Detection of Ring Structure and Its Flexibility in Periplasmic Region of FliL, Bacterial Flagellar Stator-Associate Protein, in Marine Vibrio.

Structural insight into the role of the periplasmic chaperone Skp in pathogenic Neisseria.

It is postulate that Skp in Neisseria must utilize a different mechanism than in E. coli to stabilize substrates by expanding and contracting along its long axis to accommodate substrates of differing sizes.

Structural determinants of endopilus assembly, stability, and functional specificity in bacterial type II secretion.

Key determinants of secretion specificity and endopilus stability are identified, revealing how minor sequence variations in conserved nanomachines drive functional adaptation to diverse environments.

Navigating the oligomeric landscape of the periplasmic stress response protease-chaperone DegP with charge detection mass spectrometry

DegP is a periplasmic protease-chaperone essential for protein quality control and virulence factor trafficking in Gram-negative bacteria. In its apo form, DegP adopts a dynamic ensemble of oligomers derived from trimer building blocks through two competing self-assembly pathways. Upon engaging client proteins, apo DegP oligomers redistribute into discrete cage structures inside which the clients are encapsulated. The cage ensembles formed depend on the size of the bound clients, and notably can include 12mers, 24mers, and 60mers. Previous studies mapped the DegP oligmeric landscape using dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, modalities which in general report ensemble averages and often cannot directly delineate closely related coexisting species. Here, we apply charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions, resolving the complete oligomeric distribution in the absence and presence of four clients of increasing size. We reveal previously undetected odd-numbered oligomers and quantify the relative abundance of each assembly. Through heat-cool cycling CDMS experiments, we track cage distribution changes and reveal client protection and refolding, providing a direct view of the chaperone capabilities of DegP. These results further establish CDMS as a powerful single-molecule tool for dissecting heterogeneous protein assembly landscapes.

Insights into the structure and diversity of the bacterial flagellum from cryo-EM studies.

Systematic identification of cell division proteins in haloarchaea and the discovery of a membrane anchor for the Z ring

Most archaea rely on the tubulin-like protein FtsZ for division. In the last decade, several novel cell division proteins have been discovered in the model haloarchaeon Haloferax volcanii which contains two FtsZ proteins, FtsZ1 and FtsZ2. However, the composition of this FtsZ-based archaeal divisome is largely uncharacterized. Here, using in vivo crosslinking coupled with immunoprecipitation and mass spectrometry (CLIP-MS), we identified ten proteins that localize to the division site, including wide-spread proteins predicted to be involved in DNA binding and energy metabolism. Deletion analysis indicate that most of these proteins have a modest or minor impact on cell division, but HVO_0399, renamed as Cell Division Protein C (CdpC), is important for cell division and functions as a membrane anchor for FtsZ1. CdpC consists of three domains, an N-terminal domain (NTD) containing an amphipathic helix critical for membrane binding, a long intrinsically disordered linker, and a C-terminal domain (CTD) that binds FtsZ1 with high affinity and likely promotes its polymerization. Notably, both the NTD and CTD of CdpC harbor a zinc finger that is indispensable for its function. Phylogenetic analysis indicates that CdpC sequences evolved fast across haloarchaea, but exhibited high conservation in domain structure and critical residues found in this study. Overall, our study expands the repertoire of candidate division proteins and establishes CdpC as a membrane anchor for FtsZ1 in haloarchaea. These findings pave the way for in-depth studies of arcaheal cell division and broaden the function of zinc finger proteins in archaea.

FlgJ cell-wall hydrolyzing activity enhances, but is not required, for flagellum assembly in Salmonella enterica

Salmonella can assemble functional flagella, even when the acetylglucosaminidase activity of FlgJ is genetically inactivated, showing that early steps in flagellar assembly are more diverse than previously thought and that differences in cell wall structure between gram-positive and -negative bacteria likely determine whether a cell wall hydrolyzing activity is required for flagellum assembly.