Expression profile and subcellular location of the plasmid-encoded virulence (Spv) proteins in wild-type Salmonella dublin

The plasmid-encoded virulence genes (spvABCD) in nontyphoid Salmonella strains mediate lethal infections in a variety of animals. Previous studies have shown that these genes are transcriptionally regulated by stationary-phase growth. We studied the expression profile and the subcellular locations of the SpvABCD proteins in wild-type S. dublin by using polyclonal antibodies against SpvA, SpvB, SpvC, and SpvD. The cellular levels of the individual proteins were determined during growth by quantitative immunoblotting, As expected, SpvA, SpvB, SpvC, and SpvD were not detectable before the late logarithmic growth phase and appeared in the sequence SpvA, SpvB, SpvC, and SpvD. In contrast to the transcriptional regulation, however, SpvA and SpvB reached their maximal expression shortly after induction and declined during further growth whereas SpvC and SpvD expression remained high throughout the stationary phase, indicating that the Spy proteins are individually regulated at a posttranscriptional level. To localize SpvABCD within the bacteria, the cells were fractionated into the periplasmic, cytoplasmic, inner membrane, and outer membrane components. The cell fractions and the culture supernatant were analyzed by immunoblotting. SpvA was present in the outer membrane, SpvB was present in the cytoplasm and the inner membrane, and SpvC was present in the cytoplasm. SpvD was secreted into the supernatant; however, a substantial portion of this protein was also detected in the cytoplasm and membranes. The molecular weights of SpvD in the supernatant and in the cytoplasm appeared to be equal, suggesting that SpvD is not cleaved upon secretion.