Bacterial sugar utilization gives rise to distinct single-cell behaviours

Abstract

Inducible utilization pathways reflect widespread microbial strategies to uptake and consume sugars from the environment. Despite their broad importance and extensive characterization, little is known how these pathways naturally respond to their inducing sugar in individual cells. Here, we performed single-cell analyses to probe the behaviour of representative pathways in the model bacterium Escherichia coli. We observed diverse single-cell behaviours, including uniform responses (d-lactose, d-galactose, N-acetylglucosamine, N-acetylneuraminic acid), 'all-or-none' responses (d-xylose, l-rhamnose) and complex combinations thereof (l-arabinose, d-gluconate). Mathematical modelling and probing of genetically modified pathways revealed that the simple framework underlying these pathways - inducible transport and inducible catabolism - could give rise to most of these behaviours. Sugar catabolism was also an important feature, as disruption of catabolism eliminated tunable induction as well as enhanced memory of previous conditions. For instance, disruption of catabolism in pathways that respond to endogenously synthesized sugars led to full pathway induction even in the absence of exogenous sugar. Our findings demonstrate the remarkable flexibility of this simple biological framework, with direct implications for environmental adaptation and the engineering of synthetic utilization pathways as titratable expression systems and for metabolic engineering.

Figure 1

Inducible sugar utilization. (A) Inducible utilization pathways are commonly composed of a set of transporters and catabolic enzymes whose expression is induced in the presence of the recognized sugar. (B) The pathways harbor a combination of positive (green plus) and negative (red dash) feedback. Positive feedback emerges from the sugar inducing the expression of the transporters, leading to additional import of the sugar. Negative feedback emerges from the sugar inducing the expression of the enzymes, leading to breakdown of the sugar.

Figure 2

Varying single-cell responses to different sugars in E. coli. MG1655 cells harboring a transcriptional reporter plasmid were grown in M9 glycerol with varying concentrations of the indicated sugar for 20 hours in exponential phase to a low cell density and then subjected to flow cytometry analysis. The promoter used to drive GFP expression is specified. Each dot represents the mean fluorescence and the relative number of cells in the induced (black) and uninduced (white) subpopulations (see Figure S1A). Only one dot is shown for unimodal distributions. The diameter of each dot scales with the fraction of cells in that population. Each dot plot is representative of at least three biological replicates conducted on separate days.

Figure 3

A simple model predicts varying responses from coupling inducible transport and inducible catabolism. (A) Major reactions considered by the deterministic model. Extracellular sugar (S₀) is imported into the cell by the transporter (T). The intracellular sugar (S) can reversibly bind to the sensory activator or can undergo irreversible degradation by the enzyme (E). The activator bound to the sugar (R) upregulates the expression of the transporter and the enzyme. See Supplementary Information for more details on the model. (B) Phase diagram based on the maximal activities of the fully expressed transporter (α) and enzyme (γ). See Table S3 for values of fixed parameters (with v = 0). Monostable regions represent parameter values in which the model predicts one stable enzyme level for any extracellular sugar concentration at steady-state. Bistable regions represent parameter values in which the model predicts two stable and one unstable enzyme levels for at least one extracellular sugar concentration at steady-state. The dashed blue line represents parameters that achieve 95% of the maximal dynamic range, where the dynamic range is defined as the ratio of enzyme levels for saturating extracellular sugar and no sugar. Proceeding to the right of this line further increases the dynamic range. (C) Bifurcation curves representing the steady-state relationship between enzyme levels and extracellular sugar concentration for a given set of parameter values. Different curves are shown whereby the only altered parameter value is the maximal enzyme activity (γ). Dashed red lines indicate steady-state enzyme levels that are unstable. (D) Bifurcation curves whereby the only altered parameter value is the maximal transporter activity (α). See Supplementary Information for more details about the model.

Figure 4

Single-cell responses in the absence of sugar catabolism. The specific genes deleted from each pathway and the promoter driving GFP expression are indicated. Note that the lactose utilization pathway was not evaluated because its behavior was previously probed in the absence of sugar catabolism through the use of the non-hydrolyzable inducer TMG (Ozbudak et al., 2004). The D-gluconate utilization pathway was also not evaluated because of redundancy in D-gluconate catabolism. See Figure 2 for the experimental growth conditions and an explanation of the dot plots. All dot plots are representative of at least three biological replicates conducted on separate days.

Figure 5

Impact of endogenous biosynthesis of the sugar on the predicted response. (A) The model was modified to allow for constitutive biosynthesis of the intracellular sugar (ν, yellow arrow). (B) Bifurcation curves for the predicted enzyme levels (E) in the presence of sugar catabolism (γ = 0.1) for different rates of endogenous sugar production. See Figure 2 for more information about the bifurcation curves. (C) Predicted ratio of enzyme levels without (γ = 0) or with (γ > 0) sugar catabolism in the absence of exogenous sugar (S₀ = 0). The ratio peaks at intermediate rates of endogenous sugar biosynthesis and then begins to decline as enzyme levels approach full induction (E = 1) even in the presence of sugar catabolism. All plots represent α = 0.1 and all other parameter values are reported in Table S3. See Supplementary Information for more details about the model.

Figure 6

Extent of hysteresis for the D-xylose and L-arabinose utilization pathways with intact or disabled sugar catabolism. Overnight cultures of wild type cells (WT) or cells lacking the catabolic genes (ΔxylAB or ΔaraBAD) were incubated with or without sufficient sugar to induce the entire population (1 mM of D-xylose or 100 μM of L-arabinose for WT, 0.1 mM of D-xylose for ΔxylAB, 10 μM of L-arabinose for ΔaraBAD). The cultures were then washed and grown in M9 glycerol with the indicated concentration of sugar for 20 hours. See Figure 2 for an explanation of the dot plots. The gray regions represent the extent of hysteresis based on the extrapolated sugar concentration required to induce ~50% of the population. All dot plots are representative of at least three biological replicates conducted on separate days.