(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). In order to reveal the longer-term effects of the presence of OMVs on T4 infectivity in a microenvironment, we observed the infection and reproduction
small molecule library screening of the phage in the mixture following a 1 h incubation with the titer strain. After we co-BGB324 incubated the T4 and OMVs, we added this mixture to growing cultures of the titer strain and incubated for 1 hour instead of only 5 min. This timepoint is sufficient to allow several cycles of infection and allowed us to observe whether the OMVs in the mixture have an affect beyond the initial inactivation. To use as a comparison, we first CHIR98014 purchase determined the amount of free phage (105) that produced the equivalent PFUs to the amount
of infectious phage in the mixture when it was incubated with the titer strain for only 5 min (Figure 5A, 5 min). Then we compared the amount of PFUs formed after a 60 min incubation of cells incubated with 105 T4 or with the mixture of T4 and OMVs. We found that the sample containing the mixture
of T4 and OMVs contained fewer infectious phage as compared to both the original 106 T4 as well as the 105 free T4 samples (Figure 5A, 60 min). This suggests that the addition of OMVs to T4 significantly oxyclozanide reduces the infectivity of T4 over several generations of phage infection. Finally, we used electron microscopy to determine whether complexes between T4 and OMVs could be visualized. We found many complexes between T4 and OMV (an example is shown in Figure 5C), and in these cases, T4 was in a similar orientation as was observed between T4 and bacterial cell wall . These data support the model that released OMVs and vesiculation may contribute to the innate bacterial defense against outer-membrane acting stressors. Discussion Understanding how bacteria manage to survive in hostile environments has been an important step towards understanding bacterial defense and pathogenesis. As our understanding of the bacterial world has increased, so has our appreciation of the complexity of the constant interactions that occur between bacteria and their environment. These include the well-studied interactions that occur between a pathogen and the host environment, as well as the less-appreciated interactions that occur between bacteria and the general environment.