Role of type IV pili in predation by Bdellovibrio bacteriovorus

Abstract

Bdellovibrio bacteriovorus, as an obligate predator of Gram-negative bacteria, requires contact with the surface of a prey cell in order to initiate the life cycle. After attachment, the predator penetrates the prey cell outer membrane and enters the periplasmic space. Attack phase cells of B. bacteriovorus have polar Type IV pili that are required for predation. In other bacteria, these pili have the ability to extend and retract via the PilT protein. B. bacteriovorus has two pilT genes, pilT1 and pilT2, that have been implicated in the invasion process. Markerless in-frame deletion mutants were constructed in a prey-independent mutant to assess the role of PilT1 and PilT2 in the life cycle. When predation was assessed using liquid cocultures, all mutants produced bdelloplasts of Escherichia coli. These results demonstrated that PilT1 and PilT2 are not required for invasion of prey cells. Predation of the mutants on biofilms of E. coli was also assessed. Wild type B. bacteriovorus 109JA and the pilT1 mutant decreased the mass of the biofilm to 35.4% and 27.9% respectively. The pilT1pilT2 mutant was able to prey on the biofilm, albeit less efficiently with 50.2% of the biofilm remaining. The pilT2 mutant was unable to disrupt the biofilm, leaving 92.5% of the original biofilm after predation. The lack of PilT2 function may impede the ability of B. bacteriovorus to move in the extracellular polymeric matrix and find a prey cell. The role of Type IV pili in the life cycle of B. bacteriovorus is thus for initial recognition of and attachment to a prey cell in liquid cocultures, and possibly for movement within the matrix of a biofilm.

Figure 1. Axenic growth curve of B. bacteriovorus 109JA and pilT mutants.

Growth in PY medium over a 42 h period. Results shown are an average of 20 replicates repeated in triplicate. Error bars represent standard error.

Figure 2. Effect of mutations in pilT genes on predation by B. bacteriovorus on E. coli.

The decrease in prey cell optical density was used to assess predator growth. Results shown are an average of 20 replicates repeated in triplicate. Controls included: HM Buffer alone, E. coli ML35 prey cells alone and the wild type strain 109JA. Error bars represent standard error.

Figure 3. Biofilm predation assay.

Biofilms of E. coli CO1 were pre-formed for 48 h in 96-well microtiter plates. Predator cultures containing either B. bacteriovorus 109JA or a pilT mutant were added and the plates incubated for a further 24 h. Residual biofilm cells were stained with crystal violet and the optical density at 600 nm (OD600) determined (*p<0.005). To exclude secreted factors contributing to the decrease in remaining biofilm, a 0.45 µm filtrate of a B. bacteriovorus 109JA culture used as a control. The percent biofilm remaining relative to the 109JA filtrate is shown on the secondary axis. Data presented were an average of 12-wells per replicate repeated in triplicate. Statistical significance was measured using a 1-way ANOVA with a Bonferonni corrected post-hoc Students T-test, *p<0.005.

Figure 4. Scanning electron microscopy of predation on pre-formed biofilms of E. coli.

(A) Biofilms of E. coli CO1 (arrows) were formed for 48 h on polyvinyl chloride plastic coverslips. Predator cultures (arrowheads) of (B) B. bacteriovorus 109JA, (C) ΔpilT1, (D) ΔpilT2 (E) ΔpilT1pilT2 were added for a further 24 h.