(583e) Discovery Of A Bacterial Rnai System Targeting A Tat Export Signal

RNA interference (RNAi) is a powerful regulatory mechanism in eukaryotes that enables sequence-specific RNA degradation. Until recently, such a system was not thought to exist in prokaryotes, however recent evidence suggests that bacteria may have evolved an RNAi-like system as a mode of immunity against invading viruses and plasmids. The presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), separated by unique sequences of similar length, in the genomes of both bacteria and archaea are thought to play an important role in their resistance against foreign invading DNA. These repeats together with the adjacent cas (CRISPR-associated) genes have been hypothesized to form a defense mechanism analogous to the eukaryotic RNAi system but so far only limited data has emerged to support this notion. Previous studies in our laboratory demonstrated a reporter protein consisting of a twin-arginine translocation (Tat) signal peptide fused to GFP (ssTorA-GFP) was completely undetectable when expressed in dnaKJ mutant cells. Quantitative real-time PCR revealed that the absence of the reporter protein was due to knockdown of ssTorA-GFP mRNA levels. A transposon strategy identified suppressor mutations that restored expression of ssTorA-GFP and, remarkably, the transposon insertions mapped to the cas operon. Since the cas genes encode putative RNA processing enzymes that are suspected to form the bacterial RNAi machinery, we conclude that our transposon studies provide the first experimental evidence for a bacterial RNAi-like system as well as an experimental framework for testing hypothetical models. This talk will discuss our ongoing efforts to characterize this system and to exploit the phenomenon for gene-specific knockdown in bacterial cells as a novel mode of antimicrobial therapy.