Stimulus-dependent differential regulation in the Escherichia coli PhoQ PhoP system

Abstract

In Escherichia coli, Salmonella, and related bacteria, the PhoQ-PhoP system regulates the expression of a large collection of genes in response to conditions of low magnesium or to the presence of certain antimicrobial peptides. We measured transcription of four PhoP-regulated promoters in E. coli that have significantly different PhoP-binding sites. Surprisingly, three promoters show identical responses to magnesium concentrations that range over four orders of magnitude. By analyzing and testing a simple model of transcriptional regulation, we find an explanation for this puzzle and show that these promoters are indeed differentially regulated at sufficiently high levels of stimulus. We then use this analysis to infer an effective level of phosphorylated PhoP as a function of magnesium stimulus. Our results demonstrate that differential regulation generally depends on the strength of the stimulus and highlight how quantitative analysis of stimulus-response curves can be used to infer properties of cell regulatory circuits that cannot be easily obtained from in vitro measurements.

Fig. 1.

Transcription profiles of four PhoP-regulated promoters in response to extracellular [Mg²⁺]. (a) Each reporter strain contains a chromosomal copy of yfp controlled by a PhoP-regulated promoter, and a copy of cfp, controlled by a constitutive promoter (tetA promoter), at the attachment sites of λ and HK022 phages, respectively. The expression of CFP from the tetA promoter is constant over the range of magnesium concentrations used in our experiments (22). (b) Steady-state transcription of PhoP-regulated promoters as measured by the YFP/CFP fluorescence ratio of single cells. Cultures were grown at 37°C in minimal medium containing the indicated concentrations of MgSO₄. Each point indicates the mean of two independent cultures, and each bar indicates the corresponding range. The dotted lines denote fits to saturating curves. (c) Concentration of magnesium at which YFP/CFP is halfway between the maximal and minimal values, determined for each promoter from the fitted curves in a. Bars denote the errors from the nonlinear fits. (d) Normalized curves from b. Each curve was normalized by shifting and rescaling by constants. For details of the analysis, see SI Text.

Fig. 2.

A comparison of hypothetical PhoP-P binding curves and normalized transcription profiles for three promoters with different PhoP-P dissociation constants. (a) PhoP-P binding curves for three promoters (A, B, and C) with different dissociation constants. P₁ and P₂ denote [PhoP-P] at high and low [Mg²⁺], respectively. In this study, we assume simple binding at these promoters; however, the conclusions of this analysis are the same for the cases of binding by a PhoP-P dimer or of cooperative binding. We also assume in this analysis that the rate of transcription for a particular promoter is a linear function of its fractional occupancy by PhoP-P. (b) The transcription profiles of the promoters in a normalized so that the transcription levels coincide at P₁ and P₂. The promoters with PhoP-P dissociation constants above P₂ (promoters B and C) have normalized transcription profiles that are essentially indistinguishable. In contrast, promoter A, which has a dissociation constant below P₂, has a distinct normalized transcription profile. (c) Fold increase in transcription from P₁ to P₂ for the promoters in a. Note that promoters with different PhoP-P dissociations constants greater than P₂ (promoters B and C) show the same fold increase. For details of the analysis, see SI Text.

Fig. 3.

Relative transcriptional activation for wild-type and mutated promoters. (a) Sequences of the consensus PhoP-box, the PhoP-boxes of the wild-type promoters, and the PhoP-boxes of the mutated promoters. Hybrid 1 consists of the mgtA promoter with the mgrB PhoP-box. Hybrid 2 and hybrid 3 consist of the mgrB promoter with the mgtA PhoP-box and with portions of flanking mgtA sequence, respectively. (b) Fold increase in transcription of wild-type promoters (relative to the fold increase of mgrB transcription) for cells grown in 100 μM Mg²⁺ compared with 10 mM Mg²⁺. Fold increase is defined to be the following: [(YFP/CFP)_{100 μM Mg²⁺} − (YFP/CFP)_{100 μM Mg²⁺,ΔphoQ}]/[YFP/CFP)_{10 mM Mg²⁺} − (YFP/CFP)_{10 mM Mg²⁺,ΔphoQ}]. (c) Fold increase as in b for several mutated promoters listed in a. Two different versions of the mgtA reporter are also compared. UTR1 has the native 5′-UTR for mgtA, which includes a potential riboswitch. UTR2 has a truncated 5′-UTR with only 16 base pairs of the original 263 base pairs in the mgtA 5′-UTR. (d) Fold increase in transcription of various promoters (relative to the fold increase of mgrB transcription) for cells exposed to LL-37 in 100 μM Mg²⁺ for 1 h, compared with cells in 100 μM Mg²⁺ without LL-37. Fold increases were computed by using YFP/CFP values determined from two independent cultures. The bars denote estimates of the error determined from the corresponding ranges of the YFP/CFP measurements.

Fig. 4.

Effective PhoP-P levels and resulting transcription and binding curves, inferred from the model of transcriptional activation described in Fig. 2. (a) Transcription of the mgtA (black diamonds) and phoPQ (gray triangles) promoters for various levels of PhoP_ca expression. (PhoP_ca is a constitutively active variant of PhoP.) Transcription levels corresponding to 100 μM [Mg²⁺] and stimulation with LL-37 are indicated with dotted and dashed lines, respectively. The upper (lower) pair of dotted and dashed lines is for the mgtA (phoPQ) promoter. (b) Transcription of mgrB (squares), mgtA (diamonds), hemL (circles), and phoPQ (triangles) as a function of [PhoP-P]_effective. The filled symbols denote PhoP_ca expression data, and the dotted lines are the associated fits. Note that the PhoP_ca expression data for phoPQ fall on a line by construction. The open symbols denote transcription levels from stimulation with LL-37 in strains with wild-type phoP, which were not used in determining the fits. (c) [PhoP-P]_effective as a function of [Mg²⁺] determined from the transcription data in Fig. 1b for mgtA (diamonds) and mgrB (squares). (d) Inferred [PhoP-P]_effective bound at the mgtA (diamonds) and mgrB (squares) promoters for cells grown in the levels of magnesium shown in Fig. 1b. The [PhoP-P]_effective values are the means of the corresponding values in c. The curves are derived from the fits in b. For a and b, each point is the mean of two independent cultures, and each bar, which is smaller than the data symbol in some cases, denotes the range. For c and d, bars denote the errors associated with the nonlinear fits. For details of the analysis, see SI Text.