Dataset Information

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

ABSTRACT: The EnvZ/OmpR 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.

SUBMITTER: Batchelor E

PROVIDER: S-EPMC141058 | biostudies-literature | 2003 Jan

REPOSITORIES: biostudies-literature

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

Batchelor Eric E Goulian Mark M

Proceedings of the National Academy of Sciences of the United States of America 20030109 2

The EnvZ/OmpR 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 c ...[more]

PMID: 12522261

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Project description:This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by serRK, which is believed to control the expression of the ser2003 locus in Bifidobacterium breve UCC2003. The ser2003 locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region of ser2003 and the probable recognition sequence of SerR was determined by a combinatorial approach of in vitro site-directed mutagenesis, coupled to transcriptional fusion and EMSA assays. The importance of the serRK 2CRS in the response of B. breve to protease-mediated induction was confirmed by generating B. breve-s-serR and B. breve-::serU insertion mutants, which exhibited altered ser2003 transcriptional induction patterns as compared to their parent strain UCC2003. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

Dataset Information

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

Publications

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

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