Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system

Abstract

The EnvZ͞OmpR system in Escherichia coli, which regulates the expression of the porins OmpF and OmpC, is one of the simplest and best-characterized examples of two-component signaling. Like many other histidine kinases, EnvZ is bifunctional; it phosphorylates and dephosphorylates the response regulator OmpR. We have analyzed a mathematical model of the EnvZ-mediated cycle of OmpR phosphorylation and dephosphorylation. The model predicts that when EnvZ is much less abundant than OmpR, as is the case in E. coli, the steady-state level of phosphorylated OmpR (OmpR-P) is insensitive to variations in the concentration of EnvZ. The model also predicts that the level of OmpR-P is insensitive to variations in the concentration of OmpR when the OmpR concentration is sufficiently high. To test these predictions, we have perturbed the porin regulatory circuit in E. coli by varying the expression levels of EnvZ and OmpR. We have constructed two-color fluorescent reporter strains in which ompF and ompC transcription can be easily measured in the same culture. Using these strains we have shown that, consistent with the predictions of our model, the transcription of ompC and ompF is indeed robust or insensitive to a wide range of expression levels of both EnvZ and OmpR.A mong prokaryotes, a remarkable number of cellular functions are controlled by two-component regulatory systems. Dedicated circuits transduce and interpret specific signals such as pH, temperature, osmolarity, light, nutrients, ions, pheromones, and toxins to regulate a wide range of processes including motility, virulence, metabolism, the cell cycle, developmental switches, antibiotic resistance, and stress responses (for reviews see for example refs. 1-4). Most of the detailed features of these circuits are quite varied and depend on the particular signals that are detected and the responses that are effected. However, all twocomponent systems transmit information via phosphorylation of a histidine on one protein (a histidine kinase) followed by transfer of phosphate to an aspartic acid, which is usually on a distinct protein (a response regulator). A notable feature of many of these systems is that the histidine kinase is bifunctional: it phosphorylates and dephosphorylates its cognate response regulator. This feature raises the interesting question of what is gained by implementing a regulatory circuit in this manner. Would a circuit in which, for example, phosphorylation is controlled by the histidine kinase and dephosphorylation is caused by spontaneous hydrolysis or a separate phosphatase have important differences from a circuit in which both reactions are mediated by the same enzyme?A particularly well-characterized example of a bifunctional histidine kinase is EnvZ. In Escherichia coli, EnvZ responds to changes in the extracellular osmolarity of inner-membrane impermeable compounds and controls the phosphorylation of the response regulator OmpR. OmpR, in turn, regulates the transcription of a number of genes, the best characterized of which are ompF a...

Integrative model of the response of yeast to osmotic shock

“…Fps1 closed to allow for glycerol accumulation. The metabolic response of the cell was coupled to the osmotic response (volume and pressure changes) by considering glycerol as the osmotically active compound entering equation (5). All concentrations were linked via their relative changes to the variation of the cell volume, V os , which depends on osmotic alterations.…”

“…Previously, quantitative computational simulation proved to support understanding of regulatory systems [5][6][7][8][9][10] , characterizing threshold properties and bistability 11,12 , characteristic times 13,14 and feedback effects 12,[15][16][17][18][19] . The model presented here comprises receptor stimulation, the HOG signaling pathway, activation of gene expression, adaptation of cellular metabolism, glycerol accumulation and a thermodynamic description of the control of volume and osmotic pressure.…”

Continuous Control in Bacterial Regulatory Circuits

“…1c and d). For the porin osmoregulatory system, cfp and yfp were integrated downstream of ompC and ompF, respectively, at the wild-type loci (6). Similarly, for the tetracycline resistance system cfp was integrated into the chromosome downstream of tetA (in a strain containing a chromosomal copy of tetR and tetA), and yfp was integrated downstream of ompA, which codes for an outer membrane structural protein.…”

“…This removes 537 bp from the middle of ompC. The resulting plasmid was then used to construct EPB16 [MC4100 ⌽(⌬ompC-cfp ϩ ) ⌽(ompF ϩ -yfp ϩ )] by homologous recombination as described previously (6). To construct an in-frame deletion in ompF, the primers 5Ј-TGAGGGTAATAAA TAATGATGAAGCGCAATATTCTGGTGTAGGCTGGAGCTGCTTC-3Ј and 5Ј-CTGGTAAACGATACCCACAGCAACGGTGTCGTCTGACATATG AATATCCTCCTTAG-3Ј were used to amplify the chloramphenicol resistance cassette from pKD3 (12).…”

“…The strains and plasmids used in this study are listed in Table 1. MDG147 is a fluorescent reporter strain derived from MG1655 (4) that contains chromosomal operon fusions of cfp with ompC and yfp with ompF and was constructed in a manner similar to that for MDG131 (6). To create an in-frame deletion in ompC, the plasmid pMG35 (6) was digested with PshAI and religated.…”