Polymerase Chain Reaction-Based Signature-Tagged Mutagenesis

The study of bacterial pathogenicity in vitro has identified many signals, at the molecular and cellular levels that affect expression of virulence and other factors in causing disease. Because these pathways are not necessarily reproduced in vitro, their implication and their regulation in pathogenesis in vivo remains circumstantial. Genomics-based technologies can now be used to study pathogenesis in vivo (1 ). Signature-tagged mutagenesis (STM) (2 ) is an elegant method, based on negative selection, to identify mutations in a gene, which is essential during the infection process. In STM, transposon (Tn) mutants are generated, and each unique cell clone is tagged with a specific DNA sequence (2 ). Compared to traditional pathogenicity assays, STM minimizes the number of animals to be utilized, and eliminates false-positive and false-negative results. The strategy of STM depends on tagged Tn mutants, defective in virulence, which cannot be maintained in vivo. Attenuated mutants are selected and retested to confirm attenuation; disrupted genes are cloned via the Tn marker, and the inactivated gene confirmed by DNA sequencing. Modifications of STM, allowing rapid and easy identification of attenuated mutants, have recently been described and called “polymerase chain reaction (PCR)based STM” (3 ).