HIGH THROUGHPUT IN-SITU IDENTIFICATION OF BACTERIAL PREDATORY-PREY INTERACTIONS IN COMPLEX MICROBIAL ENVIRONMENTS

Bdellovibrio and like organisms (BALOs) are obligatory, small predatory bacteria which prey upon gram-negative bacteria. They are found at an abundance of about 0.1 to 1% in complex bacterial environments. The BALO life cycle includes a free living attack phase (AP) during which the predator searches for and penetrates a prey cell, and a growth phase (GP) during which it grows and divides into new progeny within the prey cell. This study aims at identifying and analyzing microbial predatory dynamics in Waste Water Treatment Plants (WWTP) where complex microbial ecosystems efficiently remove organics, nutrients, toxic substances, and pathogens from wastewater before their release into the environment. Fluorescence in Situ Hybridization (FISH) targeting BALO populations labelled with taxa-specific 16S rRNA probes was combined with fluorescence-activated cell sorting (FACS). Pure cultures experiments with BALOs and prey bacteria showed that AP and GP cells could be discriminated and sorted, revealing that AP cells pass through a maturation stage upon release. In sludge and wastewater liquor samples, FISH-labelled BALOs constituted about 1-3% of the total bacterial load. These populations were sorted, DNA was extracted using multiple displacement amplification (MDA), and the presence of predatory bacteria was validated after sorting. Samples of sorted, unlabeled general populations showing no BALOs signal as well as of controls using antisense probes yielded no amplicons. 16S rRNA MiSeq sequencing of the sorted cells is under way. It should, for the first time reveal the in-situ predatory interaction “pool”, thereby enabling unprecedented analyses of population dynamics.