The mechanism of outer membrane penetration by the eubacterial flagellum and implications for spirochete evolution

Abstract

The rod component of the bacterial flagellum polymerizes from the inner membrane across the periplasmic space and stops at a length of 25 nm at the outer membrane. Bushing structures, the P-and L-rings, polymerize around the distal rod and form a pore in the outer membrane. The flagellar hook structure is then added to the distal rod growing outside the cell. Hook polymerization stops after the rod-hook structure reaches ∼80 nm in length. This study describes mutants in the distal rod protein FlgG that fail to terminate rod growth. The mutant FlgG subunits continue to polymerize close to the length of the normal rod-hook structure of 80 nm. These filamentous rod structures have multiple P-rings and fail to form the L-ring pore at the outer membrane. The flagella grow within the periplasm similar to spirochete flagella. This provides a simple method to evolve intracellular flagella as in spirochetes. The mechanism that couples rod growth termination to the ring assembly and outer membrane penetration exemplifies the importance of stopping points in the construction of a complex macromolecular machine that facilitate efficient coupling to the next step in the assembly pathway.

Characterization of Unique Modification of Flagellar Rod Protein FlgG by Campylobacter jejuni Lipid A Phosphoethanolamine Transferase, Linking Bacterial Locomotion and Antimicrobial Peptide Resistance

“…The current work rules out this possibility, yet the role that pEtN-modified FlgG plays in motility is still a mystery. The three-dimensional crystal structure of FlgG has not been solved, and attempts to model FlgG based on the partial crystal structure of FlgE (34), a flagellar hook protein with 38% identical residues, were only successful at partially modeling FlgG (residues 91-223), excluding the N-terminal domain modified by pEtN (34). This limits our discussion of the structural role played by pEtN modification of FlgG, currently under investigation by our laboratory.…”

Break on through to the other side: outer membrane penetration of the nascent flagellum by a stop-polymerization mechanism: Figure 1.

“…While this work outlined a logical framework for the process of coupling the assembly of one substructure to the next, the structural basis for the secretion switch was poorly understood. In an attempt to crack the molecular code of the switch, Chevance et al (2007) used an elegant genetic screen to identify mutations that bypass the FlgM secretion block exhibited by strains that lack the P-and L-ring proteins and, thus, cannot complete the HBB (Aldridge et al 2006;Chevance et al 2007). In this situation, flagellin transcription can only be relieved by mutations that inactivate FlgM or that "rewire" the regulatory circuitry of FlgM secretion.…”

“…In this situation, flagellin transcription can only be relieved by mutations that inactivate FlgM or that "rewire" the regulatory circuitry of FlgM secretion. It is the latter class of mutations that Chevance et al (2007) sought to isolate using a 28 -dependent reporter construct. A clever secondary screen was subsequently used to discriminate between FlgM loss-of-function mutations and those that bypass the FlgM secretion block.…”

“…Of these organelles, the flagellum is renowned for its use as a model system to elucidate the intricate molecular mechanisms that direct and coordinate the precise subcellular placement and step-wise assembly of macromolecular structures (Pandza et al 2000;Aldridge and Hughes 2002;Macnab 2003;Huitema et al 2006;Lam et al 2006;Murray and Kazmierczak 2006). Interrogating the latter process, Chevance et al (2007) discovered a new conceptual mechanism that underlies flagellar self-assembly and possibly that of other complex protein assemblages.…”

“…This strategy combined with cotransduction experiments to avoid reisolating previously characterized Flk mutants (Aldridge et al 2006), led to the discovery of novel mutations in FlgG (henceforth referred to as FlgG*) (Chevance et al 2007). These FlgG* mutations allow FlgM drainage in the absence of P-and L-ring proteins, indicating that mutations in the rod can deregulate the HBB checkpoint.…”