FIGURE 3.

Changes in physiological traits expressed by D. shibae ΔluxI₁ in response to external AHL addition. (A) Flow cytometrical determination of the chromosome content by measuring fluorescence intensities of SybrGreen stained cells. The distribution of log₂ SybrGreen intensity in the cells (A, left side) shows two peaks C_1n (red) and C_2n (blue) that represent one and two chromosome equivalents, respectively. Cells undergoing replication (yellow) are located in between. The distribution of the relative chromosome content in the population until 10 h post-induction is displayed for cultures induced by 3-oxo C14 HSL and the control cultures (A, right side). Asterisks indicate significant differences in the proportion of replicating cells between AHL and DMSO treated samples determined with a two-sided t-test. The experiment has been reproduced four times with three replicates each (Supplementary Figures S7–S10). (B) Second messenger c-di-GMP concentration post-induction with 500 nM 3-oxo C14 HSL. Concentrations in pmol c-di-GMP per mg protein were determined for 6 h post-induction by HPLC. ND, not detectable. (C–E) Western blot detection of the GTA major capsid protein in cell extracts of ΔluxI₁ induced with 3-oxo C14 (C14) or C18 dien HSL (C18) or non-induced as indicated. (C) Cell extracts at 0, 4, 6 h post-induction and after overnight (O/N) cultivation with 3-oxo C14 HSL and of ΔluxI₂ as positive control. (D) Major capsid detection in cell extracts of cultures induced with 500 nM or 2.5 μM AHL and incubated overnight. (E) Induction with 3-oxo C14 or C18 dien HSL.