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One hundred fifty Tn5 IS50L::phoA (TnphoA) mutants of a mouse-virulent, nalidixic acid-resistant (Nalr), prototrophic Salmonella typhimurium strain, C5 Nalr, were isolated. None of the mutants were auxotrophs. Groups of 8 to 10 BALB/c mice were infected orally with each of 95 mutants with a dose equivalent to 20-fold the 50% lethal dose of the wild-type C5 Nalr strain, and deaths were counted over the next 28 days. Fifteen of the mutants failed to kill any mice, whereas all mice died following challenge with the other mutants. Nine of the 15 attenuated mutants exhibited a defect in lipopolysaccharide biosynthesis. The remaining six mutants were smooth. The TnphoA transposon of each of the smooth attenuated mutants was moved, using P22-mediated transduction, into a fresh C5 background, and all retransductants were still attenuated. Analysis of the membrane proteins of the attenuated mutants failed to reveal any alterations in detectable major outer membrane proteins, although colonies of two of the mutants exhibited a mucoid phenotype following growth on L-agar plates. Individual attenuated mutants differed in their abilities to translocate to livers and spleens of mice following oral infection. All of the smooth TnphoA mutants exhibited increased 50% lethal doses with respect to the wild type following intravenous infection of BALB/c mice. Southern analysis of DNA prepared from each of the mutants suggested that TnphoA had inserted into a number of different sites in the S. typhimurium genome. None of the TnphoA mutants had inserts in the virulence-associated plasmid.

Isolation of orally attenuated Salmonella typhimurium following TnphoA mutagenesis