The genetic modification of the poultry virulent form of S.
typhimurium strain 82/6915, was performed using enterobacteria
phage P22 transduction of a DNA vector containing the S.
typhimurium LT2 aroA554::Tn10 transposon originating from strain
The insertion site of the transposon Tn10 is in the aroA-serC
operon. aroA specifies the enzyme
3-enolpyruvylshikimate-5-phosphate synthetase. Strains contining
this transposon cannot grow on chemically defined media unless
provided with the essential metabolites derived from chorismic
acid, i.e. the aromatic amino acids tyrosine, phenylalanine, and
tryptophan and two minor aromatic metabolites, p-aminobenzoate,
needed as a precursor of folic acid, and 2,3-dihydroxybenzoic acid,
as a precursor of the iron-chelating compound enterobactin
(enterochelin). serC encodes phosphoserine aminotransferase, an
enzyme in the biosynthesis of serine. Insertion of the transposon
also imparts tetracycline resistance.
Transduction of the receipient strain S. typhimurium 82/6915 was
carried out in the presence of tetracycline supplemented
medium. Transductant clones that were found to be resistant
to tetracycline were selected.
Colonies were isolated and further screened for loss of
tetracycline resistance. The isolation of tetracycline-sensitive
variants is facilitated by the fact that tetracycline, at
appropriate concentrations, prevents multiplication of
tetracycline-sensitive bacteria, but does not kill them, whereas
penicillin kills multiplying bacteria but spares non-multiplying
bacteria. The technique of penicillin-selection was used for
isolation of tetracycline-sensitive variants.
A tetracycline-sensitive mutant that remained auxotrophic for
aromatic metabolites and serine was identified. The resulting S.
typhimurium strain 82/6915 aroA-serC deletion mutant, was
denominated as strain STM-1.
The requirement for aromatic metabolites and serine,is not met when
S. typhimurium is present in vertebrate tissues, resulting in
retardation of growth in vivo or outside the host. Deficient
lineages of this modified-live vaccine remain in host tissue for
several days, but without causing symptoms and eventually be
eliminated by immunological defence mechanisms while still
imparting protection against salmonella.