Characterization of the groESL operon in Listeria monocytogenes: Utilization of two reporter systems (gfp and hly) for evaluating in vivo expression

The ability of intracellular pathogens to sense and adapt to the hostile en vironment of the host is an important factor governing virulence. We have s equenced 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 an d in the opposite orientation is a gene encoding a homologue of the Bacillu s subtilis protein YdiL, while downstream of groEL is a gene encoding a put ative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and t ranscriptional fusions to the UV-optimized Aequorea victoria green fluoresc ent protein (GFP(UV)) to analyze expression of groESL under various environ mental stress conditions, including heat shock, ethanol stress, and acid sh ack, and during infection of J774 mouse macrophage cells. Strains harboring GFP(UV) transcriptional fusions to the promoter region of groESL demonstra ted a significant increase in fluorescence following heat shock that was de tected by both fluorimetry and fluorescence microscopy, Using both RT-PCR a nd GFP technology we detected expression of groESL following internalizatio n by J774 cells. Increased intracellular expression of dnaK was also determ ined using RT-PCR, We have recently described a system which utilizes L. mo nocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C, G, M, Gahan and C, HiII, Mel. Microbiol. 36:498-507 , 2000), In this study a strain was constructed in which hemolysin expressi on was placed under the control of the groESL promoter, In this strain hemo lysin expression during infection also confirms transcription from the groE SL promoter during J774 and murine infection, albeit at lower levels than t he known virulence factor plcA.