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.
Molnos
J
J
F. Hoffmann-La Roche Ltd., Pharmaceutical Research Preclinical Infectious Diseases, CH-4070 Basel, Switzerland.
Lange
R
R
Amrein
K E
KE
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics*; D011995:Recombination, Genetic; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000254:growth & development
D001426:Bacterial Proteins; D012329:RNA, Bacterial; C096222:tmRNA