Regulation of plasmid virulence gene expression in Salmonella dublin involves an unusual operon structure

The 80-kb plasmid pSDL2 of Salmonella dublin Lane is essential for lethal systemic infection in experimental mice. A cluster of five plasmid genes, designated spvR, spvA, spvB, spvC, and spvD, is sufficient to express the plasmid-related virulent phenotype. The spvR gene product has recently been identified as a positive regulator of spvB expression in the stationary phase of bacterial growth (F. C. Fang, M. Krause, C. Roudier, J. Fierer, and D. G. Guiney, J. Bacteriol. 173:6783-6789, 1991). In this study, we evaluated the role of SpvR in the transcription of the downstream virulence genes spvABCD. Analysis of mRNA synthesis revealed that SpvR promotes transcription of the downstream spvABCD genes in the stationary growth phase. Transcript mapping of the spv region demonstrated an unusual operon structure involving messages for spvA, spvAB, spvABC, and spvABCD. Quantitative measurement of transcription and of gene expression by use of translational spv-lacZ fusions suggested that SpvA, SpvB, SpvC, and SpvD are produced in decreasing abundance. Primer extension assays identified two transcriptional start sites 70 and 98 bp upstream of the start codon of spvA, but none upstream of spvB, spvC, or spvD. Deletion of a 320-bp EcoRI-ApaI segment that contains both start sites abolished expression of the downstream spvB and spvC genes. Our results establish a central function of SpvR as a positive regulator of the downstream spvABCD genes in the stationary phase of bacterial growth and indicate that the primary mechanism of regulation is by activation of promoters upstream of spvA.