Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus

Abstract

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.

Fig. 1

Electron micrograph of B. bacteriovorus wild-type strain 109J inside a bdelloplast of a small E. coli DFB225 flagellar minus cell upon which it is preying, 20 min after predators were added to prey. The Bdellovibrio has modified the cell wall of the prey, which has rounded up, and it is attached to the cytoplasmic membrane of the prey, consuming its cytoplasm. The flagellum of the Bdellovibrio is still clearly visible protruding from the bdelloplast, 1% PTA stain. Bar = 1 µm.

Fig. 2

A. Arrangement of fliC genes in both HD100 and 109J genomes, and sites used to insert an interrupting Kanamycin resistance cassette for construction of each fliC mutant strain. B. Reverse transcription PCR using fliC-specific primers on total RNA from B. bacteriovorus 109J predatory culture. Products are seen for all fliC genes. L: 100 bp DNA ladder; 1–6: fliC1–fliC6 each with no reverse transcriptase negative control in the right-side lane labelled b. Further control reactions of no template were also carried out and gave no products. C. Quantitative PCR for expression by wild-type B. bacteriovorus 109J of fliCs 2, 3 and 5, normalized by comparison with rnpO expression. Expression levels are shown for mRNA prepared from free-swimming, attack-phase Bdellovibrio, with no prey, and for Bdellovibrio during infection of prey, after 15 min (attachment and early invasion), after 1 h (establishment and growth within the bdelloplast) and after 4 h (lysis of the bdelloplast and release of attack-phase cells).

Fig. 3

Morphologies of flagella from wild type and fliC mutant strains derived from B. bacteriovorus 109J. A. Electron micrograph of B. bacteriovorus 109JK, a wild-type kanamycin-resistant strain, showing wild-type flagellar length and waveform. 5 k magnification, 1% PTA stain. B. 109J fliC6 strain; inactivation of this flagellin gene had no appreciable effect on the waveform or length of the flagellum. This is representative of strains with inactivated fliC1, fliC2 or fliC4, which also had the same visible phenotypes in 109J. 5 k magnification, 1% PTA stain. C. 109J fliC5 strain. Inactivation of this flagellin resulted in shorter flagella, with the absence of the distal miniature waveform, and the strain also showed reduced mean swimming speed and reduced predation efficiencies compared with wild-type. 5 k magnification, 1% PTA stain. D. Host-independent 109J fliC 3 strain. Deletion of fliC3 caused the absence of a flagellum and a non-motile phenotype but a disordered flagellar sheath was still seen. 7 k magnification, 1% PTA. stain. Bars = 1 µm. E. Host-independent 109J HIK3 strain showing typical, normal sheathed flagella and the diversity of cell size and shape typical for an HI strain. 7 k magnification, 1% PTA. stain. Bars = 1 µm.

Fig. 4

A. SDS-PAGE of sheared flagella/flagellar sheath preparations, mechanically removed from the surface of B. bacteriovorus fliC3 HI mutant (lane 1) and control ‘wild-type’ HIK3 (lane 2) strains. L is BenchMark protein ladder. B. Semi-quantitative reverse transcription PCR to demonstrate approximate fliC expression levels using fliC-specific primers and 25 cycles of amplification on total RNA from B. bacteriovorus fliC3 host-independent culture and HIK3: a 109JK-derived host-independent culture that is wild-type for flagella. Products are seen for all fliC genes with the exception of fliC3 in the fliC3 mutant strain. Lanes: L, 100 bp DNA ladder; 1–6: fliC1–fliC6 primer pairs, respectively, in the HI fliC3 mutant strain; 7–12: fliC1–fliC6 primer pairs, respectively, in the HIK3 flagellate ‘wild type’ strain.

Fig. 5

Spherical bdelloplasts and rod-shaped hosts picked from solid agar overlays containing YFP-labelled E. coli S17-1:pZMR100 hosts incubated with Bdellovibrio HI fliC3 mutant strain. A. A typical view including a spherical bdelloplast and a HI fliC3 cell attached to an E. coli prey cell. B. A gallery of rounded, yellow, and therefore definitively E. coli-derived, bdelloplasts are seen, which contain a growing black coencytic HI fliC3 mutant Bdellovibrio, indicating that this fliC3 strain, although lacking a functional flagellum, will still predate when placed in close proximity to prey on a surface.