Bdellovibrio attachment assay protocol

Attachment assays were performed according to Milner et al.^{20 (link)} with slight modifications. HI and HI flp mutants were grown in PYE medium to an OD₆₀₀ of 0.5, washed twice in DNB, and mixed with E. coli. The experiments were carried out at the final cell concentrations of 10⁶ and of 10⁷ ml⁻¹. The Bdellovibrio - E. coli co-culture was incubated for recovery of 30 min in 28 °C, then concentrated by low speed centrifugation (4000 rpm for 10 min), the pellet was re-suspended in fresh DNB and re-incubated in 28 °C. 10 µl were taken at different time points, placed on positively charged polylysine-coated adhesive microscope slide (X-tra slides, Leica) and examined under phase contrast microscopy. As a negative control, E. coli was replaced with Bacillus subtilis, a gram-positive bacterium that Bdellovibrio does not attach to.