TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins

Abstract A transposon, Tn blaM , designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage λ delivery vectors. Tn blaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature β-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing Tn blaM into bacterial cells and selecting ampicillin-resistant (Ap R ) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing Tn blaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. Tn blaM , like Tn phoA , can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but is has the advantage over Tn phoA that the desired mutants can be isolated by direct selection (as Ap R colonies) rather than by phenotypic screening. Isolates in which Tn blaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute Tn blaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the Ap R selection that is used with Tn blaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.