Fig. 3: C-di-GMP levels are negatively correlated with T3SS activity and positively correlated with H1-T6SS activity on the single-cell level.

A FRET measurement of c-di-GMP in T3SS-negative and -positive populations (T3SS-, T3SS+). Data is shown as corrected FRET efficiency as in Fig. 2, detailed calculations, see “Methods”. The microscopy image below with merged fluorescence channels illustrates the classification of T3SS-positive cells by fluorescence of the P_exoS-mCherry reporter for T3SS activity. B FRET measurement of c-di-GMP in T6SS-negative and -positive populations (T6SS-, T6SS + ). The microscopy image below indicates the classification of T6SS-positive cells by the presence of TssB1-mCherry structures. Cell outlines are indicated by white lines. In both graphs, 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 number of cells tested for each group is indicated below. See Suppl. Fig. 9 for larger fields of view. Statistical analysis by Student’s t -test, ***, p < 0.001; ****, p < 0.0001.