Comprehensive Analysis of the Cytoskeletal Protein Bactofilin in Caulobacter crescentus

Most bacteria contain protein filaments or filament systems collectively known as “bacterial cytoskeleton”. Similar to their counterparts in eukaryotic cells, bacterial cytoskeletal proteins are essential in temporal and spatial organization of cellular machineries. They have been implicated in a wide range of fundamental processes, such as cell division, morphogenesis, DNA segregation, and polarity establishment. In the stalked model organism Caulobacter crescentus (C. crescentus), there are structural homologues of typical eukaryotic cytoskeletal proteins, such as the tubulin homologue FtsZ, the actin homologue MreB, and the intermediate filament-like protein crescentin. In addition, an increasing number of bacteria-specific cytoskeletal proteins have been discovered in recent years, among them the bactofilins. Previous studies have demonstrated that bactofilins adopt beta-helical structures and polymerize spontaneously into stable filaments that have a variety of roles in bacteria. However, so far, many aspects of their biology remain unclear. In this work, we comprehensively investigated the properties of bactofilin using the homologue BacA from C. crescentus as a model. The aim is to elucidate its polymerization mechanism and its interaction with other proteins. The results showed that several conserved hydrophobic residues at the ends of the beta-helical bactofilin domain were crucial for the formation of protofilaments, which can further coalese into higher-order structures via lateral interactions, mediated by electrostatic forces. Moreover, studies of the membrane association of bactofilin not only indentified important amino acid residues for membrane-targeting but also revealed that polymerization and membrane binding are closely interdependent processes that promote each other. Additionally, the interaction between BacA and the penicillin-binding protein PbpC was analyzed in detail. Both interaction regions and the binding affinity were elucidated, which provides insights into the mode of interaction of bactofilins with client proteins. In an attempt to search for potential regulators of polymerization, various proteins were identified to interact with bactofilins in C. crescentus in vivo, indicating that bactofilins may play roles that go beyond their previously reported function in stalk biogenesis in this species. However, future studies are required to unravel the biological relevance of these interactions. Collectively, the findings made in this study for the first time shed light on the mode of action of bactofilins in C. crescentus, thus setting the basis for in-depth analyses of bactofilin function in other organisms.