Bdellovibrio and like organisms (BALOs): fluorescence in situ hybridization (FISH), type IV pili and predation

Bdellovibrio and like organisms (BALOs) are ubiquitous in terrestrial and aquatic environments and have the unusual property of preying on other Gram-negative Bacteria. The life cycle of these predators has two major stages: a free swimming stage spent searching for prey (the attack phase) and a stage spent inside the bdelloplast (the growth phase). There are also strains of Bdellovibrio which exhibit an epibiotic life cycle.;The contribution made by type IV pili genes to predation of Bdellovibrio on prey cells was analyzed. Genes involved in producing type IV pili were identified in the periplasmic strain Bdellovibrio bacteriovorus 109J and an epibiotic Bdellovibrio sp. strain JSS. The presence of polar fibres on attack phase cells was confirmed by examining negative stains of cells fixed with 10% buffered formalin. To confirm that these fibers were type IV pili, an antibody against the major subunit of type IV pili, PilA, was produced. Immunoelectron microscopy showed that polar fibers present on the cell surface of the predator were composed of PilA. Immunofluorescence microscopy confirmed the immunogold studies and showed the presence of PilA on attack phase cells of Bd. bacteriovorus during attachment to prey cells and just after penetration, inside the bdelloplast. The role of PilA in the life cycle of the predator was studied. Antibodies against PilA delayed and inhibited predation in co-cultures of Bd. bacteriovorus 109J, HD100 and Bdellovibrio sp. strain JSS. The pilA gene in Bd. bacteriovorus 109J was inactivated by an in-frame deletion. This knockout mutant was not able to prey. This study confirms that type IV pili play an important role in the life cycle of Bdellovibrio.;By understanding the mechanisms of invasion of Gram-negative bacteria by BALDs, and surveying their habitats, BALDs may be used as bacterial control agents in specific environmental niches.;Key words: Bdellovibrio, predation, fluorescence in situ hybridization (FISH), type IV pili;A culture-independent method for the direct detection and identification of Bdellovibrio in environmental samples was developed. A 16S rRNA oligonucleotide probe, named BDE525, specific for the genus Bdellovibrio, was designed and used in a fluorescence in situ hybridization (FISH) protocol. FISH detected cells in both stages of the life cycle. However, Bdellovibrio was not detected directly by FISH in environmental samples due to the low cell numbers. These results support the observations that BALOs are not dominant populations in natural habitats. Therefore to detect Bdellovibrio in the environment, the enrichment of environmental samples is required.