title: Construction of a conditional lethal Salmonella mutant via genetic recombination using the ara system and asd gene.
abstract: In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned into a plasmid carrying a p15A replication origin to create the recombinant plasmid pMMP55. The growth of Salmonella MMP10 harboring pMMP55 was dependent on the presence of arabinose. In the presence of arabinose, the Asd deficiency due to chromosomal deletion of asd in the Salmonella host was complemented by the asd gene transcribed and translated under the P(araBAD) promoter and araBAD Shine-Dalgarno (SD) sequence in pMMP55. Growth inhibition of the strain was demonstrated by arabinose depletion in M9 minimal medium, indicating that the strain were unable to grow in an arabinose-limited environment. In addition, the analysis of a 50% lethal dose (LD50) using mice revealed that the strain MMP10 exhibited attenuation by approximately 100-fold relative to that of the unmodified strain. In conclusion, these data suggest that the araC P(araBAD)::asd system developed in this study can be used to construct conditional lethal Salmonella mutants for application as safe, live-attenuated Salmonella vaccines.
authors.affiliation: Swine Science & Technology Center, Gyeongnam National University of Science and Technology, 150 Chilam-dong, Jinju, Gyeongnam, 660-758, South Korea.
authors.forename: Sam Woong
authors.initials: SW
authors.lastname: Kim
authors.forename: Ho Young
authors.initials: HY
authors.lastname: Kang
authors.forename: Jin
authors.initials: J
authors.lastname: Hur
authors.forename: Sang Wan
authors.initials: SW
authors.lastname: Gal
authors.forename: Woo Young
authors.initials: WY
authors.lastname: Bang
authors.forename: Kwang-Keun
authors.initials: KK
authors.lastname: Cho
authors.forename: Chul Wook
authors.initials: CW
authors.lastname: Kim
authors.forename: Jeong Dong
authors.initials: JD
authors.lastname: Bahk
authors.forename: John Hwa
authors.initials: JH
authors.lastname: Lee
mesh_terms: D000818:Animals; D051777:AraC Transcription Factor / Q000235:genetics* / Q000378:metabolism; D001089:Arabinose / Q000378:metabolism; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012475:Salmonella / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D012480:Salmonella Infections / Q000382:microbiology* / Q000401:mortality; D022562:Salmonella Vaccines / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014774:Virulence
chemical_list: D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D022562:Salmonella Vaccines; D001089:Arabinose; D001223:Aspartate-Semialdehyde Dehydrogenase