Developing an assay system for screening inhibitors of Hfq-mediated RNA metabolism.

Abstract

Hfq is an RNA chaperone conserved across diverse bacterial lineages that regulates gene expression by facilitating the pairing of small RNAs with their target mRNAs. It influences both the translation and stability of specific transcripts, thereby contributing to complex post-transcriptional regulatory networks. We have previously shown that overproduction of Hfq inhibits cell division by suppressing the expression of the essential cell division protein FtsZ. Based on this observation, we developed a novel antibiotic screening system using an Escherichia coli strain harboring an IPTG-inducible hfq gene to identify compounds that interfere with Hfq-mediated RNA metabolism. We tested representative inhibitors of DNA synthesis, protein synthesis, and cell wall synthesis; however, none restored colony formation. In contrast, rifampicin, an inhibitor of RNA polymerase, restored colony formation, as expected for a compound interfering with Hfq function. Sublethal concentrations of rifampicin restored colony formation of the Hfq-overproducing cells. Western blot analysis revealed that Hfq expression levels decreased upon rifampicin treatment, indicating that rifampicin was a false-positive hit. To eliminate such false-positive hits, a rifampicin-resistant rpoB mutation was introduced into the indicator strain. As expected, rifampicin did not restore growth of the rifampicin-resistant rpoB strain. This assay provides a useful platform for identifying compounds that interfere with Hfq-mediated RNA metabolism.