FLAGELLAR FILAMENT OF CAULOBACTER CRESCENTUS: SUBUNIT CHARACTERIZATION AND ORGANIZATION

Abstract

Two flagellins, Fla A (25,000 daltons) and Fla B (27,500), are isolated in a 4:1 ratio from populations of flagella shed into the culture medium by the gram-negative bacterium Caulobacter crescentus. I have purified Fla A and Fla B by ion exchange chromatography and have verified the immunological and compositional similarity of the two proteins. I have also demonstrated their similarity by tryptic peptide mapping, However the differences in the tryptic peptide maps were greater than could be explained by a precursor-product relationship between the two flagellins. Analysis of the amino acid composition of purified tryptic peptides indicated that at least 33 percent of the two proteins contained identical sequences. However about 20 percent of the sequence of the two proteins apparently was homologous except for eight amino acids. Seven of the putative amino acid substitutions can be explained by a single base change in the DNA sequence. These results suggest that C. crescentus has two genes for flagellin and that the two genes arose by duplication of an ancestral sequence.;It was not known whether an individual filament was made up entirely of either Fla A or Fla B or whether both Fla A and Fla B are found in each individual filament. In vitro reaggregation experiments, which are repeated here, showed that monomers of either Fla A or Fla B were capable of assembling into filament-like structures. When I analyzed the flagellin composition of flagellar fragments of different lengths after they were precipitated with hook antibody, I found that the proportion of Fla B in the immune precipitate increased sharply as the length of the filament attached to the hook decreased. A filament about one-fifteenth the full length was composed of 80 percent Fla B. In contrast, the filaments from two linkage groups of non-motile mutants were normally this short but contained over 90 percent Fla A. The tryptic peptide maps of Fla A from these strains were the same as the wild type Fla A map. The following conclusions were drawn: (a) both flagellins are found in each filament, (b) the region of the filament proximal to the hook is highly enriched for Fla B, and (c) the ordered polymerization of Fla B and Fla A may be required for the assembly of a functional filament.