FIG 6.

Transwell cultures demonstrate that C. acetobutylicum-ZapA-FAST and C. ljungdahlii-DeepRed do not exchange proteins when physically separated. Comparison of transwell unseparated versus separated cocultures (and monocultures of) between C. acetobutylicum-ZapA-FAST and wild-type C. ljungdahlii labeled with the CellTracker Deep Red dye over 20 h. The 100-nm membranes separating the two organisms allowed exchange of small molecules, but prevented C. acetobutylicum and C. ljungdahlii cells from physically interacting. In unseparated coculture, C. ljungdahlii and C. acetobutylicum cells interacted and exchanged fluorescent material, producing double-positive (labeled) cells. In separated coculture, no double-positive (labeled) cells developed. The Deep Red dye was fully retained in C. ljungdahlii cells and did not leak out over time, and C. acetobutylicum cells remained green without any red contamination. Pure Red C. ljungdahlii or C. acetobutylicum-ZapA-FAST control monocultures retained the red or green color throughout the time course, respectively. The values in parentheses represent normalized fluorescent populations in each sample, determined by dividing each fluorescent percentage by the total fluorescent population, i.e., the sum of the green, red, and double-positive cells. Green C. acetobutylicum-ZapA-FAST cells were not 100% labeled from the beginning, as has been documented (23). Quadrangle gates were determined using pure green- and red-fluorescing cells (Fig. S7).