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

Abstract

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.

Comprehensive Identification of Salmonella enterica Serovar Typhimurium Genes Required for Infection of BALB/c Mice

“…Beside SPI-2, other well established virulence-related genes that are identified in our dataset include: the aromatic amino acid biosynthesis (or pre-chorismate) pathway genes aroA , aroC and aroD , mutants of which are prototype live attenuated Salmonella vaccines [24],[33],[34]; the purine biosynthesis genes purA , purD , purF , purG , purH , guaA and guaB [3],[35],[36]; LPS core biosynthesis genes [37]; O-antigen biosynthesis genes [38]; and the virulence plasmid spv genes [39]. Presumably because of the requirement for the spv operon, plasmid partitioning is required for virulence, and both parA and parB mutants are attenuated.…”

Expression of disulphide-bridge-dependent conformational epitopes and immunogenicity of the carboxy-terminal 19 kDa domain of Plasmodium yoelii merozoite surface protein-1 in live attenuated Salmonella vaccine strains

“…Oligonucleotide primers containing sites for BamHI and SpeI cohesive ends (forward primer, 5' TAGG 3' ; reverse primer, 5' CACACTAGTCTCGAGGCT-GGAAGAACTACAGAATAC 3') were used to amplify the DNA, allowing directional cloning of the MSPl,, EGF-like modules into pTECH2 that had been previously digested with BamHI and SpeI. The bacterial strains used were E. coli Topp 10 (Stratagene), E. coli TG2 (recA) (Sambrook et al, 1989), Salmonella typhimurium SL5338 (galE r-m+) (Brown et al, 1987) and the S. typhimurium vaccine strains : SL3261(aroA) (Hoiseth & Stocker, 1981), BRD726 (AhtrA) (Chatfield et al, 1992), CSaroD (Miller et al, 1989) and C.5046 (htrA::TnphoA) (Johnson et al, 1991) (referred to in this study as C5htrA). Bacteria were grown aerobically in Luria-Bertani (LB) broth supplemented with 50 pg ampicillin ml-l as required.…”

A new chromosomal locus associated with gut-modulated phenotypes in Salmonella enterica serotype Typhimurium

“…Recombinants were packaged into ë particles and transfected into Escherichia coli HU835. Because smooth strains of S. Typhimurium such as LT7 cannot be infected with ë it was necessary to repackage the library into Typhimurium bacteriophage P22 via S. Typhimurium strain AS68 which has both ë and P22 receptors, before transduction into LT7 and selection for Cb-resistant colonies [24].…”