Affiliation:
1. Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, CNRS UMR 5577, Institut National des Sciences Appliquées, F-69621 Villeurbanne Cedex, France
Abstract
ABSTRACT
The divergent structural operons
caiTABCDE
and
fixABCX
of
Escherichia coli
are required for anaerobic carnitine metabolism. Transcriptional monocopy
lacZ
fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an
fnr
mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at −41.5 of the
cai
transcriptional start site, plays a direct role in the sole
cai
activation. In contrast, mutation in CRP site 2, positioned at −69.5 of the
fix
promoter, caused only a threefold reduction in
fix
expression. Thus, the role of the third CRP site, located at −126.5 of
fix
, might be to reinforce the action of site 2. A critical 50-bp
cis
-acting sequence overlapping the
fix
mRNA start site was found, by deletion analysis, to be necessary for
cai
transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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