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Infection and Immunity, June 2001, p. 3924-3932, Vol. 69, No. 6
Department of Microbiology and National Food
Biotechnology Centre, University College Cork, Cork, Ireland
Received 8 January 2001/Returned for modification 13 February
2001/Accepted 26 March 2001
The ability of intracellular pathogens to sense and adapt to the
hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock
proteins GroES and GroEL in the gram-positive food-borne pathogen
Listeria monocytogenes. The operon has a conserved
orientation in the order groES groEL. Upstream of
groES and in the opposite orientation is a gene encoding a
homologue of the Bacillus subtilis protein YdiL, while
downstream of groEL is a gene encoding a putative bile
hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and
transcriptional fusions to the UV-optimized Aequorea
victoria green fluorescent protein (GFPUV) to analyze
expression of groESL under various environmental stress
conditions, including heat shock, ethanol stress, and acid shock, and
during infection of J774 mouse macrophage cells. Strains harboring
GFPUV transcriptional fusions to the promoter region of
groESL demonstrated a significant increase in fluorescence
following heat shock that was detected by both fluorimetry and
fluorescence microscopy. Using both RT-PCR and GFP technology we
detected expression of groESL following internalization by
J774 cells. Increased intracellular expression of dnaK was
also determined using RT-PCR. We have recently described a system which
utilizes L. monocytogenes hemolysin as an in vivo reporter
of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol. 36:498-507, 2000). In this
study a strain was constructed in which hemolysin expression was placed
under the control of the groESL promoter. In this strain hemolysin expression during infection also confirms transcription from
the groESL promoter during J774 and murine infection,
albeit at lower levels than the known virulence factor
plcA.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.3924-3932.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of the groESL Operon in
Listeria monocytogenes: Utilization of Two Reporter Systems
(gfp and hly) for Evaluating In Vivo
Expression
*
Corresponding author. Mailing address: Department of
Microbiology, University College Cork, Cork, Ireland. Phone:
353-21-4902080. Fax: 353-21-4903101. E-mail:
c.gahan{at}ucc.ie.
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