FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

Abstract

The bacterial flagellum is a predominantly cell-external supermacromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB C ), which is consistently and specifically cleaved into two subdomains (FlhB CN and FlhB CC ) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB C (P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB C and FlhB C (P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.A large percentage of the bacterial flagellar structure lies outside of the cell envelope, thus requiring that the vast majority of the subunits that compose the flagellum be exported from the cytosol across both the inner and outer membranes. Salmonella employs a type III export pathway to accomplish this (1, 2). It is a Sec-independent pathway that utilizes a flagellum-specific export apparatus to transport flagellar components across the inner membrane. These exported proteins then travel the length of the developing flagellum within an interior channel prior to their incorporation at the structure's cell-distal end (3-6) (the developing structure therefore facilitates export across the outer membrane). At least nine flagellar proteins are involved in the flagellumspecific export apparatus (7). Six are integral membrane components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) postulated to be located within the basal body MS ring (8, 9), and three are soluble components: an ATPase (FliI) that drives export (10), a regulator of the ATPase (FliH) (11-13), and a general chaperone (FliJ) (14, 15).One of the integral membrane proteins, FlhB, has been found to play a central role in export substrate-specificity switching; i.e. regulation of the order in which flagellar subunits are exported, such that proteins incorporated into the cell-proximal rod and hook structure...

Organization and coordinated assembly of the type III secretion export apparatus

“…In addition to the structural components of the inner and outer rings of the NC (PrgH, PrgK, and InvG) (6) and the needle and inner rod proteins (PrgI and PrgJ) (17,18), we detected the export apparatus components InvA (19), SpaP, and SpaS (20) (Table S1). To validate the interaction of the export apparatus components with the NC base, we immunoprecipitated the complex from a strain carrying a carboxy terminal FLAG-epitope tagged allele of SpaS (SpaS N258A ), which carries a mutation in its catalytic, autoprocessing site, thus preventing the cleavage of its carboxyl terminus (21,22). Proteins affinity purified with an anti-FLAG antibody were separated by BN-PAGE, and the band corresponding to the NC was analyzed by LC-MS/MS.…”

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

“…15 Ferris et al proposed a cleavage mechanism for the flagellar paralog of YscU, FlhB, in which the protein undergoes auto-cleavage of the peptide bond between asparagine and proline at the conserved NPTH site, and Zarivach et al have recently provided structural evidence to support such an intein-like, auto-cleavage mechanism involving asparagine cyclization by determining the crystal structure of the E. coli YscU ortholog EscU in the cleaved form along with several structures of uncleaved mutants. 15,18 The autocleavage mechanism requires a specific conformation of the NPTH loop in which the NPTH sequence adopts a type II b-turn, giving rise to a strained conformation that is required for efficient catalysis to occur. The cleavage reaction is initiated by the lone pair of electrons associated with the side chain Nd atom of N263 attacking the partially positively charged P264 carbonyl carbon atom to create a cyclized tetrahedral intermediate.…”

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

“…has been shown earlier that YscU auto-proteolysis followed by YscU CC dissociation constitute critical steps for regulation of the substrate specificity switch (14,29,30). In addition, we recently showed that YscU CC is secreted by the T3SS when bacteria are grown in conditions allowing Yop secretion (26).…”

“…Several studies have demonstrated the involvement of the YscU/FlhB family of protein in the substrate specificity switch (11,13,14,34). YscU is anchored in the inner membrane via four transmembrane segments.…”

“…After activation of the T3SS, the secretion of early substrates is modulated in favor of the secretion of translocator and effector proteins, the so called middle and late substrates, respectively (11,12). This phenomenon is described as the substrate specificity switch that was first identified by Macnab and coworkers in the flagellum (13,14).…”