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J Bacteriol, June 1998, p. 3070-3079, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Promoter Region of the Escherichia coli O7-Specific
Lipopolysaccharide Gene Cluster: Structural and Functional
Characterization of an Upstream Untranslated mRNA Sequence
Cristina L.
Marolda and
Miguel A.
Valvano*
Department of Microbiology and Immunology,
The University of Western Ontario, London, Ontario N6A 5C1, Canada
Received 1 December 1997/Accepted 15 April 1998
We report the identification of the promoter region of the
Escherichia coli O7-specific lipopolysaccharide (LPS) gene
cluster (wbEcO7). Typical
10 and
35
sequences were found to be located in the intervening region between
galF and rlmB, the first gene of the
wbEcO7 cluster. Data from RNase protection
experiments revealed the existence of an untranslated leader mRNA
segment of 173 bp, including the JUMPStart and two ops
sequences. We characterized the structure of this leader mRNA by using
the program Mfold and a combination of nested and internal deletions
transcriptionally fused to a promoterless lac operon. Our
results indicated that the leader mRNA may fold into a series of
complex stem-loop structures, one of which includes the JUMPStart
element. We have also found that one of the ops sequences
resides on the predicted stem and the other resides on the loop region,
and we confirmed that these sequences are essential for the
RfaH-mediated regulation of the O polysaccharide cluster. A very
similar stem-loop structure could be predicted in the promoter region
of the LPS core operon encoding the waaQGPSBIJYZK genes. We
observed another predicted stem-loop, located immediately downstream
from the wbEcO7 transcription initiation site,
which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O
polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional
fusions in single copy numbers were also used to determine the effects
of various environmental cues in the transcriptional regulation of O
polysaccharide synthesis. No effects were detected with temperature,
osmolarity, Mg2+ concentration, and drugs inducing changes
in DNA supercoiling. We therefore conclude that the
wbEcO7 promoter activity may be constitutive
and that regulation takes place at the level of elongation of the mRNA
in a RfaH-mediated manner.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, The University of Western Ontario, London, Ontario N6A 5C1, Canada. Phone: (519) 661-3996. Fax: (519) 661-3499. E-mail: mvalvano{at}uwo.ca.
J Bacteriol, June 1998, p. 3070-3079, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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