Abstract
Bacterial flagella contain a rotor-mounted protein complex termed the switch complex that functions in flagellar assembly, rotation, and clockwise/counterclockwise direction control. In Escherichia coli and Salmonella, the switch complex contains the proteins FliG, FliM, and FliN and corresponds structurally with the C-ring in the flagellar basal body. Certain features of subunit organization in the switch complex have been deduced previously, but details of subunit organization in the lower part of the C-ring and the molecular movements responsible for motor switching remain unclear. In this study, we use cross-linking, binding, and mutational experiments to examine subunit organization in the bottom of the C-ring and to probe movements that occur upon switching. The results show that FliN tetramers alternate with FliM C-terminal domains to form the bottom of the C-ring in an arrangement that closely reproduces the major features observed in electron microscopic reconstructions. When motors were switched to clockwise rotation by a repellent stimulus, cross-link yields were altered in a pattern indicating relative movement of FliN and FliM(C). These results are discussed in the framework of a structurally grounded hypothesis for the switching mechanism.
Highlights
The direction of flagellar rotation is regulated by a structure at the bottom of the basal body called the switch complex, constructed from the proteins FliG, FliM, and FliN
The development of molecular hypotheses for switching has been hampered by a shortage of detailed structural information on the switch complex as well as insufficient information on the molecular movements occurring upon motor reversal
The present results cast significant new light on the switching process by clarifying aspects of subunit organization in the lower portion of the switch complex, a part that includes binding sites for the signaling molecule CheY-P, and by providing a glimpse of subunit movements that occur within the complex upon switching
Summary
CheY-P, phospho-CheY; EM, electron microscopic; IPTG, isopropyl -D-thiogalactopyranoside; GST, glutathione S-transferase. The other domain of FliG present in the crystal structure displays another well conserved surface feature, termed the EHPQR motif for its constituent residues, that was found to interact with FliM [32]. In this model, FliG is at the top of the C-ring, where it can interact with the stator; FliM is just below FliG and forms the relatively. Structurally grounded model for switching is proposed that accords with present knowledge of switch complex organization and function
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