Transposon Tn 10

Abstract

Classical transposons applicable to Haemophilus influenzae have had their limitations. Recently, however, advances have been made in the development of transposon systems and their applications, such as signature tagged mutagenesis, to identify in-vivo survival genes, and the GAMBIT strategy, to identify in-vitro essential genes. Over the last two decades, transposon mutagenesis has been developed mainly for Escherichia coli. During that time, several transposable gene fusion vector and phage systems were constructed, which targeted different aspects of gene expression, protein subcellular localization, and membrane protein topology (1). For example, operon and gene fusion systems were established to study transcriptional/translational regulation of target genes. These systems consist of “reporter” genes, such as lacZ, blaM, phoA, gfp, luxA, or cat, lacking either their own transcriptional or translational signals. The reporters are embedded within transposons in such a way that they form transcriptional or translational hybrids with the target gene. For example, systems such as TnphoA (2) or Tnbla (3) carry the phoA or blaM reporter genes, encoding alkaline phosphatase or TEM1 β-lactamase, respectively, but lacking signal sequences. Both reporter genes express activity only after an insertion occurs in the reading frame of an expressed gene encoding for an exported or membrane associated protein. A potential advantage of the blaM reporter is that gene fusion events can be selected directly after transposition by demanding various levels of resistance to ampicillin or other β-lactam antibiotics.