Mutations that Affect Separate Functions of OmpR the Phosphorylated Regulator of Porin Transcription in Escherichia coli

Russo, Frank D.; Slauch, James M.; Silhavy, Thomas J.

doi:10.1006/jmbi.1993.1281

Cited by 75 publications

(68 citation statements)

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“…Sequence Changes in the Linker Affect OmpR Phosphorylation and Dephosphorylation by the Kinase EnvZ-Since two previously identified point mutants (G129D and P131S) of the OmpR linker region were OmpF ϩ OmpC Ϫ , it was of interest that the GGK linker substitution also resulted in this phenotype (17,18). To characterize the OmpF ϩ OmpC Ϫ phenotype observed in OmpR mutants with a defective linker, we expressed and purified OmpRG129D, OmpRP131S, and OmpRGGK for in vitro analysis.…”

Section: Changes In the Sequence Of The Linker Region Of Ompr Results mentioning

confidence: 99%

“…Binding reactions were prepared, DNase I-treated, and run on gels as described (22). (17,18,28). To test this hypothesis more directly, we constructed ompR mutants containing non-native amino acids in place of the endogenous linker sequence.…”

Section: Methodsmentioning

confidence: 99%

“…These authors suggested that the Q-linkers serve the simple role of tethering the two domains to allow functional interactions to occur. However, point mutants have been isolated in the linker region of both OmpR and the homologous regulator DmsR that disrupt protein function (17)(18)(19). It was therefore of interest to determine whether specific amino acids were required for interdomain communication of OmpR.…”

mentioning

confidence: 99%

“…As such, although the linker sequences are conserved, it is impossible to determine whether this has functional significance, as the entire protein varies by only 1-8 residues. Among more divergent species such as Xenorhabdus nematophilus, Vibrio cholerae, and Pseudomonas aeruginosa, it appears that a central PGAP sequence (residues 128 -131 in OmpR) is more highly conserved than the rest of the linker; this is where the previously isolated point mutants are located (17)(18)(19).…”

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confidence: 99%

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The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

Mattison¹,

Oropeza²,

Kenney

2002

Journal of Biological Chemistry

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OmpR is the response regulator of a two-component regulatory system that controls the expression of the porin genes ompF and ompC in Escherichia coli. This regulator consists of two domains joined by a flexible linker region. The amino-terminal domain is phosphorylated by the sensor kinase EnvZ, and the carboxylterminal domain binds DNA via a winged helix-turn-helix motif. In vitro studies have shown that amino-terminal phosphorylation enhances the DNA binding affinity of OmpR and, conversely, that DNA binding by the carboxyl terminus increases OmpR phosphorylation. In the present work, we demonstrate that the linker region contributes to this communication between the two domains of OmpR. Changing the specific amino acid composition of the linker alters OmpR function, as does increasing or decreasing its length. Three linker mutants give rise to an OmpF ؉ OmpC ؊ phenotype, but the defects are not due to a shared molecular mechanism. Currently, functional homology between response regulators is predicted based on similarities in the amino and carboxylterminal domains. The results presented here indicate that linker length and composition should also be considered. Furthermore, classification of response regulators in the same subfamily does not necessarily imply that they share a common response mechanism.

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Section: Changes In the Sequence Of The Linker Region Of Ompr Results mentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

Mattison¹,

Oropeza²,

Kenney

2002

Journal of Biological Chemistry

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“…For example, envZ mutations were created by hydroxylamine (Tokishita and Mizuno, 1994) or nitrosoguanadine mutagenesis (Russo et al, 1993) or by UV irradiation (Hsing et al, 1998). Another study isolated missense mutations in envZ that were better able than wildtype E. coli MG1655 to colonize the streptomycin-treated mouse (Adediran et al, 2014;Leatham-Jensen et al, 2012).…”

Section: Tm Domains Modulate Envz Activity But Are Not Essentialmentioning

confidence: 99%

Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

Foo

Gao

Zhang

et al. 2015

Progress in Biophysics and Molecular Biology

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Two-component regulatory systems drive signal transduction in bacteria. The simplest of these employs a membrane sensor kinase and a cytoplasmic response regulator. Environmental sensing is typically coupled to gene regulation. The histidine kinase EnvZ and its cognate response regulator OmpR regulate expression of outer membrane proteins (porins) in response to osmotic stress. We used hydrogen:deuterium exchange mass spectrometry to identify conformational changes in the cytoplasmic domain of EnvZ (EnvZc) that were associated with osmosensing. The osmosensor localized to a seventeen amino acid region of the four-helix bundle of the cytoplasmic domain and flanked the His 243 autophosphorylation site. High osmolality increased autophosphorylation of His 243 , suggesting that these two events were linked. The transmembrane domains were not required for osmosensing, but mutants in the transmembrane domains altered EnvZ activity. A photoactivatable fusion protein composed of EnvZc fused to the fluorophore mEos2 (EnvZc-mEos2) was as capable as EnvZc in supporting OmpR-dependent ompF and ompC transcription. Over-expression of EnvZc reduced activity, indicating that the EnvZ/OmpR system is not robust. Our results support a model in which osmolytes stabilize helix one in the four-helix bundle of EnvZ by increased hydrogen bonding of the peptide backbone, increasing autophosphorylation and downstream signaling. The likelihood that additional histidine kinases use similar cytoplasmic sensing mechanisms is discussed.

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PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains.

1993

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The SecY protein of Escherichia coli and its homologues in other organisms, are integral components of the cellular protein translocation machinery. Suppressor mutations that alter SecY (the prlA alleles) broaden the specificity of this machinery and allow secretion of precursor proteins with defective signal sequences. Twenty‐five prlA alleles have been characterized. These suppressor mutations were found to cluster in regions corresponding to three distinct topological domains of SecY. Based on the nature and position of the prlA mutations, we propose that transmembrane domain 7 of SecY functions in signal sequence recognition. Results suggest that this interaction may involve a right‐handed supercoil of alpha‐helices. Suppressor mutations that alter this domain appear to prevent signal sequence recognition, and this novel mechanism of suppression suggests a proofreading function for SecY. We propose that suppressor mutations that alter a second domain of SecY, transmembrane helix 10, also affect this proof‐reading function, but indirectly. Based on the synthetic phenotypes exhibited by double mutants, we propose that these mutations strengthen the interaction with another component of the translocation machinery, SecE. Suppressor mutations were also found to cluster in a region corresponding to an amino‐terminal periplasmic domain. Possible explanations for this unexpected finding are discussed.

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Mutations that Affect Separate Functions of OmpR the Phosphorylated Regulator of Porin Transcription in Escherichia coli

Cited by 75 publications

References 0 publications

The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains.

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