Plasticity of the domain structure in FlgJ, a bacterial protein involved in flagellar rod formation

In Salmonella, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ϳ22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis. The bacterial flagellum exemplifies nanoscale engineering that microbes have been perfecting over billions of years (1). This supramolecular structure is composed of approximately 25 distinct protein subunits and self-assembles to final lengths of up to 20 m, several times the length of the cell itself (2). The flagellum enables a bacterium to colonize surfaces, search for food, and avoid noxious substances in its environment (3). To orchestrate the ordered assembly of a flagellum, flagellated species of bacteria have evolved numerous regulatory mechanisms. Flagellar biosynthesis is regulated at the genetic level by mechanisms that couple gene expression to assembly (4, 5) and at the biomechanical level with many flagellar proteins possessing inherent self-assembly characteristics (6-8).Synthesis of the flagellum begins with the assembly of a basal body composed of several substructures (9). First to assemble is the MS-ring within the cytoplasmic membrane, followed by the cytoplasm-facing C-ring, which serves as both the rotor for the flagellar motor and as an affinity site for the secretion and assembly of other flagellar structures (10). The flagellum-specific type three secretion (T3S) system assembles in the inner membrane within the MS-and C-rings. The flagellar T3S system secretes flagellar subunits from the cytoplasm to be assembled at the tip of the growing flagellum (11, 12). The rod assembles through the periplasmic space to the outer membrane and transmits the torque generated by the basal body to the extracellular hook and filament (13).The rod initially assembles into a proximal rod structure that lies between the MS-ring and the cell wall and is com...

Section: Resultsmentioning

confidence: 99%

Rod-to-Hook Transition for Extracellular Flagellum Assembly Is Catalyzed by the L-Ring-Dependent Rod Scaffold Removal

Cohen

Hughes

2014

“…Recently, Nambu et al (37) found that various alphaproteobacteria, including R. sphaeroides, possess proteins homologous to FlgJ that lack the lytic transglycosylase domain, whereas beta-and gammaproteobacteria possess FlgJ that contains the N-terminal domain fused to the lytic C-terminal moiety. It should be noted that this catalytic domain does not show muramidase enzymatic activity (21).…”

Section: Fig 5 Affinity Blot and Coimmunoprecipitation (A)mentioning

confidence: 99%

The Flagellar Muramidase from the Photosynthetic Bacterium Rhodobacter sphaeroides

Mora¹,

Ballado²,

González‐Pedrajo³

et al. 2007

We have characterized open reading frame RSP0072, which is located within the flgG operon in Rhodobacter sphaeroides. The amino acid sequence analysis of this gene product showed the presence of a soluble lytic transglycosylase domain. The deletion of the N-terminal region (90 amino acids) of the product of RSP0072 yields a leaky nonmotile phenotype, as determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagella in mutant cells. The purified wild-type protein showed lytic activity on extracts of Micrococcus luteus. In contrast, no lytic activity was observed when the residues E57 or E83 were replaced by alanine. Affinity blotting suggests that the protein encoded by RSP0072 interacts with the flagellar rod-scaffolding protein FlgJ, which lacks the muramidase domain present in FlgJ of many bacteria. We propose that the product of RSP0072 is a flagellar muramidase that is exported to the periplasm via the Sec pathway, where it interacts with FlgJ to open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar structure.

“…Similar to FlgJ Bb , the FlgJ homologs in other sequenced spirochetes also belong to the family of singledomain FlgJ; they all lack the C-terminal PGase domain, suggesting that the spirochetes may have evolved another route to process the passage of elongating rods through the PG layer. It has been postulated that the class of dual-domain FlgJ proteins has evolved by merging two individual domains together (44). Consistent with this assumption, the absent PGase domain has been identified in the genome of R. sphaeroides, which is encoded by an independent gene (14).…”

Section: Figmentioning

confidence: 83%

“…FlgJ homologs are present in many bacteria (44). However, their functions in flagellation and motility have only been studied in S. Typhimurium and Rhodobacter sphaeroides (14,24,25,45).…”

Section: Discussionmentioning

confidence: 99%

“…In S. Typhimurium, flgJ null mutants are aflagellated and nonmotile, while mutants that do not express the PGase domain produce fewer flagella and show poor motility (25,45). However, the PGase domain is absent in the FlgJ homologs from several bacterial phyla, including Alphaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria and Spirochaetes (44). As there is only one domain, these homologs are referred to as "single-domain FlgJ."…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Single-Domain FlgJ Contributes to Flagellar Hook and Filament Formation in the Lyme Disease Spirochete Borrelia burgdorferi

Zhang

Tong²,

Li³

et al. 2012

T he bacterial flagellum is a sophisticated macromolecular complex. Its structure and assembly have been well studied in two model organisms, Escherichia coli and Salmonella enterica serovar Typhimurium (for reviews, see references 5, 13, 36, and 58). The flagellum is composed of at least 25 different proteins that can be grouped into three physical parts: the basal body, the flagellar hook, and the filament. The basal body is imbedded within the cell envelope and works as a reversible rotary motor; the flagellar hook and filament extend outwards to the cell exterior and function as a universal joint and a propeller, respectively. The basal body is very complex and consists of several functional units: the membranesupramembrane (MS)-C ring (rotor), the rod (driveshaft), the L-P rings (bushings), the stator (torque generator), and the flagellar export apparatus. The motor is driven by an inward-directed electrochemical gradient of protons or sodium. The torque generated by the motor is mechanically transmitted to the filament via the rod-hook complex, leading to the rotation of flagellar filament, which propels the bacterial cells forward.Flagellar assembly is a sequential process (for reviews, see references 1 and 13). It begins with the MS ring assembly. Built onto the MS ring is a hollow rod that spans the periplasmic space. After formation of the MS ring/rod complex, the FlgI and FlgH proteins assemble around the rod to form the P and L rings, respectively. The hook and filament proteins are subsequently assembled on the rod. The flagellar rod begins with the MS ring and stops at the flagellar hook. Thus, it needs to penetrate the peptidoglycan (PG) layer during flagellar formation. It has been postulated that FlgJ is essential for flagellar rod formation (25,45), with the N-terminal domain (rod-capping) acting as a scaffold for rod assembly and the C-terminal domain functioning as a PG hydrolase (PGase), which makes a hole in the PG layer to allow rod penetration. In S. Typhimurium, flgJ null mutants are aflagellated and nonmotile, while mutants that do not express the PGase domain produce fewer flagella and show poor motility (25, 45). However, the PGase domain is absent in the FlgJ homologs from several bacterial phyla, including Alphaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria and Spirochaetes (44). As there is only one domain, these homologs are referred to as "single-domain FlgJ." The function of these FlgJ homologs remains unknown.Spirochetes are a group of motile bacteria that have a unique morphology and are able to swim in highly viscous gel-like environments (for reviews, see references 11 and 31). It is believed that motility plays a critical role in the biology of spirochetes and in the processes of diseases caused by pathogenic spirochetes (9,11,16,32,55). Spirochetes swim by means of two rotating bundles of periplasmic flagella (PFs) that reside between the outer membrane and cell cylinder (23,32,38,49). PFs are structurally similar to the flagella of other bacteria, as each ...