Occurrence and characterization of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of gene disruption

Abstract

Mercury resistance mediated by mercuric reductase (MerA) is widespread among bacteria and operates under the control of MerR. MerR represents a unique class of transcription factors that exert both positive and negative regulation on gene expression. Archaea and bacteria are prokaryotes, yet little is known about the biological role of mercury in archaea or whether a resistance mechanism occurs in these organisms. The archaeon Sulfolobus solfataricus was sensitive to mercuric chloride, and low-level adaptive resistance could be induced by metal preconditioning. Protein phylogenetic analysis of open reading frames SSO2689 and SSO2688 clarified their identity as orthologs of MerA and MerR. Northern analysis established that merA transcription responded to mercury challenge, since mRNA levels were transiently induced and, when normalized to 7S RNA, approximated values for other highly expressed transcripts. Primer extension analysis of merA mRNA predicted a noncanonical TATA box with nonstandard transcription start site spacing. The functional roles of merA and merR were clarified further by gene disruption. The merA mutant exhibited mercury sensitivity relative to wild type and was defective in elemental mercury volatilization, while the merR mutant was mercury resistant. Northern analysis of the merR mutant revealed merA transcription was constitutive and that transcript abundance was at maximum levels. These findings constitute the first report of an archaeal heavy metal resistance system; however, unlike bacteria the level of resistance is much lower. The archaeal system employs a divergent MerR protein that acts only as a negative transcriptional regulator of merA expression.

FIG. 1.

Adaptive resistance to mercuric chloride. Wild-type S. solfataricus was grown in SM and challenged with mercuric chloride. Open symbols, unadapted cultures; closed symbols, adapted culture; inverted triangles, untreated control. Unadapted cells challenged with 0.3 (squares), 0.5 (triangles), or 1.5 μM (open circles). The arrow indicates the time of addition of mercuric chloride.

FIG. 2.

S. solfataricus mer locus. Lengths are in nucleotides.

FIG. 3.

Protein phylogenies of MerA and MerR. (A) MerA tree; (B) MerR tree. Neighbor-joining distance trees are based on comparison of near-full-length protein sequences of 422 residues for MerA and 104 residues for MerR. Distances are indicated by the bar in the lower left corner and represent 10 substitutions per 100 residues. Percent occurrence among 100 trees is given for all nodes. Accession numbers are indicated in the text.

FIG.4.

Multiple sequence alignments of MerA and MerR. Sequence conservation is indicated by boxshading. (A) MerA alignment. Multiple sequence alignment is shown for Bacillus sp. strain RC607 (607), Tn501 (501), Tn21 (21), and S. solfataricus (Sso) MerA proteins. Numbering refers to the position of the protein relative to its amino-terminal end. Conserved catalytically active residues are outlined in boxes and labeled. (B) MerR alignment. Multiple sequence alignment is shown for Tn501 (501), Tn21 (21), and S. lividans (Slv) and S. solfataricus (Sso) MerR proteins. The conserved glutamate required for DNA binding and the catalytically active cysteines are outlined in boxes and labeled.

FIG. 5.

Northern analysis of merA following mercuric chloride challenge. Cells in exponential phase were treated with 0.3 mM mercuric chloride, and RNA was extracted at the times indicated beneath the figures for analysis. Blots were probed simultaneously using merA and 7S RNA riboprobes. The data in panels A and B were prepared from independent experiments.

FIG. 6.

Primer extension analysis and DNA sequence of merAp. (A) Primer extension analysis of RNA prepared from mercuric chloride-treated cells. The sequencing ladder is on the left, and the extension reaction is in lane 1. The start codon is boxed. The location of the start site is indicated by the arrow. (B) Location and composition of the TATA box and BRE for canonical and merAp promoters. (C) DNA sequence of merAp with underlines indicating the positions of the BRE, TATA box, and start codon. The large A indicates the transcription start site.

FIG. 7.

Disruption of merA and merR. Schematic representations of the disrupted loci indicating the location of the disrupting copy of lacS (black region) in the target genes (grey regions). The direction of transcription is indicated by the arrows; merA (A) and merR (B) are divergently transcribed. The primers used in the analysis of the disrupted and wild-type alleles are indicated beneath the schematic.

FIG. 8.

Response of the merA disruption mutant to mercuric chloride. (A and B) Response of the wild type (closed symbols) and merA disruption mutant (open symbols) to mercuric chloride challenge during growth in SM liquid medium. (A) 0.3 μM mercuric chloride challenge (circles); (B) 0.5 μM mercuric chloride challenge (circles). Inverted triangles (A and B), no addition. The arrow indicates the time of addition of mercuric chloride. (C) EOP of the wild type (filled bars) and the merA disruption mutant (grey bars) on RM plates containing mercuric chloride. Values are a percentage of the EOP observed with no added mercuric chloride. Data are averages from duplicate plates.

FIG. 9.

Response of the merR disruption mutant to mercuric chloride. Cells were grown in SM liquid medium and challenged with 0.75 μM mercuric chloride (arrow). Cultures were merR disruption mutant (triangles) or wild type (circles). Open symbols, untreated cultures; closed symbols, treated cultures.

FIG. 10.

Northern analysis of merA in the merR disruption mutant following mercuric chloride challenge. Levels of merA in the merR disruption mutant and the wild type (PBL2025) were determined in response to mercuric chloride challenge (0.3 μM). Lanes 1, 3, 5, 7, 9, and 11, wild type; lanes 2, 4, 6, 8, and 10, merR disruption mutant. Sample times and lane numbers were as follows: lanes 1 and 2, 0 h; lanes 3 and 4, 0.5 h; lanes 5 and 6, 1 h; lanes 7 and 8, 2 h; lanes 9 and 10, 4 h; lanes 11 and 12, 9 h. The positions of the merA and 7S RNA are indicated. A larger transcript possibly encoding merA and SSO2690 is indicated by the arrow.