Résumé
The bacterial flagellar motor, which spins a helical propeller for propulsion, has undergone evolutionary diversification across bacterial species, often involving the addition of structures associated with increasing torque for motility in viscous environments. Understanding how such structures function and have evolved is hampered by challenges in visualizing motors in situ. Here we developed a Campylobacter jejuni minicell system for in situ cryogenic electron microscopy imaging and single-particle analysis of its motor, one of the most complex flagellar motors known, to subnanometre resolution. Focusing on the large periplasmic structures which are essential for increasing torque, our structural data, interpreted with molecular models, show that the basal disk comprises concentric rings of FlgP. The medial disk is a lattice of PflC with PflD, while the proximal disk is a rim of PflB attached to spokes of PflA. PflAB dimerization is essential for proximal disk assembly, recruiting FliL to scaffold more stator complexes at a wider radius which increases torque. We also acquired insights into universal principles of flagellar torque generation. This in situ approach is broadly applicable to other membrane-residing bacterial molecular machines.
How evolution innovates remains a fundamental question. While innovations in eukaryotes often arise by rewiring existing gene transcriptional netwoks 1 , examples of the emergence of evolutionary novelty at the molecular-scale focus on small protein complexes 2-5 . What is needed is a comprehensive case study of a family of molecular machines that have diversified, so we can infer how their diversity evolved.
Bacterial flagella, helical propellers rotated by cell-envelopeembedded rotary motors, are icons of the evolution of molecular novelty 6 (Fig. 1a). The flagellum, best studied in model organisms Salmonella enterica serovar Typhimurium and Escherichia coli, is composed of a ring of inner-membrane motor proteins ('stator complexes') that harness ion flux to rotate a large cytoplasmic rotor ring (the 'C-ring'). Torque is transmitted through a chassis (the 'MS-ring') and periplasm-spanning axial driveshaft (the 'rod') to an extracellular propeller structure that generates thrust. Structures of the C-ring 7-9 , parts of stator complexes 10,11 , MS-ring and rod 12,13 , and axial structures 14,15 have recently been determined from purified subcomplexes.
Because flagella may pre-date the emergence of the bacteria, their age renders understanding how these core structures first evolved challenging. More tractable is the promise of understanding how recent diversifications evolved. Many flagellar motors have recruited additional proteins that scaffold more stator complexes than the ~11 seen in