Fig. 4: T3SS and H1-T6SS show antagonistic behavior on the single-cell level.

A Microscopy analysis of H1-T6SS assembly in LB, non-secreting (LB + 5 mM CaCl₂) and secreting medium (LB + 5 mM EGTA, see Methods for details). Data is shown as a fraction of T6SS-positive bacteria (presence of TssB1-mCherry foci), with exact numbers shown above each bar. Samples were taken from >10 different fields in 3 different experiments. Statistical analysis was done via Student’s t-test, ***, p < 0.001; ns, non-significant. B Microscopy analysis T6SS expression levels in EGFP-SctQ TssB1-mCherry ΔsctW ΔretS bacteria with or without T3SS assembly. Data is shown as background-subtracted and normalized TssB1-mCherry overall fluorescence intensity. Each data point represents the background-corrected TssB1-mCherry overall fluorescence intensity from one cell. Samples were taken from >10 different fields of view from 3 different experiments. Highest and lowest end of each bar represent maximum and minimum value, respectively; boxes indicate median and 25th–75th percentile. Statistical analysis was done via Student’s t-test, ****, p < 0.0001. Microscopy images below show an overlay of the T3SS channel (green) and the H1-T6SS signal (red), as well as the individual channels (left, green; right, red). C Quantification of co-occurrence of T3SS and H1-T6SS in EGFP-SctQ, TssB1-mCherry, ΔretS, ΔsctW cultured in LB medium. Data is shown as a fraction of bacteria with the indicated presence or absence of the T3SS and T6SS, as evaluated by the presence of EGFP-SctQ and TssB1-mCherry foci; the exact fraction for each group is shown at the top, and the total number of cells tested is indicated at the bottom. Inner histograms show the actual ratio of cells with both T3SS and H1-T6SS, and its comparison to the theoretical ratio based on the individual fractions, assuming no correlation. Samples were taken from >50 different fields in 8 different experiments. Statistical analysis was done via χ² test, ***, p < 0.001.