The Dynamic Bacterial Secretosome

: A Trans-Envelope Energy Transduction System?

Abstract

Gram-negative bacteria are enclosed by a multi-layer envelope, composed of an inner and an outer membrane (OM) separated by a periplasmic space. The OM provides the contact point between the cell and its environment, as such is vital in defending the cell against insults such as antibiotics. Outer membrane proteins (OMPs) form a vital component of the OM, but like all other bacterial proteins are synthesised in the cytosol. The transport of OMPs to the OM relies on a complex network of membrane-bound transporters and periplasmic chaperones, specifically the Sec translocon at the inner membrane and the BAM complex at the outer membrane. Sec acts as a passageway across the inner membrane, delivering OMPs into the periplasm. The BAM complex handles the integration of OMPs into the OM, achieved in the absence of ATP. Sec interacts with ancillary factors SecDF-YajC and YidC forming the holo-translocon (HTL), the periplasmic domains of which form a trans-envelope interaction with the BAM complex, giving rise to the secretosome. This work describes a biochemical exploration of the interactions and dynamics of the secretosome, utilising structural modelling and cross-linking to identify new interaction interfaces. Through proteomic analysis, the periplasmic domain of BAM is shown to form novel interactions with machineries involved in cell division, peptidoglycan-remodelling and OMP biogenesis. Finally, a series of cross-linking mutants mapping to regions in BAM that are dynamic in response to HTL were designed. The development of an in vivo cross-linking assay, in addition to electron paramagnetic resonance studies, highlight disruption of the PMF at the inner membrane is communicated to BAM at these dynamic domains– likely via Sec interactors – suggesting the formation of an energy transduction system spanning the envelope. Overall, this work highlights the dynamic nature of the secretosome and introduces a PMF-dependent model of OMP biogenesis by the secretosome.

Date of Award	18 Mar 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Ian R Collinson (Supervisor) & Vicki Gold (Supervisor)