1887

Abstract

and PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels of - and PrfA-dependent genes in exponential-phase wild-type and Δ strains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposed showed -dependent increases in (10- and 17-fold higher, respectively) and transcript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected by transcript levels, was up to 95-fold higher in intracellular as compared to non-stressed bacteria, activity was only slightly higher in intracellular than in non-stressed bacteria. Increased transcript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in both expression and PrfA activity. qRT-PCR assays were designed to measure expression of from each of its three promoter regions. Under all conditions, readthrough transcription from the upstream promoter was very low. The relative contribution to total transcription from the -dependent P1 promoter ranged from ∼17 % to 30 %, while the contribution of the P2 region, which appears to be transcribed by both and , ranged from ∼70 % to 82 % of total transcript levels. In summary (i) is primarily activated during environmental stress and does not contribute to PrfA activation in intracellular and (ii) the partially -dependent P2 promoter region contributes the majority of transcripts in both intra- and extracellular bacteria.

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2006-06-01
2019-10-17
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