The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life. The OM and the IM are separated by the periplasm, a compartment that contains the peptidoglycan. The OM is tethered to the peptidoglycan via the lipoprotein, Lpp. However, the importance of the envelope’s multilayered architecture remains unknown. Here, when we removed physical coupling between the OM and the peptidoglycan, cells lost the ability to sense defects in envelope integrity. Further experiments revealed that the critical parameter for the transmission of stress signals from the envelope to the cytoplasm, where cellular behaviour is controlled, is the IM-to-OM distance. Augmenting this distance by increasing the length of the lipoprotein Lpp destroyed signalling, whereas simultaneously increasing the length of the stress-sensing lipoprotein RcsF restored signalling. Our results demonstrate the physiological importance of the size of the periplasm. They also reveal that strict control over the IM-to-OM distance is required for effective envelope surveillance and protection, suggesting that cellular architecture and the structure of transenvelope protein complexes have been evolutionarily co-optimised for correct function. Similar strategies are likely at play in cellular compartments surrounded by 2 concentric membranes, such as chloroplasts and mitochondria.

The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun’s lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer.

The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic b-barrel Outer-Membrane Proteins (OMPs) – are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones e.g. SurA, which prevent aggregation. OMPs are then offloaded to the b-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL) – an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane 'insertase' YidC – contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive-force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG; and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.

Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts' gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni's flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni's opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by highspeed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrappedmode swimming.

In Salmonella, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ϳ22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis. The bacterial flagellum exemplifies nanoscale engineering that microbes have been perfecting over billions of years (1). This supramolecular structure is composed of approximately 25 distinct protein subunits and self-assembles to final lengths of up to 20 m, several times the length of the cell itself (2). The flagellum enables a bacterium to colonize surfaces, search for food, and avoid noxious substances in its environment (3). To orchestrate the ordered assembly of a flagellum, flagellated species of bacteria have evolved numerous regulatory mechanisms. Flagellar biosynthesis is regulated at the genetic level by mechanisms that couple gene expression to assembly (4, 5) and at the biomechanical level with many flagellar proteins possessing inherent self-assembly characteristics (6-8).Synthesis of the flagellum begins with the assembly of a basal body composed of several substructures (9). First to assemble is the MS-ring within the cytoplasmic membrane, followed by the cytoplasm-facing C-ring, which serves as both the rotor for the flagellar motor and as an affinity site for the secretion and assembly of other flagellar structures (10). The flagellum-specific type three secretion (T3S) system assembles in the inner membrane within the MS-and C-rings. The flagellar T3S system secretes flagellar subunits from the cytoplasm to be assembled at the tip of the growing flagellum (11, 12). The rod assembles through the periplasmic space to the outer membrane and transmits the torque generated by the basal body to the extracellular hook and filament (13).The rod initially assembles into a proximal rod structure that lies between the MS-ring and the cell wall and is com...