Fig. 5: Presence of flagella positively correlates with presence of the T3SS, but negatively with H1-T6SS presence and c-di-GMP distribution.

A FRET measurement of c-di-GMP within flagellum on/off populations. Data is shown as corrected FRET efficiency as in Fig. 2, detailed calculations, see “Methods”. Each data point represents a measurement of a single cell. Experiments were repeated on multiple dates, with each color set representing data points from one day. Highest and lowest end of each bar represent maximum and minimum value, respectively; boxes indicate median and 25th–75th percentile. The total cells tested for each group is labeled under the respective bars. Statistical analysis was done via Student’s t -test, ****, p < 0.0001. The microscopy image below indicates flagellum fluorescence label at the cell terminal. Cell outlines are labeled. B Quantification of co-existence of T3SS and flagellum (presence of EGFP-SctQ and FliM-mCherry foci, respectively, bacteria cultured in T3SS-secreting medium). Data is shown as a ratio of cells with indicated status (flagellum on/off and T3SS on/off) to total cells the exact ratio for each group is shown at the top, and the total cell amount tested is marked at the bottom. Inner histograms show the actual ratio of cells with both T3SS and flagellum, and its comparison to the theoretical ratio based on the individual fractions. Representative microscopy image below. Samples were taken from >30 different fields and in 3 different experiments. Statistical analysis was done via χ² test, ****, p < 0.0001. C Quantification of co-existence of H1-T6SS and flagellum (presence of TssB1-Ypet and FliM-mCherry foci, respectively, in a ΔretS background, bacteria cultured in LB medium). Graph structure as in (B). Representative microscopy image below. Samples were taken from >30 different fields and in 5 different experiments. See Suppl. Fig. 9 for larger fields of view. Statistical analysis was done via χ² test, ****, p < 0.0001.