Tn-seq of ssrA deletion mutant of Bacillus subtilis

In bacteria, the trans-translation system serves as the primary rescue mechanism for ribosomes stalled on a non-stop mRNA. Bacterial species that can survive in the absence of trans-translation often possess alternative ribosome rescue factors. We conducted a Tn-seq analysis to identify genes whose dysfunction compromise cell growth in B. subtilis cells that lacks ssrA, a gene for tmRNA that act as a major component of the trans-translation system. Transposon that harbors kanamycin-resistance gene were transposed into a chromosomal DNA of the wild-type as well as an ssrA-deleted derivative. Cells harboring transposon were selected based on kanamycin-resistance. Subsequently, chromosomal DNA was purified, and DNA libraries were prepared to sequence the genomic region adjacent to the inserted transposon.