Bdellovibrio and like organisms (BALOs) are obligate prokaryotic predators of other Gramnegative bacteria. Bdellovibrio bacteriovorus 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 Bdellovibrio, there are also strains exhibiting an epibiotic life cycle. The genome sequence of the type strain B. bacteriovorus HD100T revealed the presence of multiple dispersed pil 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 Bdellovibrio. In this study, genes involved in producing type IV pili were identified in the periplasmic strain B. bacteriovorus 109J and an epibiotic Bdellovibrio sp. strain JSS. The presence of fibres on attackphase 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 Escherichia coli 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 B. bacteriovorus 109J during attachment to prey cells and just after penetration, inside the bdelloplast. Antibodies against PilA delayed and inhibited predation in co-cultures of Bdellovibrio. This study confirms that type IV pili play a role in invasion of prey cells by Bdellovibrio.

A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment.Bdellovibrio and like organisms (BALOs) are predatory gram-negative bacteria that are ubiquitous in terrestrial and aquatic environments. Prey include free-living bacteria as well as plant, animal, and human pathogens (12,13,17). The life cycle of Bdellovibrio has two major stages: a motile, nonreplicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the gram-negative prey cell after the formation of an osmotically stable body termed the bdelloplast (the growth phase) (15,27). Until recently, all bacteria that exhibited these life cycle traits were included in the genus Bdellovibrio. Phylogenetic studies based on 16S rRNA sequences have demonstrated extensive diversity among the organisms in this genus, and two new genera have been established: Bacteriovorax (4) and Peredibacter (8). To date, the genus Bdellovibrio comprises only one species, Bdellovibrio bacteriovorus. However, a Bdellovibrio-like organism (BLO) with a different life cycle was isolated from sewage on lawns of Caulobacter crescentus cells. This epibiotic strain, JSS, does not enter the periplasmic space of the prey cell (20,26). Phylogenetic studies show that it belongs to the genus Bdellovibrio (8) and is a new species (S. F. Koval, unpublished data). Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a commonly used technique for the direct identification of prokaryotes and is widely used to determine bacterial community composition (2, 10). Previously designed BALO-targeted oligonucleotides for use in FISH were not specific enough for the identification of the genus Bdellovibrio, as the targets detected included non-BALOs (8, 16). However, Bdellovibrio-specific primers were used to detect Bdellovibrio sequences in soil (9). In this study, an oligonucleotide probe designated BDE525, targeting a region of the 16S rRNA gene sequence of Bdellovibrio, was designed. The probe was used in a FISH procedure with B. bacteriovorus strains, other BLO isolates, and environmental samples.Cultures. B. bacteriovorus strain 109J was cultured on Escherichia coli ML35 according to the method of Flannagan et al.(11). B. bacteriovorus 6-5-S was cultured on Aquaspirillum serpens VHL and BLO strains JSS and KL1 were cultured on C. crescentus CB2A as described previously by Koval and Hynes (20). BLO strains DM7C, DM8A, and DM11A were cultured on Burkholderia cenocepacia strains grown in Lennox LuriaBertani medium (Difco) for 24 h at 30°C. For the maintenance of predators, 3 ml of prey cells and 1 ml of predators were mixed in 20 ml of HM buffer (3 mM HEPES [Sigma], pH 7.6, with 1 mM CaCl 2 and 0.1 mM MgSO 4 ...

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