Characterization of two sets of subpolar flagella in Bradyrhizobium japonicum

Abstract

Bradyrhizobium japonicum is one of the soil bacteria that form nodules on soybean roots. The cell has two sets of flagellar systems, one thick flagellum and a few thin flagella, uniquely growing at subpolar positions. The thick flagellum appears to be semicoiled in morphology, and the thin flagella were in a tight-curly form as observed by dark-field microscopy. Flagellin genes were identified from the amino acid sequence of each flagellin. Flagellar genes for the thick flagellum are scattered into several clusters on the genome, while those genes for the thin flagellum are compactly organized in one cluster. Both types of flagella are powered by proton-driven motors. The swimming propulsion is supplied mainly by the thick flagellum. B. japonicum flagellar systems resemble the polar-lateral flagellar systems of Vibrio species but differ in several aspects.

FIG. 1.

Electron micrographs of B. japonicum wild-type cells. (A) The wild-type cell typically has two types of flagella, a thick flagellum and a few thin flagella, both at the cell subpole. Enlarged images of (B) the thick flagellum, (C) the thin filament, (D) the complex flagellum from Sinorhizobium meliloti, and (E) the sheathed flagellum from V. parahaemolyticus are shown. A part of the sheath is broken, exposing the filament inside. Cells were negatively stained with 1% phosphotungstic acid (pH 7.0). Bars indicate 1 μm (A) and 100 nm (B to E).

FIG. 2.

Genome map of B. japonicum. The total size of the B. japonicum genome is 9,105,828 bp, containing 8,317 genes. The arrows under the genes show the directions of transcription, but the genes may not necessarily form an operon. The dashes among the flagellar genes indicate other genes or open reading frames that are not related to the flagella.

FIG. 3.

Electron microscopic images of flagellar mutants. (A) Mutant BJDΔ283 has a thick flagellum without thin flagella. (B) Mutant BJDΔ293 has thin flagella without a thick flagellum. (C) Double mutant BJDΔ289-Δ293 has no filaments. Bars, 1 μm.

FIG. 4.

SDS-PAGE of flagellins of B. japonicum. The molecular mass of the thick flagellin was 65 kDa, and that of the thin flagellin was 33 kDa. Lane 1, protein marker; lane 2, wild-type strain 110spc4 (both types of flagella exist); lane 3, BJDΔ283 (thin flagella are missing); lane 4, BJDΔ293 (thick flagellum is missing); lane 5, BJDΔ289-Δ293 (both types of flagella are missing).

FIG. 5.

Amino acid analysis of the N-terminal region of the thick flagellin. The first row is the determined sequence from the 33-kDa-band protein. The lower two columns are the deduced sequences of bll6865 flagellin (FliCI) and bll6866 flagellin (FliCII). The amino acids in boldface type give a strong signal in the analysis.

FIG. 6.

Electron microscopic images of flagellar basal structures. The intact flagella attached to the hook-basal body were isolated according to a conventional method (2). The appearances of the hook-basal body of thin flagellum (A) and thick flagellum (B) are similar in terms of the number of rings and their sizes. The “thick” hook looks larger than the “thin” hook. Bars, 100 nm.

FIG. 7.

Colony types of B. japonicum mutants on an agar plate. Cells were inoculated on an agar plate (0.4% agar in nutrient broth) and incubated at 30°C for 3 weeks. BJDΔ293 cells made a swarm ring that was larger than that made by BJDΔ283 cells on the plate.