An improved vector system for insertional gene inactivation inspired by the tmRNA-tagging system of S. pneumoniae.

Insertional mutagenesis is a technique often used to inactivate genes in Streptococcus pneumoniae. Using conventional vectors, a 5' segment of the targeted gene remains under the control of the gene's authentic promoter following gene disruption. Thus, the expression of a functional peptide and the misinterpretation of results in consequence cannot be excluded. To circumvent this problem, we have developed a plasmid for insertional mutagenesis based on the tmRNA-tagging system of S. pneumoniae which ensures that any protein expressed after gene disruption is degraded. Insertional mutagenesis using this vector results in the targeted gene being tagged with a tmRNA-derived sequence coding for a proteolysis tag. Here we show that the translation product of a gene tagged by this method is not detectable by Western blotting, suggesting that the protein was degraded. This modified vector allows total inactivation of genes with a reliability that cannot be achieved by conventional vectors for insertional mutagenesis. This approach can be applied to other bacterial species.