title: Signature-tagged mutagenesis of Vibrio vulnificus.
abstract: Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.
authors.affiliation: Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Takashige
authors.initials: T
authors.lastname: Kashimoto
authors.forename: Ping
authors.initials: P
authors.lastname: Tong
authors.forename: Jianbo
authors.initials: J
authors.lastname: Xiao
authors.forename: Michiko
authors.initials: M
authors.lastname: Sugiyama
authors.forename: Miyuki
authors.initials: M
authors.lastname: Inoue
authors.forename: Rie
authors.initials: R
authors.lastname: Matsunaga
authors.forename: Kohei
authors.initials: K
authors.lastname: Hosohara
authors.forename: Kazue
authors.initials: K
authors.lastname: Nakata
authors.forename: Kenji
authors.initials: K
authors.lastname: Yokota
authors.forename: Keiji
authors.initials: K
authors.lastname: Oguma
authors.forename: Koichiro
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D015342:DNA Probes; D005260:Female; D005798:Genes, Bacterial / Q000235:genetics; D051379:Mice; D008813:Mice, Inbred ICR; D016254:Mutagenesis, Insertional / Q000379:methods; D041261:Vibrio vulnificus / Q000235:genetics* / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D015342:DNA Probes; D037521:Virulence Factors