Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria

We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides far tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA str ands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screenin g. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutan ts; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. Th is simple STM method can be adapted to any bacterial system and used for ge nome scanning in various growth conditions.