A chain mechanism for flagellum growth.

Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction

Conformational adaptability of the terminal regions of flagellin.

Secondary structure formation in the disordered terminal regions of flagellin were studied by circular dichroic (CD) spectroscopy, Fourier transform infrared spectroscopy, and x-ray diffraction. The terminal regions of flagellin are known to form alpha-helical bundles upon polymerization into flagellar filaments. We found from comparative CD studies of flagellin and its F40 tryptic fragment that a highly alpha-helical conformation can be induced and stabilized in the terminal regions in 2,2,2-trifluoroethanol (TFE) containing solutions, which is known to promote intra-molecular hydrogen bonding. Two oligopeptides, N(37-61) and C(470-494), each corresponding to a portion of terminal regions and predicted to have a high alpha-helix forming potential, were synthesized and studied. Both peptides were disordered in an aqueous environment, but they showed a strong tendency to assume alpha-helical structure in solutions containing TFE. On the other hand, peptides were found to form transparent gels at high concentrations (> 15 mg/ml) and all three methods confirmed that the peptides become ordered into a predominantly beta structure upon gel formation. Our results show that large segments of the disordered terminal regions of flagellin can adopt alpha-helical as well as beta structure depending on the environmental conditions. This high degree of conformational adaptability may be reflecting some unique characteristics of the flagellin termini, which are involved in self-assembly and polymorphism of flagellar filament

A partial atomic structure for the flagellar hook of Salmonella typhimurium

The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The N- and C-terminal sequences of the eight axial proteins were predicted to form interlocking α-domains generating an axial tube. We report on an ≈1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an ≈2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook