Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains.
A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB lambda receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.
Miller
I A
IA
Department of Pathology, University of Cambridge, UK.
Chatfield
S
S
Dougan
G
G
Desilva
L
L
Joysey
H S
HS
Hormaeche
C
C
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D003360:Cosmids; D005798:Genes, Bacterial; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D012480:Salmonella Infections / Q000382:microbiology; D012483:Salmonella Phages / Q000235:genetics*; D012486:Salmonella typhimurium / Q000235:genetics* / Q000472:pathogenicity; D014161:Transduction, Genetic; D014774:Virulence