RT Journal Article SR Electronic T1 A genetic screen suggests an alternative mechanism for inhibition of SecA by azide JF bioRxiv FD Cold Spring Harbor Laboratory SP 173039 DO 10.1101/173039 A1 Mohammed Jamshad A1 Rachael Chandler A1 Mark Jeeves A1 Ashley Robinson A1 Farhana Alam A1 Tamar Cranford Smith A1 Anokhi Shah A1 Oliver J. Daubney A1 Karl A. Dunne A1 Naomi Nabi A1 Aadil Iqbal A1 Anna F. A. Peacock A1 Janet E. Lovett A1 Timothy Knowles A1 Ian Henderson A1 Damon Huber YR 2017 UL http://biorxiv.org/content/early/2017/08/06/173039.abstract AB Sodium azide inhibits bacterial growth by inhibiting the activity of SecA, an ATPase required for translocation of proteins across the cytoplasmic membrane. To investigate the mechanism of action of azide, we used transposon directed insertion-site sequencing (TraDIS) to screen a high-density library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions in genes encoding most components of the Sec machinery increased susceptibility to azide. However, insertions truncating the C-terminal extension (CTE) of SecA decreased susceptibility of E. coli to azide. Insertions in genes encoding many metal binding proteins also increased susceptibility to azide, and transcriptional profiling suggested that treatment with azide disrupted iron homeostasis. The presence of iron in the media decreased the susceptibility of E. coli to azide, and mutations in the secA gene that confer resistance to azide altered the response of E. coli to iron limitation, suggesting a connection between iron metabolism and protein translocation. Although previous work suggests that SecA binds to zinc, SecA copurified with iron when expressed at physiological levels, and azide disrupted the interaction of the C-terminal metal-binding domain (MeBD) with iron in vivo. Biophysical analysis of metal binding by the MeBD using isothermal titration calorimetry and 1H-nuclear magnetic resonance indicated a clear binding preference for Fe2+ over Zn2+. These results indicate that the physiological ligand of SecA is iron and that azide inhibits SecA by disrupting iron binding.