Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

Abstract

The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N-and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCEDespite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled. The bacterial flagellum is a highly efficient nanomachine that propels the cell in liquid and semisolid media and over surfaces. It is composed of approximately 30 different proteins, whose numbers range from a few to several thousand copies (1). Its structure includes three major components, namely, the basal body, the hook, and the filament.The basal body contains the flagellum-specific type III secretion system (T3SS) housed in a bell-like structure, named the C ring, which is also the input for chemotactic signals that control the direction of rotation and consequently cell movement (2). The basal body also contains an inner membrane ring (MS ring) and a periplasmic ring (P ring); in addition, Gram-negative bacteria possess an outer membrane ring (L ring). This structure includes the rod that expands from the MS ring through the L and P rings, as well as the rotary motor driven by proton or sodium ions, which is surrounded by stator subunits that are located around the MS ring and harness energy from the electrochemical gradient.The hook is composed of about 120 copies of FlgE and is connected proximally to the rod and distally to the filament, by means of two hook-associated proteins (HAPs) (HAP1 and HAP3). The filament is composed of thousands of subunits of flagellin (FliC) and is the most abundant component of this organelle. HAP2 acts as a scaffold protein that helps flagellin subunits to polymerize at the distal end of the filament (3).Th...

Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF

“…Despite the overall structural similarities between these proteins as predicted earlier by in silico modeling based on amino acid sequence alignments (25), SltF appeared to bind to only the two proximal rod proteins FlgB (15 kDa) and FlgF (27 kDa) under the conditions used (Fig. 5).…”

“…FlgF and FlgG were purified under native conditions (25). Cells were harvested by centrifugation (10,000 ϫ g, 10 min, 4°C) and then frozen at Ϫ20°C.…”

“…FliE, FlgB, and FlgC were purified under denaturing conditions as previously described (25). For FliE and FlgB, cells were harvested and lysed as described above for FlgF and FlgG.…”

Assembly Order of Flagellar Rod Subunits in Bacillus subtilis

“…FlgG is thought to be the last rod protein polymerized, as particular mutants of FliF cause the rod to shear such that FlgG is released with the flagellar hook (10,11,24). Thus, the inferred rod order from cell proximal to cell distal is FlgB, FlgC/FlgF, and FlgG with recent evidence that FlgF may precede FlgC (25) (Fig. 1A, left).…”

“…First, FliE may be structural and serve as a geometric adaptor protein that connects the intramembrane, ring-like polymer of the basal body (14). The position of FlgC in the Gram-negative rod has traditionally been considered cell proximal to FlgF, but a recent publication suggests that the orders may be reversed (25). The order of the rod components in B. subtilis is indicated based on the information in the present manuscript.…”