General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats.
A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.
Bloch
C A
CA
Department of Genetics, Harvard Medical School, Boston, Massachusetts.
Thorne
G M
GM
Ausubel
F M
FM
D000818:Animals; D000831:Animals, Newborn / Q000235:genetics / Q000382:microbiology; D001426:Bacterial Proteins / Q000235:genetics; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000201:enzymology / Q000235:genetics* / Q000382:microbiology; D009154:Mutation; D051381:Rats; D018805:Sepsis / Q000201:enzymology / Q000235:genetics* / Q000382:microbiology; D013482:Superoxide Dismutase / Q000235:genetics*; D013604:T-Phages / Q000235:genetics; D014161:Transduction, Genetic
D001426:Bacterial Proteins; D013482:Superoxide Dismutase