Figure 5. SitA toxins are serially transferred by OME.

(A) Viable cells (CFU) of a target population as a function of inhibitor to target cell ratio quantifies the efficiency of SitA1 and OME delivery. Strains were co-cultured on agar for 48 hr at indicated ratios before determining CFU of the marked (Km^r) target strain. (B) Experimental design to test serial transmissibility of SitA1. The grey cell produces the SitA1 toxin and contains traAB^Mf alleles. The target cells (green) are susceptible, but carry incompatible traAB^DK1622 alleles that preclude OME with inhibitors. Intermediary cells (red) express both traAB alleles. (C–F) Competitive indices of intermediary (red) and target (green) strains from two- and three-strain co-cultures. (G) Three-strain competition when the target strain expresses SitI1. (H) Three strain competition when the intermediary strain is ∆traA. Competition outcomes were determined at 24 hr by fluorescent microscopy. Competitive index was calculated relative to the inhibitor (C–E, G–H) or relative to intermediary strain (F). Starting ratio was 1:5:5 inhibitor to intermediary to target. (I) Serial transfer of the SitA1-mCherry fusion. The left panel shows a 10:1:1 mixture of sitA1-mCherry cells to intermediary to target visualized at 0 and 6 hr. Red arrow indicates a representative example of an intermediary cell that expresses cytoplasmic tdTomato (which does not transfer). Yellow arrows indicate GFP-labeled target cells that have acquired an OM-localized mCherry signal at 6 hr. Boxes represent the number of mCherry positive GFP cells out of 100. Right panel: otherwise identical experiment omitting the intermediary strain. Bar, 5 μm.

DOI: http://dx.doi.org/10.7554/eLife.29397.011

Figure 5—figure supplement 1. TraA is not transferred during OME.

(A) TraA-mCherry fusion protein is functional. Cells expressing TraA-mCherry were mixed with either traA⁺ (top) or ∆traA (bottom) donors that express an OM lipoprotein reporter, SS_OM-sfGFP. Arrows indicate a TraA-mCherry cell that has acquired SS_OM-sfGFP after 6 hr on agar media. White insets indicate the number of recipient cells where transfer was detected from a total of 100 scored recipient cells. (B) TraA-mCherry is not transferred during OME. Top: positive control in which donors that express an OM lipoprotein reporter, SS_OM-mCherry, were mixed with cytoplasmic GFP expressing cells for 24 hr on agar media. Arrows indicate a GFP cells that has acquired the mCherry signal. Bottom: TraA-mCherry expressing cells were mixed with cells expressing cytoplasmic GFP for 24 hr. No mCherry signal was visible in the GFP cells. Bar, 5 μm.

DOI: http://dx.doi.org/10.7554/eLife.29397.012