A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.

We cured a strain of Salmonella typhimurium of its cryptic plasmid and confirmed that orally administered cured strains lost virulence for mice. Loss of the cryptic plasmid rendered the S. typhimurium strain sensitive to the bactericidal action of normal human serum. However, loss of the plasmid did not change the ability of the strain to associate with HeLa cells in tissue culture. Furthermore, when administered orally to mice, both the plasmid-containing and plasmid-free strains invaded the Peyer's patches of the small intestine to the same extent, and both were capable of inducing resistance to oral challenge with virulent S. typhimurium. When injected intraperitoneally, the cured strain was eliminated rapidly, whereas the parental strain persisted. We also showed that the cured strain did not contain a plasmid copy in the chromosome. We propose that although the plasmid-cured strain of S. typhimurium is able to colonize Peyer's patches, it cannot survive when administered intraperitoneally because it is susceptible to elimination by macrophages.