Characterization of three proteins expressed from the virulence region of plasmid pSDL2 in Salmonella dublin.
Infection of both cattle and humans with Salmonella dublin can result in septicemia and death. Like many nontyphoid Salmonella species that cause disease, S. dublin contains a cryptic plasmid (pSDL2) that is required for the full expression of virulence. Transposon mutagenesis of pSDL2 defined a 4.1-kb EcoRI region that is necessary for the development of a systemic infection in BALB/c mice. This EcoRI fragment was cloned into an expression vector (pEL11), and three proteins produced from this region with apparent molecular weights of 30,500, 76,000, and 27,000 were identified. Because bacterial proteins that play a role in virulence are often associated with the outer membrane, we were interested in establishing whether the proteins expressed from the EcoRI fragment are located in the membrane. Transposon mutagenesis of pEL11 with TnphoA defined the order of the genes along the fragment and suggested that the proteins may be exported out of the cytoplasm. Sucrose gradient cell fractionation was done to identify the cellular location of each of the three proteins. The 30-kDa protein was identified in the outer membrane fraction, and the 76-kDa protein was located in the cytosolic fraction. The 27-kDa protein was identified in both the cytosolic and the outer membrane fractions. The outer membrane contained less than 10% of the activity of enzymes known to be located in the cytoplasm, periplasm, and inner membrane. Sequence data of the 4.1-kb EcoRI region revealed that both the 30- and the 27-kDa proteins lack a typical signal sequence for export out of the cytoplasm (M. Krause, C. Roudier, J. Fierer, J. Harwood, and D. G. Guiney, Mol. Microbiol. 5:307, 1991). The outer membrane location of these proteins suggests that they may be exported out of the cytoplasm by an unusual mechanism.
Valone
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Department of Medicine, School of Medicine, University of California, Los Angeles 90024-1736.
Chikami
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis; D010957:Plasmids; D012475:Salmonella / Q000235:genetics / Q000472:pathogenicity*; D014774:Virulence
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D009097:Multienzyme Complexes; D007770:L-Lactate Dehydrogenase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases