Role of MerH in mercury resistance in the archaeon Sulfolobus solfataricus.

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.

Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus.

Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices.

Regulation of mercury resistance in the crenarchaeote Sulfolobus solfataricus.

Mercuric ion, Hg(II), inactivates generalized transcription in the crenarchaeote Sulfolobus solfataricus. Metal challenge simultaneously derepresses transcription of mercuric reductase (merA) by interacting with the archaeal transcription factor aMerR. Northern blot and primer extension analyses identified two additional Hg(II)-inducible S. solfataricus genes, merH and merI (SSO2690), located on either side of merA. Transcription initiating upstream of merH at promoter merHp was metal inducible and extended through merA and merI, producing a merHAI transcript. Northern analysis of a merRA double mutant produced by linear DNA recombination demonstrated merHp promoter activity was dependent on aMerR to overcome Hg(II) transcriptional inhibition. Unexpectedly, in a merA disruption mutant, the merH transcript was transiently induced after an initial period of Hg(II)-mediated transcription inhibition, indicating continued Hg(II) detoxification. Metal challenge experiments using mutants created by markerless exchange verified the identity of the MerR binding site as an inverted repeat (IR) sequence overlapping the transcription factor B binding recognition element of merHp. The interaction of recombinant aMerR with merHp DNA, studied using electrophoretic mobility shift analysis, demonstrated that complex formation was template specific and dependent on the presence of the IR sequence but insensitive to Hg(II) addition and site-specific IR mutations that relieved in vivo merHp repression. Despite containing a motif resembling a distant ArsR homolog, these results indicate aMerR remains continuously DNA bound to protect and coordinate Hg(II)-responsive control over merHAI transcription. The new genetic methods developed in this work will promote experimental studies on S. solfataricus and other Crenarchaeota.

Community analysis of a mercury hot spring supports occurrence of domain-specific forms of mercuric reductase.

Mercury is a redox-active heavy metal that reacts with active thiols and depletes cellular antioxidants. Active resistance to the mercuric ion is a widely distributed trait among bacteria and results from the action of mercuric reductase (MerA). Protein phylogenetic analysis of MerA in bacteria indicated the occurrence of a second distinctive form of MerA among the archaea, which lacked an N-terminal metal recruitment domain and a C-terminal active tyrosine. To assess the distribution of the forms of MerA in an interacting community comprising members of both prokaryotic domains, studies were conducted at a naturally occurring mercury-rich geothermal environment. Geochemical analyses of Coso Hot Springs indicated that mercury ore (cinnabar) was present at concentrations of parts per thousand. Under high-temperature and acid conditions, cinnabar may be oxidized to the toxic form Hg2+, necessitating mercury resistance in resident prokaryotes. Culture-independent analysis combined with culture-based methods indicated the presence of thermophilic crenarchaeal and gram-positive bacterial taxa. Fluorescence in situ hybridization analysis provided quantitative data for community composition. DNA sequence analysis of archaeal and bacterial merA sequences derived from cultured pool isolates and from community DNA supported the hypothesis that both forms of MerA were present. Competition experiments were performed to assess the role of archaeal merA in biological fitness. An essential role for this protein was evident during growth in a mercury-contaminated environment. Despite environmental selection for mercury resistance and the proximity of community members, MerA retains the two distinct prokaryotic forms and avoids genetic homogenization.

A freestanding proofreading domain is required for protein synthesis quality control in Archaea.

Threonyl-tRNA synthetase (ThrRS) participates in protein synthesis quality control by selectively editing the misacylated species Ser-tRNA(Thr). In bacteria and eukaryotes the editing function of ThrRS resides in a highly conserved N-terminal domain distant from the active site. Most archaeal ThrRS proteins are devoid of this editing domain, suggesting evolutionary divergence of quality-control mechanisms. Here we show that archaeal editing of Ser-tRNAThr is catalyzed by a domain unrelated to, and absent from, bacterial and eukaryotic ThrRSs. Despite the lack of sequence homology, the archaeal and bacterial editing domains are both reliant on a pair of essential histidine residues suggestive of a common catalytic mechanism. Whereas the archaeal editing module is most commonly part of full-length ThrRS, several crenarchaeal species contain individual genes encoding the catalytic (ThrRS-cat) and editing domains (ThrRS-ed). Sulfolobus solfataricus ThrRS-cat was shown to synthesize both Thr-tRNAThr and Ser-tRNAThr and to lack editing activity against Ser-tRNAThr. In contrast, ThrRS-ed lacks aminoacylation activity but can act as an autonomous protein in trans to hydrolyze specifically Ser-tRNAThr, or it can be fused to ThrRS-cat to provide the same function in cis. Deletion analyses indicate that ThrRS-ed is dispensable for growth of S. solfataricus under standard conditions but is required for normal growth in media with elevated serine levels. The growth phenotype of the ThrRS-ed deletion strain suggests that retention of the discontinuous ThrRS quaternary structure relates to specific physiological requirements still evident in certain Archaea.

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

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.