In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD 50 ] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10 ؊5 to 10 ؊7 to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD 50 , >10 7 CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10-to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.The three pathogenic Yersinia species, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, are closely related but differ in the mode of infection. Both Y. enterocolitica and Y. pseudotuberculosis are fecal-oral pathogens that cause invasive gastrointestinal diseases. On the other hand, infection with Y. pestis, the causative agent of plague, which is transmitted either by an infected-flea bite or as an inhaled aerosol, culminates in a fatal disease. Most of the documented Y. pestis virulence factors are encoded by the 70-kb plasmid common to all three Yersinia pathogenic species (5,12,15,16,36). Only a few virulence factors that are unique to Y. pestis are known to reside on the strain-specific plasmids (pMT1 and pPCP1) (35). The pMT1 virulence plasmid (19,26,34) harbors, in addition to the sequences encoding the known virulence factors murine toxin and fraction 1 (F1) capsular antigen, sequences encoding several hypothetical proteins whose relevance to Y. pestis pathogenesis remains to be determined. Moreover, the recently completed genome sequences of two Y. pestis strains, CO92 and KIM (14, 34), revealed the dynamic nature of the genome, the presence of many pseudogenes, and the existence of genes encoding hypothetical proteins residing in several putative pathogenicity islands, whose effects on Y. pestis pathogenicity remain to be carefully studied (14,34).During the last deca...