Purification and characterization of the flagellar basal body of Rhodobacter sphaeroides

Abstract

Flagellar hook-basal body (HBB) complexes were purified from Rhodobacter sphaeroides. The HBB was more acid labile but more heat stable than that of Salmonella species, and protein identification revealed that HBB components were expressed only from one of the two sets of flagellar gene clusters on the R. sphaeroides genome, under the heterotrophic growth conditions tested here.

FIG. 1.

Electron micrograph of intact flagella purified by the modified method. Shown are two flagella aligned side by side (A) and a larger image of the basal structure (B). The hook (arrows) is straight. The HAP region (arrowheads) extends beyond the thickness of the filament and hook. Bars, 100 nm.

FIG. 1.

Electron micrograph of intact flagella purified by the modified method. Shown are two flagella aligned side by side (A) and a larger image of the basal structure (B). The hook (arrows) is straight. The HAP region (arrowheads) extends beyond the thickness of the filament and hook. Bars, 100 nm.

FIG. 2.

Electron micrographs of partial structure of HBB. The stability of flagella against acid and heat was tested at pH 3.3 (A), pH 3.2 (B), and 55°C (C). Bars, 100 nm.

FIG. 3.

(A) SDS gel showing the component proteins of HBB purified from R. sphaeroides. Lane 1, Salmonella HBB as molecular mass protein markers (arrows indicate 65, 58, 42, 30, and 27 kDa from top to bottom); lane 2, R. sphaeroides HBB (each band was assigned by apparent molecular size and proteins if identified). (B) Western blotting with anti-FliF antibody. Lane 1, Salmonella HBB was probed with polyclonal anti-R. sphaeroides FliC antibody, and the arrow indicates flagellin; lane 2, R. sphaeroides HBB was reacted with anti-R. sphaeroides FliF antibody, and the arrow indicates FliF. Minor bands are nonspecific.

FIG. 4.

Physical map summarizing the position of R. sphaeroides flagellar genes. Details may be found in the data bank of the Oak Ridge National Laboratory KEGG Analysis for DJGI (http://genome.ornl.gov/microbial/rsph/1/kegg_fc.html).