-methionine (l-Met), α-ketoglutarate (alpha-KG). red, reduction; ox, oxidation; L, low dose; H, high dose; N, no metal added control. The scale bar correlates to the LSM value. Violet, white, and green represent above average, average, and below average transcription levels, respectively.