Activation of the P fnrS promoter was measured in vivo. (a) Initial in vitro profiling of the P fnrs promoter was performed by flow cytometry. Aerobic (+O 2 , left histogram) and anaerobic (-O 2 , right histogram) cultures were analyzed with a negative control strain of EcN without GFP (red), a positive control strain expressing GFP constitutively (blue), and a strain expressing GFP under control of the P fnrS promoter (green). As expected, the negative control showed no detectable GFP signal in either aerobic or anaerobic conditions, the positive control strain showed equivalent GFP expression in either aerobic or anaerobic conditions, and the P fnrS -GFP fusion strain only expressed GFP under anaerobic conditions (Note: While the constitutive control was expressed from the chromosome, P fnrS -GFP was expressed from a plasmid, which may have led to more variable and higher level expression observed in the historgrams). This experiment was repeated independently with similar results (b) The aerobically grown strains analyzed in (a) were dosed orally to C57BL/6 mice and recovered 4h post-dose by flushing excised cecal tissue with PBS As demonstrated in (a), the P fnrS -GFP fusion strain was off at the time of dosing. Cecal effluents were analyzed by flow cytometry. For each plot, 40,000 counted events are shown. The y-axis represents the SSC and the x-axis the GFP channel. Plot 1 shows analysis of the background and autofluorescence of cecal effluent in a mouse that did not receive cells. Plot 2 shows analysis of cecal effluent from a mouse dosed with control EcN (no GFP), and no fluorescence is observed. Plot 3 shows analysis of cecal effluent after dosing with EcN expressing GFP constitutively, and a GFP fluorescent population is observed and gated on. This gate was applied to all plots. Plot 4 shows analysis of cecal effluent after dosing with EcN containing a P fnrS -GFP fusion that were OFF at the time of dosing. GFP fluorescence was observed in cecal effluent (increased abundance of events counted in the GFP-positive gate). When normalized to CFU counts recovered from the effluent and by subtracting cecal effluent background (Plot 1), the gated populations account for ~76 % (constitutive GFP control) and ~64 % (P fnrS -GFP) of the cells recovered by plating. Though considerable variability exists in CFU counting, especially from biological samples, the conclusion is that a majority of the P fnrS -controlled cells appear to enter the “ON” state post-dosing. This experiment was repeated independently with similar results