The flagellar filament of Rhodobacter sphaeroides: pH-induced polymorphic transitions and analysis of the fliC gene

Abstract

Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.

FIG. 1

Phase diagram showing the predominant polymorphic forms of isolated R. sphaeroides flagellar filaments in buffers of different pHs and ionic strengths.

FIG. 2

High-intensity dark-field microscope images of typical polymorphic forms of R. sphaeroides flagella. (A) Straight; (B) normal with some open coils; (C) open coils; (D) curly (the inset shows greater magnification of the curly form). The open coils and the normal forms were faint and highly susceptible to Brownian motion, and therefore it was difficult to obtain good micrographs of these from the videotape; some examples are shown.

FIG. 3

Map of the region encoding R. sphaeroides FliC. The putative ς²⁸ promoter region and the site of cloning of the luxCDEAB reporter cassette used in expression studies are shown.

FIG. 4

Prettybox multiple sequence alignment of R. sphaeroides FliC (flic_rsph) with FliCs from enteric and soil bacteria. For reasons of space, much of the variable central region is not shown. The regions predicted to form coiled coils are shown using serovar Typhimurium coordinates (heavy bars) or R. sphaeroides coordinates (dotted line). Accession numbers are as follows: S. meliloti FlaA (flaa_rhime), P13118; S. meliloti FlaB (flab_rhime), P13119; P. aeruginosa FlaA (flaa_pseae), P21184; B. subtilis FliC (flic_bacsu), P02868; serovar Typhimurium FliC (flic_salty), P06179; E. coli FliC (flic_ecoli), P04949.

FIG. 5

Promoter activity of the region upstream from the fliC ORF as indicated by the luxCDEAB reporter system. OD600, optical density at 600 nm.

FIG. 6

Consensus ς²⁸ recognition sequences compared to the putative promoter sequence of R. sphaeroides fliC. The B. subtilis ς²⁸ is also known as ς^D, and that of E. coli is known as ς^F (4, 12, 21).

FIG. 7

Prediction of coiled-coil structures at the termini of flagellin proteins. The predictions were carried out using the COILS program (22) with a window size of 28 residues. Solid line, serovar Typhimurium FliC; dashed line, R. sphaeroides FliC.