Role of MerH in mercury resistance in the archaeon Sulfolobus solfataricus

Abstract

Crenarchaeota include extremely thermoacidophilic organisms that thrive in geothermal environments dominated by sulfidic ores and heavy metals such as mercury. Mercuric ion, Hg(II), inactivates transcription in the crenarchaeote Sulfolobus solfataricus and simultaneously derepresses transcription of a resistance operon, merHAI, through interaction with the MerR transcription factor. While mercuric reductase (MerA) is required for metal resistance, the role of MerH, an adjacent small and predicted product of an ORF, has not been explored. Inactivation of MerH either by nonsense mutation or by in-frame deletion diminished Hg(II) resistance of mutant cells. Promoter mapping studies indicated that Hg(II) sensitivity of the merH nonsense mutant arose through transcriptional polarity, and its metal resistance was restored partially by single copy merH complementation. Since MerH was not required in vitro for MerA-catalysed Hg(II) reduction, MerH may play an alternative role in metal resistance. Inductively coupled plasma-mass spectrometry analysis of the MerH deletion strain following metal challenge indicated that there was prolonged retention of intracellular Hg(II). Finally, a reduced rate of mer operon induction in the merH deletion mutant suggested that the requirement for MerH could result from metal trafficking to the MerR transcription factor.

Fig. 1.

Introduction to the mutations in the mer operon. (a) Schematic of the mer operon. Solid arrows indicate transcribed ORFs and open rectangles indicate intergenic regions. Nucleotide lengths are indicated. Promoters are indicated by right angle arrows. (b) Schematic of the complemented merH_TAG mutant by single copy merH⁺ integration. Location of the merH_TAG point mutation in the merH locus (top). Reintroduction of WT merH at the trbE locus (bottom); the merA gene is disrupted by lacS. (c) DNA sequence of the merH_TAG mutation and addition of new diagnostic restriction site. (d) Schematic of the merH in-frame deletion mutant. Location of the in-frame deletion in the merH locus is indicated by an open box. (e) DNA sequence of the merH in-frame deletion mutation including retention of start and stop codons. (f) DNA sequences of mer promoter mutations for merHp_TATA, merRp_TATA and merAp_TATA.

Fig. 2.

Response of mer operon mutants to mercuric chloride. All strains were treated with mercuric chloride (arrows) at 0.5 µM unless otherwise indicated. Closed symbols (treated cultures), open symbols (untreated cultures). (a) merH_TAG mutant (squares), merA mutant (triangles) or WT (circles). (b) merH_TAG (triangles), merH_TAG/merH⁺ (squares) and WT (circles). (c) ΔmerH in-frame deletion mutant (inverted triangles), merA mutant (squares) and WT (circles). RNA was extracted for qRT-PCR analysis of merA at 32 h post-challenge (arrow). (d) merHp_TATA mutant (triangles), merA disruption mutant (squares) and WT (circles) treated with 0.75 µM mercuric chloride. (e) merRp_TATA mutant (squares), merR mutant (triangles) or WT (circles). Cultivation experiments were repeated at least three times.

Fig. 3.

MerA protein purification and effect of merH mutations on mer transcript abundance. (a) Expression and purification of polyhistidine-tagged MerA from an S. solfataricus MerA expression strain. Ni-NTA purified samples were analysed by 1D SDS-PAGE on a 12.5 % acrylamide gel. Lanes: 1, single passaged Ni-NTA eluant; 2, double passaged Ni-NTA eluant (3.6 µg protein loaded). (b) qRT-PCR primer locations. (c) qRT-PCR analysis of merH and merA mRNA. merH and merA transcript abundance normalized to 7S RNA were determined in total RNA 4 h after challenge with 0.3 µM Hg(II) from the WT, merHp_TATA and merH_TAG strains. Technical repeats produced less than 10 % variation between results.

Fig. 4.

Intracellular Hg measurement by ICP-MS analysis. (a) ICP-MS analysis of cell-associated mercury in parts per billion (p.p.b.). WT (closed circles), merH deletion mutant (inverted triangles) and merA disruption mutant (closed squares). (b) RNA was isolated from the ΔmerH mutant and WT 32 h after 0.5 µM Hg(II) challenge and the relative merA transcript abundance was determined after normalization to tbp mRNA. Technical repeats produced less than 10 % variation between results.