Journal of Bacteriology, October 2003, p. 5722-5734, Vol. 185, No. 19
0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.19.5722-5734.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Listeria monocytogenes
B Regulates Stress Response and Virulence Functions
Mark J. Kazmierczak, Sharon C. Mithoe, Kathryn J. Boor, and Martin Wiedmann*
Department of Food Science, Cornell University, Ithaca, New York
Received 18 April 2003/
Accepted 15 July 2003
While
the stress-responsive alternative sigma factor
B
has been identified in different species of Bacillus,
Listeria, and Staphylococcus, the
B regulon has been extensively characterized only
in B. subtilis. We combined biocomputing and microarray-based
strategies to identify
B-dependent genes in the
facultative intracellular pathogen Listeria monocytogenes.
Hidden Markov model (HMM)-based searches identified 170 candidate
B-dependent promoter sequences in the strain EGD-e
genome sequence. These data were used to develop a specialized,
208-gene microarray, which included 166 genes downstream of
HMM-predicted
B-dependent promoters as well as
selected virulence and stress response genes. RNA for the microarray
experiments was isolated from both wild-type and
sigB
null mutant L. monocytogenes cells grown to stationary phase
or exposed to osmotic stress (0.5 M KCl). Microarray analyses
identified a total of 55 genes with statistically significant
B-dependent expression under the conditions used in
these experiments, with at least 1.5-fold-higher expression in the wild
type over the sigB mutant under either stress condition (51
genes showed at least 2.0-fold-higher expression in the wild type). Of
the 55 genes exhibiting
B-dependent expression, 54
were preceded by a sequence resembling the
B
promoter consensus sequence. Rapid amplification of cDNA ends-PCR was
used to confirm the
B-dependent nature of a subset
of eight selected promoter regions. Notably, the
B-dependent L. monocytogenes genes
identified through this HMM/microarray strategy included both stress
response genes (e.g., gadB, ctc, and the glutathione
reductase gene lmo1433) and virulence genes (e.g.,
inlA, inlB, and bsh). Our data demonstrate
that, in addition to regulating expression of genes important for
survival under environmental stress conditions,
B
also contributes to regulation of virulence gene expression in L.
monocytogenes. These findings strongly suggest that
B contributes to L. monocytogenes gene
expression during
infection.
* Corresponding author. Mailing address: Department of Food Science, 412 Stocking Hall, Cornell University, Ithaca, NY 14853. Phone: (607) 254-2838. Fax: (607) 254-4868. E-mail: mw16{at}cornell.edu.
Journal of Bacteriology, October 2003, p. 5722-5734, Vol. 185, No. 19
0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.19.5722-5734.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
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Copyright © 2003 by the American Society for Microbiology. All rights reserved.