A Single-Cell View of the BtsSR/YpdAB Pyruvate Sensing Network in Escherichia coli and Its Biological Relevance

Abstract

Fluctuating environments and individual physiological diversity force bacteria to constantly adapt and optimize the uptake of substrates. We focus here on two very similar two-component systems (TCSs) of Escherichia coli belonging to the LytS/LytTR family: BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB. Both TCSs respond to extracellular pyruvate, albeit with different affinities, typically during postexponential growth, and each system regulates expression of a single transporter gene, yjiY and yhjX, respectively. To obtain insights into the biological significance of these TCSs, we analyzed the activation of the target promoters at the single-cell level. We found unimodal cell-to-cell variability; however, the degree of variance was strongly influenced by the available nutrients and differed between the two TCSs. We hypothesized that activation of either of the TCSs helps individual cells to replenish carbon resources. To test this hypothesis, we compared wild-type cells with the btsSR ypdAB mutant under two metabolically modulated conditions: protein overproduction and persister formation. Although all wild-type cells were able to overproduce green fluorescent protein (GFP), about half of the btsSR ypdAB population was unable to overexpress GFP. Moreover, the percentage of persister cells, which tolerate antibiotic stress, was significantly lower in the wild-type cells than in the btsSR ypdAB population. Hence, we suggest that the BtsS/BtsR and YpdA/YpdB network contributes to a balancing of the physiological state of all cells within a population.IMPORTANCE Histidine kinase/response regulator (HK/RR) systems enable bacteria to respond to environmental and physiological fluctuations. Escherichia coli and other members of the Enterobacteriaceae possess two similar LytS/LytTR-type HK/RRs, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which form a functional network. Both systems are activated in response to external pyruvate, typically when cells face overflow metabolism during post-exponential growth. Single-cell analysis of the activation of their respective target genes yjiY and yhjX revealed cell-to-cell variability, and the range of variation was strongly influenced by externally available nutrients. Based on the phenotypic characterization of a btsSR ypdAB mutant compared to the parental strain, we suggest that this TCS network supports an optimization of the physiological state of the individuals within the population.

Keywords: histidine kinase; nutrient limitation; overflow metabolism; persister cells.

FIG 1

Model of the nutrient-sensing BtsS/BtsR and YpdA/YpdB network in E. coli. The scheme summarizes the signal transduction cascades triggered by the BtsS/BtsR and YpdA/YpdB systems and the influence of other regulatory elements. Activating (→) and inhibitory (⊦) effects are indicated. PP, periplasm; CM, cytoplasmic membrane; CP, cytoplasm. See the text for details.

FIG 2

Single-cell analysis of P_yhjX and P_yjiY activation during growth in LB medium. E. coli cells expressing gfp under the control of the yhjX or yjiY promoter, respectively, were grown in LB medium, and fluorescence micrographs were taken before (exponential growth phase) and after activation (post-exponential growth phase) of the two TCSs. Representative fluorescence and phase-contrast images of P_yhjX-gfp and P_yjiY-gfp reporter strains are shown in panels A and C, respectively. The corresponding distributions of the fluorescence intensity of the P_yhjX-gfp and P_yjiY-gfp reporter strains are depicted in panels B and D. Unfilled bars refer to values prior to activation, and filled bars refer to values observed after activation. Dashed lines represent the threshold of activation for each of the reporter strains. A total of 200 cells were analyzed in each experiment, and frequency refers to the percentage of cells with the indicated intensity (see Materials and Methods for details). The continuous curves represent Gaussian fits based on the histograms of the fluorescence intensity. PH, phase contrast; AU, arbitrary units. Scale bar, 2 μm. Experiments were performed independently three times.

FIG 3

Effects of different external pyruvate concentrations on P_yhjX-gfp activation at the single-cell level. E. coli cells expressing gfp under the control of the P_yhjX promoter were grown in M9 minimal medium containing increasing concentrations of pyruvate (supplemented with succinate; final carbon concentration, 20 mM) and analyzed by fluorescence microscopy. A total of 200 cells was analyzed in each experiment at the time point of maximal expression, and frequency refers to the percentage of cells with the indicated intensity (see Materials and Methods). Histograms of the fluorescence intensities of cells were fitted using a Gaussian distribution (solid line). The dashed line represents the threshold of activation for the reporter strain. AU, arbitrary units. Experiments were performed independently three times.

FIG 4

Effects of different external pyruvate concentrations on P_yjiY-gfp activation at the single-cell level. E. coli cells expressing gfp under the control of the P_yjiY promoter were grown in a nutrient-poor environment (0.1× LB medium) for 1 h. The medium was then supplemented with 20 mM pyruvate (A) or with increasing pyruvate concentrations (B), and the cells were subsequently analyzed by fluorescence microscopy. A total of 200 cells were analyzed for each experiment, and frequency is represented as a percentage of the cells (refer to Materials and Methods for a detailed explanation). Histograms of the fluorescence intensities of cells were fitted using a Gaussian distribution (solid line). Dashed lines represent the threshold of activation for the reporter strain. AU, arbitrary units. Experiments were performed three independent times. For further details, see the legends to Fig. 2 and 3.

FIG 5

In the absence of the BtsSR/YpdAB network, rrnB P1 promoter activity is low and bistable. Wild-type E. coli MG1655 (blue) or mutant btsSR ypdAB (red) cells harboring a chromosomally encoded rrnB P1-gfp fusion were grown in LB medium and examined by fluorescence microscopy. For further details, see the legends to Fig. 2 and 3. A total of 200 cells were analyzed for each experiment at the post-exponential growth phase, and frequency is represented as a percentage of the cells (refer to Materials and Methods for detailed explanation). Histograms of the fluorescence intensities of cells were fitted using a Gaussian distribution (solid line). AU, arbitrary units. Experiments were performed three independent times.

FIG 6

The BtsSR/YpdAB network promotes homogeneous protein overproduction in all cells. Wild-type (WT) or btsSR ypdAB mutant cells harboring the overproduction vector pBAD24-gfp were grown in LB medium. Samples were taken before and after the addition of the inducer arabinose (Ara) (0.2% [wt/vol]). The cells were analyzed by fluorescence microscopy and flow cytometry. Distributions of fluorescent cell counts and representative views of WT cells before and after addition of arabinose are shown in panels A and B, while the corresponding data for the btsSR ypdAB mutant are depicted in panels C and D. About 2,000 events were recorded for each plot. Cell counts represent the numbers of cells, and fluorescence intensity is expressed in arbitrary units (AU). Scale bar, 2 μm. Experiments were performed independently three times.

FIG 7

The BtsSR/YpdAB network reduces the proportion of persister cells in populations. Either WT (blue lines) or mutant btsSR ypdAB (red lines) cells were grown in LB medium. Before (exponential growth phase) (A) and after (B) activation (post-exponential growth phase) of the systems, the cells were exposed to ampicillin (200 μg/ml). Samples were taken and analyzed for CFU. Three independent experiments were performed, and error bars indicate the standard deviations of the means.