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.
Schelert
James
J
George Beadle Center for Genetics, University of Nebraska, Lincoln, NE 68588-0666, USA.
Drozda
Melissa
M
Dixit
Vidula
V
Dillman
Amanda
A
Blum
Paul
P
D001665:Binding Sites / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D019848:Gene Expression Regulation, Archaeal; D019766:Genes, Archaeal; D008628:Mercury / Q000633:toxicity*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D019642:RNA, Archaeal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D048229:Sulfolobus solfataricus / Q000187:drug effects* / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D019642:RNA, Archaeal; D012333:RNA, Messenger; D014157:Transcription Factors; D008628:Mercury