Fine‐tuning regulation of (p) <scp>ppGpp</scp> ‐driven outer membrane vesicle formation in <i>Acinetobacter baumannii</i>

The roles of (p)ppGpp in regulating cytosolic proteins are well established; however, their effects on membrane remodeling remain elusive. The translocation of signal recognition particle (SRP)-dependent proteins can be modulated through (p)ppGpp binding to two key GTPase components: FtsY, which interacts with SecYEG, and Ffh, a homolog of SRP54. A (p)ppGpp-specific Broccoli RNA aptamer and the chemometer PyDPA were used to quantify the (p)ppGpp levels in the ΔrelA and ΔrelA/ΔspoT strains of Acinetobacter baumannii, confirming a stepwise reduction in (p)ppGpp levels in the following order: wild-type > ΔrelA > ΔrelA/ΔspoT. The ΔrelA strain, with intermediate (p)ppGpp levels, exhibited increased outer membrane vesicle (OMV) production, whereas the ΔrelA/ΔspoT strain exhibited reduced production, highlighting a non-linear relationship between OMV production and (p)ppGpp levels. Scanning and transmission electron microscopy revealed (p)ppGpp-dependent changes in cell envelope integrity: the relA mutant exhibited outer membrane disruption leading to OMV formation, whereas the relA/spoT mutant maintained an intact outer membrane, suggesting that membrane stability is modulated by fine-tuned (p)ppGpp levels. Western blotting and proteomic analyses identified significant OmpA accumulation in the inner membrane of the ΔrelA/ΔspoT strain, and an accumulation of SRP-dependent inner membrane proteins, including NuoB, NuoL, and TolA, in the ΔrelA strain. These findings indicate that (p)ppGpp levels are crucial for regulating membrane protein incorporation in A. baumannii. Regulation of (p)ppGpp levels using the CRISPRi system revealed that outer membrane disruption and OMV formation peaked at intermediate (p)ppGpp concentrations, highlighting the importance of precise (p)ppGpp adjustment in regulating bacterial phenotypes.