Circuit-level input integration in bacterial gene regulation

Abstract

Gene regulatory circuits can receive multiple simultaneous inputs, which can enter the system through different locations. It is thus necessary to establish how these genetic circuits integrate multiple inputs as a function of their relative entry points. Here, we use the dynamic circuit regulating competence for DNA uptake in Bacillus subtilis as a model system to investigate this issue. Specifically, we map the response of single cells in vivo to a combination of (i) a chemical signal controlling the constitutive expression of key competence genes, and (ii) a genetic perturbation in the form of copy number variation of one of these genes, which mimics the level of stress signals sensed by the bacteria. Quantitative time-lapse fluorescence microscopy shows that a variety of dynamical behaviors can be reached by the combination of the two inputs. Additionally, the integration depends strongly on the relative locations where the two perturbations enter the circuit. Specifically, when the two inputs act upon different circuit elements, their integration generates novel dynamical behavior, whereas inputs affecting the same element do not. An in silico bidimensional bifurcation analysis of a mathematical model of the circuit offers good quantitative agreement with the experimental observations, and sheds light on the dynamical mechanisms leading to the different integrated responses exhibited by the gene regulatory circuit.

Fig. 1.

Dynamical features of genetic competence. (A) Typical filmstrip of a cell undergoing two consecutive competence events, as labeled by the activity of the comG promoter fused to cfp (in red in the figure). The comS promoter activity, fused to yfp, is shown in green. (B) Time traces of comG and comS fluorescence levels for the cell highlighted in A. The conditions for this experiment are those of Fig. 5H below. See Materials and Methods and SI Materials and Methods, Growth Conditions for Microscopy, for details of the microscopy procedures used.

Fig. 2.

Scheme of the circuit underlying genetic competence in B. subtilis. The two types of inputs whose integration is considered in this paper are also shown.

Fig. 3.

Statistical analysis of competence dynamics in the presence of two inputs. The initiation (A, D), exit (B, E), and reinitiation (C, F) probabilities are plotted as the constitutive expressions of ComS (upper row) and ComK (lower row) are varied. Three different copy numbers of the natural ComS gene are considered in both cases: 1 (black), 6.5 (red), and 75 (green). The error bars are calculated via the standard deviation of the means taken in different movies.

Fig. 4.

Phase diagrams of the competence circuit. The upper row corresponds to the joint variation of the constitutive expression of ComS, , and the ComS copy number, , whereas in the lower row, the constitutive expression of ComK, , is varied together with . The symbols represent the experimental observations, with green circles corresponding to excitable dynamics (here defined by and ), blue squares to bistable behavior ( if : a fraction of the cells turn on competence and stay there, representative of spatial heterogeneity between two stable states; or if : most cells turn on competence and come back, representative of temporal switching between two stable states), black triangles to monostable competence ( and ), and red diamonds to oscillatory dynamics (). Lines represent bifurcation boundaries of a deterministic mathematical model of the competence circuit (see text), as computed with the numerical continuation software AUTO through XPP; solid lines correspond to saddle-node bifurcations and dashed lines to Hopf bifurcations. The symbols and lines are the same among the three columns, which differ in the quantities plotted in grayscale, obtained from discrete simulations of the competence circuit: (A and D), (B and E), and (C and F). Parameters of the deterministic model are , , , , , , , , and . Parameters of the discrete model are given in Table S6.

Fig. 5.

Dynamical phenotypes arising from input integration. The four upper panels correspond to joint variation of the ComS copy number (increasing vertically toward the right) and the ComS constitutive expression level (increasing horizontally toward the right): (A) , ; (B) , ; (C) , ; and (D) , . The four lower panels correspond to joint variation of the ComS copy number (increasing vertically toward the right) and the ComK constitutive expression level (increasing horizontally toward the right): (E) , ; (F) , ; (G) , ; and (H) , . In each panel, the left plot shows a collection of single-cell time traces of CFP levels quantifying P_comG activity, and thus acting as ComK reporter. Color coding corresponds to the one used in Fig. 4, and a particular time trace is highlighted with a thicker line. The right plots show histograms of CFP levels as measured from a typical frame in each movie analyzed, and the Insets display selected snapshots from these movies.

Fig. 6.

Schematic summary of the two integration modes observed experimentally. Background colors have the same meaning as in Figs. 4 and 5. Additionally, green cells denote vegetative cells, red cells represent functional competence, and white cells denote sporulation.