T4 phage bind OMVs, reducing their capacity to infect E. coli. (A) 106 T4 phage were co-incubated with 1 μg purified WT OMVs (106 T4+OMV) for 2 h. As controls, 106 T4, 1 μg of purified WT OMVs, and 105 T4 were also incubated under the same conditions for 2 h. For the 5 min panel, samples were mixed with MK496 cells and allowed to incubate for 5 min, PFU were then determined and compared to the PFU produced by the 106 T4 sample (% PFU Remaining). For the 60 min panel, the phage and WT OMV preparations were incubated for 1 h with mid log-phase MK496 cells, PFU were determined, and compared to the PFU produced by the 106 T4 sample (% PFU Remaining). (n = 9) (B) 106 T4 phage were mixed with 1 μg purified WT OMVs, then immediately ("0" min), and at 5 min intervals thereafter, samples were taken and chloroform was added to disrupt the OMVs and allow reversibly bound phage to be released. The T4 activity in each sample was determined by PFU titration and compared to the PFU produced by 106 T4 (% PFU Remaining). (n = 6) (C) Negative stain electron micrograph of the T4-OMV complex (size bar = 50 nm).