An aroA mutant of Yersinia pestis is attenuated in guinea-pigs, but virulent in mice

This study describes a PCR-based approach for the production of a rationally attenuated mutant of Yersinia pestis. Degenerate primers were used to amplify a fragment encoding 91.45% of the aroA gene of Y. pestis MP6 which was cloned into pUC18. The remainder of the gene was isolated by inverse PCR. The gene was sequenced and a restriction map was generated. The Y. pestis aroA gene had 75.9% identity with the aroA gene of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain CB using the suicide vector pGP704. A stable are-defective mutant, Y. pestis GB Delta aroA, was isolated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response against challenge with the virulent Y. pestis strain GB. Unusually for an are-defective mutant, the Y. pestis aroA mutant was virulent in mice, with a median dose which induced morbidity or death similar to that of the wildtype, although time to death was significantly prolonged.