ABSTRACT
While
the stress-responsive alternative sigma factor σB
has been identified in different species of Bacillus,
Listeria, and Staphylococcus, theσ
B regulon has been extensively characterized only
in B. subtilis. We combined biocomputing and microarray-based
strategies to identify σB-dependent genes in the
facultative intracellular pathogen Listeria monocytogenes.
Hidden Markov model (HMM)-based searches identified 170 candidateσ
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 σB-dependent promoters as well as
selected virulence and stress response genes. RNA for the microarray
experiments was isolated from both wild-type and ΔsigB
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 significantσ
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 σB-dependent expression, 54
were preceded by a sequence resembling the σB
promoter consensus sequence. Rapid amplification of cDNA ends-PCR was
used to confirm the σB-dependent nature of a subset
of eight selected promoter regions. Notably, theσ
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, σB
also contributes to regulation of virulence gene expression in L.
monocytogenes. These findings strongly suggest thatσ
B contributes to L. monocytogenes gene
expression during
infection.