Roles of multiple flagellins in flagellar formation and flagellar growth post bdelloplast lysis in Bdellovibrio bacteriovorus

Abstract

Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5.

Fig. 1

Alignment of terminal regions of B. bacteriovorus multiple flagellins. The first 60 and last 40 amino acids of each flagellin were aligned. Identical amino acids are marked by stars.

Fig. 2

EM images of flagella from flagellin knockout mutants: (a) wild-type 109J, (b) fliC1, (c) fliC2, (d) fliC3, (e) fliC4, (f) fliC5, and (g) fliC6 mutants and (h) a hook–basal body complex in the fliC3 mutant. Bars represent 1 μm except for (h) which is 100 nm. Histograms showing the distribution of flagellar lengths at 24 h of incubation for each strain are shown to the right of EM images. Fluorescent microscope images of (i) Bdellovibrio and E. coli RP437 (both highly motile in this culture) mixed together and stained with FM464 membrane stain. Only the Bdellovibrio (which are attached to the E. coli, preying on them) show flagellar staining, despite the E. coli having many non-sheathed flagella that do not stain. (j) B. bacteriovorus 109J fliC3 mutant cells stained with the FM464, showing disordered membrane bleb at the pole of the cell.

Fig. 2

EM images of flagella from flagellin knockout mutants: (a) wild-type 109J, (b) fliC1, (c) fliC2, (d) fliC3, (e) fliC4, (f) fliC5, and (g) fliC6 mutants and (h) a hook–basal body complex in the fliC3 mutant. Bars represent 1 μm except for (h) which is 100 nm. Histograms showing the distribution of flagellar lengths at 24 h of incubation for each strain are shown to the right of EM images. Fluorescent microscope images of (i) Bdellovibrio and E. coli RP437 (both highly motile in this culture) mixed together and stained with FM464 membrane stain. Only the Bdellovibrio (which are attached to the E. coli, preying on them) show flagellar staining, despite the E. coli having many non-sheathed flagella that do not stain. (j) B. bacteriovorus 109J fliC3 mutant cells stained with the FM464, showing disordered membrane bleb at the pole of the cell.

Fig. 3

One-dimensional SDS gels of flagellin bands from isolated filaments. Lane 1, crude filaments; M, molecular markers; lane 2, purified filaments from wild-type 109J; lane 3, fliC1; lane 4, fliC2; lane 5, fliC4; lane 6, fliC5; lane 7, fliC6 mutants. Thick arrows indicate flagellins, and thin arrows (25 and 36 kDa) indicate contaminants. Lower panels, enlarged images of flagellin bands from upper panel. Three major bands were marked as 29, 30, 31 kDa according to their molecular sizes.

Fig. 4

(a) Two-dimensional gels of flagellins in filament purified from wild-type 109J, fliC1, fliC2, fliC4, fliC5, and fliC6 mutants. Three major spots were labeled as I, II, III, and two small spots as a and b. Missing spots in mutant preparations are marked by dotted circles. (b) 1D (left) and 2D (right) gels of flagellins recovered from spent culture media from the fliC3 mutant. Thick arrows indicate flagellin bands and the dotted arrow indicates a contaminant. (c) Summary of 2D gel analysis of flagellins, FliC1–6.

Fig. 5

EM images of B. bacteriovorus wild-type strain 109J and fliC5 mutant during starvation. (a) Wild-type cells, 24 h, 3 days, and 5 days and (b) fliC5 mutant cells, 24 h, 3 days, and 5 days after start of experiment. Cells were stained with 0.5% uranyl acetate. Scale bars represent 1 μm. (c) Average filament lengths of wild type (shaded bars) and fliC5 mutant (empty bars) during starvation. Error bars show the 95% CI around the mean for each sample. Fifty cells of each sample were analyzed from each of two biological replicates, giving sample sizes of 100 for each data set. (d) 1D gel patterns of flagellin bands from wild type (left) and fliC5 mutant (right).

Fig. 6

RT-PCR using FliC-specific primers and matched total RNA concentrations for B. bacteriovorus wild-type (WT) strain 109J and fliC5 mutant strains cultured as attack-phase motile cells in calcium Hepes buffer for 1, 3, or 5 days after lysis of prey cells. Lanes: M, 100-bp marker (NEB); lane 1, WT day 1; lane 2, WT day 3; lane 3, WT day 5; lane 4, fliC5 day 1; lane 5, fliC5 day 3; lane 6, fliC5 day 5; lane 7, E. coli DFB225 as a negative control; lane 8, no template; lane 9, WT genomic DNA as a positive control (25 cycles of amplification were carried out for fliC1 and fliC2 and 30 cycles for fliC3 to fliC6).

Fig. 7

A cartoon presentation of a filament showing the relative location of each flagellin species.