Affiliation:
1. George Beadle Center for Genetics, University of Nebraska—Lincoln, Lincoln, Nebraska
Abstract
ABSTRACT
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
.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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