Self-sensing in Bacillus subtilis quorum-sensing systems

Abstract

Bacterial cell-cell signalling, or quorum sensing, is characterized by the secretion and groupwide detection of small diffusible signal molecules called autoinducers. This mechanism allows cells to coordinate their behaviour in a density-dependent manner. A quorum-sensing cell may directly respond to the autoinducers it produces in a cell-autonomous and quorum-independent manner, but the strength of this self-sensing effect and its impact on bacterial physiology are unclear. Here, we explore the existence and impact of self-sensing in the Bacillus subtilis ComQXP and Rap-Phr quorum-sensing systems. By comparing the quorum-sensing response of autoinducer-secreting and non-secreting cells in co-culture, we find that secreting cells consistently show a stronger response than non-secreting cells. Combining genetic and quantitative analyses, we demonstrate this effect to be a direct result of self-sensing and rule out an indirect regulatory effect of the autoinducer production genes on response sensitivity. In addition, self-sensing in the ComQXP system affects persistence to antibiotic treatment. Together, these findings indicate the existence of self-sensing in the two most common designs of quorum-sensing systems of Gram-positive bacteria.

Figure 1. In co-culture, ComX-secreting cells have a stronger quorum-sensing response than non-secreting cells.

(a) Quorum- and self-sensing can be decoupled by co-culturing fluorescently distinguishable secreting and non-secreting strains, both encoding for a fluorescent reporter of quorum sensing response. Secreting and non-secreting strains are correspondingly denoted by a red and blue colors throughout the paper. (b) ComX-secreting (ΔQ^kanR;Q⁺) and non-secreting (ΔQ^kanR) P_srfA-YFP reporter expression, measured simultaneously at different optical densities during co-culture, by introducing distinguishing fluorescent markers into the strains (Supplementary Fig. 4). (c) Quorum-sensing response ratios of ComX- secreting and non-secreting variants in co-culture as a function of YFP fluorescence of the non-secreting strain. Shown are results for the overexpressed system (dark green) and physiologically expressed system (light green). Additionally, results are shown for a control co-culture comprised of a pair of wild-type strains (gray). (d) Maximal response ratio (mean±50^th percentile expected variation) for six co-cultures of ComQXP variants, which differ in the presence or absence of the comQ and comX genes, as described in the table. Maximal values are calculated by interpolation from response profiles presented in Supplementary Fig. 6. Conditioned medium from a ComX-producing E. coli strain was added to co-cultures of two non-secreting strains. Asterisk mark results which are statistically different from a ratio of one (strict quorum-sensing null hypothesis). Results in (c) were taken from ≥4 biological repeats for each co-culture pair. In (c,d), each co-culture pair was measured over ≥5 time points at different optical densities (All data is given in Supplementary File 1).

Figure 2. The cell autonomous effect of ComX-secretion fits a self-sensing model with no over-reception.

(a,b) Schemes of the self-sensing (a) and over-reception (b) models and the expected difference between the responses of secreting and non-secreting strains to an external autoinducer under the two models. (c,d) YFP expression of each strain during co-culture, as a function of the relative volume of ComX-conditioned medium added (methods). (c) ComX-secreting (ΔQ^kanR;Q⁺, red) and Non-secreting (ΔQ^kanR, blue). (d) Non-secreting strains with High (ΔQ^kanR, dark blue) and low (P^ind, light blue) ComP levels. (e) Optimal slope ratio and x-offset between co-cultured strains, under a model that allows the two parameters to vary independently. Shown is the fit for the ΔQ^kanR;Q⁺ (ComX-secreting) and ΔQ^kanR (non-secreting) pair (purple, n=8) and for the ΔQ^kanR (High ComP levels) and P^ind (Low ComP levels) pair (green, n=6). Average ± St. Err. marks are shown for each pair of strains in a darker tone. Each point in (e) arises from a biological repeat of a series of measurements as shown in (c,d). Experiments were repeated over multiple days. All data points are given in Supplementary File 1.

Figure 3. Self-sensing contributes to antibiotic persistence.

The relative fitness of co-cultured strains is plotted as a function of the P_srfA-YFP expression of genotype #1 prior to administration of antibiotics (see legend). Relative fitness was calculated as the ratio of relative frequency of the strains at the end of the experiment to that prior to the addition of antibiotics [19]. Shown are results for two differentially marked P^ind;Q⁺X⁺-secreting strains (gray), and between the physiological ComX-secreting (P^ind;Q⁺X⁺) and the corresponding non-secreting (P^ind) strains (green). A value of one (dashed line) indicates no change in frequency. Each data point represents a separate measurement. Data was collected over ≥3 different optical densities in each series of experiments. Experiments were repeated multiple times over ≥3 days for each co-culture type. All data points are given in Supplementary File 1.

Figure 4. Self-sensing in also apparent in the Rap-Phr system.

(a) Co-culture response ratio of PhrP secreting (rapP⁺;phrP⁺) and non-secreting (rapP⁺) strains as a function of YFP response of the latter strain. Shown are results with (dark green) or without (light green) the addition of conditioned medium collected from a ComX-producing E. coli. Response ratio of a control co-culture of two phrP⁺ secreting strains is also shown (gray). Each data point represents a separate measurement. Series of experiments over ≥3 varying optical densities were repeated ≥3 times, on different days. Line at response ratio of 1 represents the null hypothesis of no cell-autonomous response. (b) Fluorescence levels (mean ± st. err.) of a P_srfA-YFP reporter integrated into PhrP secreting (red) and non-secreting (blue) strains. Response was measured at very low densities with ComX conditioned medium added to the culture (methods). The response is measured for pure cultures of the two strains and in co-culture. Also shown is the auto-flurescence of a similarly measured wild-type with no YFP reporter (gray). Note that auto-fluorescence here is larger than that measured in Supplementary Fig. 7, due to the addition of conditioned medium. Asterisks and N.S. mark statistically significant and non-significant differences accordingly. All data points are given in Supplementary File 1.