1887

Abstract

and like organisms (BALOs) are obligate prokaryotic predators of other Gram-negative bacteria. is the most studied organism among BALOs. It has a periplasmic life cycle with two major stages: a motile, non-replicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the Gram-negative prey cell (the growth phase) after forming an osmotically stable body termed the bdelloplast. Within , there are also strains exhibiting an epibiotic life cycle. The genome sequence of the type strain HD100 revealed the presence of multiple dispersed genes encoding type IV pili. Type IV pili in other bacteria are involved in adherence to and invasion of host cells and therefore can be considered to play a role in invasion of prey cells by . In this study, genes involved in producing type IV pili were identified in the periplasmic strain 109J and an epibiotic sp. strain JSS. The presence of fibres on attack-phase cells was confirmed by examining negative stains of cells fixed with 10 % buffered formalin. Fibres were at the non-flagellated pole on approximately 25 % of attack-phase cells. To confirm that these fibres were type IV pili, a truncated form of PilA lacking the first 35 amino acids was designed to facilitate purification of the protein. The truncated PilA fused to a His-tag was overexpressed in BL21(DE3) plysS. The fusion protein, accumulated in the insoluble fraction, was purified under denaturing conditions and used to produce polyclonal antisera. Immunoelectron microscopy showed that polar fibres present on the cell surface of the predator were composed of PilA, the major subunit of type IV pili. Immunofluorescence microscopy showed the presence of pilin on attack-phase cells of 109J during attachment to prey cells and just after penetration, inside the bdelloplast. Antibodies against PilA delayed and inhibited predation in co-cultures of . This study confirms that type IV pili play a role in invasion of prey cells by

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2010-04-01
2019-12-07
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Electron micrographs of negative stains of (A) the width (7.7 nm) of a broken pilus of 109J; (B) prey-independent 109JA showing a broken pilus; (C) an example of pleomorphic prey-independent 109JA cells grown on 1% PY agar. Bars, 0.5 μm. [ PDF] (540 kb) Detection of PilA in 109J by immunofluorescence. Phase-contrast images (left) and epifluorescence images (right). Arrows point to attack-phase cells attached to . (A) Fluorescent signals detected after incubation with anti-PilA antiserum. (B) No fluorescent signals are detected without permeabilization of cells prior to immunostaining. Bars, 10 μm. [ PDF] (458 kb) Growth curves of predators in liquid co-cultures. Predation was determined by assessing the reduction in turbidity of the co-culture over time. Data points represent the mean of three independent experiments. Predation of after pre-incubation with 1:10 diluted anti-PilA antiserum: (A) 109J; (B) HD100; (C) sp. strain JSS. Predation of after pre-incubation with 1:50 diluted anti-PilA antiserum: (D) 109J; (E) HD100; (F) sp. strain JSS. [ PDF] (1107 kb)

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Electron micrographs of negative stains of (A) the width (7.7 nm) of a broken pilus of 109J; (B) prey-independent 109JA showing a broken pilus; (C) an example of pleomorphic prey-independent 109JA cells grown on 1% PY agar. Bars, 0.5 μm. [ PDF] (540 kb) Detection of PilA in 109J by immunofluorescence. Phase-contrast images (left) and epifluorescence images (right). Arrows point to attack-phase cells attached to . (A) Fluorescent signals detected after incubation with anti-PilA antiserum. (B) No fluorescent signals are detected without permeabilization of cells prior to immunostaining. Bars, 10 μm. [ PDF] (458 kb) Growth curves of predators in liquid co-cultures. Predation was determined by assessing the reduction in turbidity of the co-culture over time. Data points represent the mean of three independent experiments. Predation of after pre-incubation with 1:10 diluted anti-PilA antiserum: (A) 109J; (B) HD100; (C) sp. strain JSS. Predation of after pre-incubation with 1:50 diluted anti-PilA antiserum: (D) 109J; (E) HD100; (F) sp. strain JSS. [ PDF] (1107 kb)

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Electron micrographs of negative stains of (A) the width (7.7 nm) of a broken pilus of 109J; (B) prey-independent 109JA showing a broken pilus; (C) an example of pleomorphic prey-independent 109JA cells grown on 1% PY agar. Bars, 0.5 μm. [ PDF] (540 kb) Detection of PilA in 109J by immunofluorescence. Phase-contrast images (left) and epifluorescence images (right). Arrows point to attack-phase cells attached to . (A) Fluorescent signals detected after incubation with anti-PilA antiserum. (B) No fluorescent signals are detected without permeabilization of cells prior to immunostaining. Bars, 10 μm. [ PDF] (458 kb) Growth curves of predators in liquid co-cultures. Predation was determined by assessing the reduction in turbidity of the co-culture over time. Data points represent the mean of three independent experiments. Predation of after pre-incubation with 1:10 diluted anti-PilA antiserum: (A) 109J; (B) HD100; (C) sp. strain JSS. Predation of after pre-incubation with 1:50 diluted anti-PilA antiserum: (D) 109J; (E) HD100; (F) sp. strain JSS. [ PDF] (1107 kb)

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