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Journal of Bacteriology, November 2000, p. 6347-6357, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Silencing and Activation of ClyA Cytotoxin
Expression in Escherichia coli
Marie
Westermark,1
Jan
Oscarsson,1
Yoshimitsu
Mizunoe,2
Jurate
Urbonaviciene,1 and
Bernt Eric
Uhlin1,*
Department of Microbiology, Umeå University,
S-90187 Umeå, Sweden,1 and Department
of Bacteriology, Graduate School of Medical Sciences, Kyushu
University, Fukuoka 812-8582, Japan2
Received 8 May 2000/Accepted 21 August 2000
Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by
the clyA gene of Escherichia coli K-12. Genetic
analysis suggested that clyA is silenced by the nucleoid
protein H-NS. Purified H-NS protein showed preferential binding to
clyA sequences in the promoter region, as evidenced by
DNase I footprinting and gel mobility shift assays. Transcriptional
derepression and activation of a chromosomal
clyA::luxAB operon fusion were seen
under conditions of H-NS deficiency and SlyA overproduction,
respectively. In H-NS-deficient bacteria neither the absence nor the
overproduction of SlyA affected the derepressed ClyA expression any
further. Therefore, we suggest that overproduction of SlyA in
hns+ E. coli derepresses clyA
transcription by counteracting H-NS. The cyclic AMP receptor protein
(CRP) was required for ClyA expression, and it interacted with a
predicted, albeit suboptimal, CRP binding site in the clyA
upstream region. Site-specific alterations of the CRP binding site to
match the consensus resulted in substantially higher levels of ClyA
expression, while alterations that were predicted to reduce CRP binding
reduced ClyA expression. During anaerobic growth the fumarate and
nitrate reduction regulator (FNR) was important for ClyA expression,
and the clyA gene could be activated by overexpression of
FNR. A major clyA transcript having its 5' end (+1) located
72 bp upstream of the translational start codon and 61 bp downstream of
the CRP-FNR binding site was detected in the absence of H-NS. The
clyA promoter was characterized as a class I promoter that
could be transcriptionally activated by CRP and/or FNR. According to
DNA bending analyses, the clyA promoter region has high
intrinsic curvature. We suggest that it represents a regulatory region
which is particularly susceptible to H-NS silencing, and its features
are discussed in relation to regulation of other silenced operons.
*
Corresponding author. Mailing address: Department
of Microbiology, Umeå University, S-90187 Umeå, Sweden. Phone:
46-90-7856731. Fax: 46-90-772630. E-mail:
bernt.eric.uhlin{at}micro.umu.se.
Journal of Bacteriology, November 2000, p. 6347-6357, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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