Fig 4. HilD induction activates the stringent response.

(A) HilD-activation results in decreased growth rate and decreased translation rate. Strains were grown in the presence of AnTc as an inducer for HilD production. The expression of pBAD-GFP was achieved by addition of arabinose. The kymographs illustrate a single lineage of HilD-induced or control cells. Images from every second frame from the time-lapse were used to create the shown sequence. (B) Changes in cell length through time as determined using time-lapse microscopy for individual lineages. Traces of a sample of ten lineages are plotted in translucent colour and a sample trace is overlaid in opaque colour for each strain. (C) Probability density function of generation times (left) and GFP translation levels (right) in single cells as determined by time-lapse microscopy. Strains analysed in (A), (B) and (C) were EM12802 (control) and EM12803 (hilD↑). Violin plots represent at least 300 single-cell data values from a representative experiment. hilD↑: strain expressing hilD under an inducible promoter. a.u.: arbitrary units. (D) HilD induction activates the expression of rpoS; a reporter for the stringent response. Fluorescence intensities of a rpoS-mCherry translational fusion were quantified using fluorescence microscopy (left). Individual data points represent the averages of the single-cell values of independent experiments. Violin plots represent data values from at least 700 analysed single cells. Horizontal bars (bold) represent the mean of the calculated average of three independent experiments. The error bars represent the standard error of the mean and statistical significances were determined using a two-tailed Student’s t-test (**, P < 0.01; ns, P > 0.05). Representative microscopy images are shown (right). Scale bar is 2 μm. Strains analysed were EM13017 (control), EM13065 (hilD↑) and EM13276 (hilD↑ ΔSPI-1). hilD↑: strain expressing hilD under an inducible promoter. a.u.: arbitrary units.