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A library of tagged mutants of the virulent<jats:italic>Y. pestis<\/jats:italic>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<jats:sub>50<\/jats:sub>] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10<jats:sup>\u22125<\/jats:sup>to 10<jats:sup>\u22127<\/jats:sup>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.,<jats:italic>purH<\/jats:italic>,<jats:italic>purK<\/jats:italic>,<jats:italic>dnaE<\/jats:italic>, and<jats:italic>greA<\/jats:italic>) or for hypothetical polypeptides, as well as for the virulence regulator gene<jats:italic>lcrF<\/jats:italic>. One of the avirulent mutant strains (LD<jats:sub>50<\/jats:sub>, &gt;10<jats:sup>7<\/jats:sup>CFU) was found to be disrupted in the<jats:italic>pcm<\/jats:italic>locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53<jats:italic>pcm<\/jats:italic>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.<\/jats:p>","DOI":"10.1128\/iai.72.2.908-915.2004","type":"journal-article","created":{"date-parts":[[2004,1,24]],"date-time":"2004-01-24T23:46:23Z","timestamp":1074987983000},"page":"908-915","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":89,"title":["Generation of<i>Yersinia pestis<\/i>Attenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates"],"prefix":"10.1128","volume":"72","author":[{"given":"Yehuda","family":"Flashner","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emanuelle","family":"Mamroud","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avital","family":"Tidhar","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raphael","family":"Ber","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moshe","family":"Aftalion","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Gur","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shirley","family":"Lazar","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anat","family":"Zvi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamar","family":"Bino","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naomi","family":"Ariel","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baruch","family":"Velan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avigdor","family":"Shafferman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Cohen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.1998.58.793"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.71.8.4563-4579.2003"},{"key":"e_1_3_2_4_2","doi-asserted-by":"crossref","first-page":"131","DOI":"10.4049\/jimmunol.68.2.131","volume":"68","year":"1952","unstructured":"Baker, E. 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