Molecular analysis of OmpR binding sequences involved in the regulation of ompF in Escherichia coli (original) (raw)
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Proceedings of the National Academy of Sciences, 1989
EnvZ and OmpR, the regulatory proteins for ompF and ompC expression in Escherichia coli, belong to a modulator-effector family of regulatory proteins which are essential for the response to environmental signals. We show that the soluble cytoplasmic domain of the transmembrane modulator protein EnvZ is phosphorylated in vitro by [7-32P]-ATP. We also demonstrate that the phosphate group can, in turn, be transferred to the transcription activator protein OmpR. The pH stability properties of the phosphate groups linked to EnvZ indicate that this molecule contains histidyl phosphate. The invariant His-243 of EnvZ corresponds to the phosphorylated His-48 of the chemotactic modulator protein CheA. Substitution of His-243 with valine produces an EnvZ that is refractory to phosphorylation and can no longer catalyze the transfer of phosphate to OmpR. Furthermore, in a AenvZ strain of E. coli, containing the envZ Val-243 plasmid, ompC expression is elevated 7-fold relative to that found in cells carrying the wild-type envZ plasmid. Based on these results we propose a model in which the phosphorylated state of OmpR modulates the expression of the ompF and ompC genes.
Journal of Bacteriology, 1999
Expression of the Escherichia coli OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in Salmonella typhi is not influenced by osmolarity, while OmpF is regulated as in E. coli. To better understand the lack of osmoregulation of OmpC expression in S. typhi, we compared the expression of the ompC gene in S. typhi and E. coli, using ompC-lacZ fusions and outer membrane protein (OMP) electrophoretic profiles. S. typhi ompC expression levels in S. typhi were similar at low and high osmolarity along the growth curve, whereas osmoregulation of E. coli ompC in E. coli was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in S. typhi, while expression of both was regulated by osmolarity in E. coli. Complementation experiments with either the S. typhi or E. coli ompB operon in an S. typhi ⌬ompB strain carrying the ompC-lacZ fusions showed that both S. typhi and E. coli ompC were not regulated by osmolarity when they were under the control of S. typhi ompB. Interestingly, in the same strain, both genes were osmoregulated under E. coli ompB. Surprisingly, in E. coli ⌬ompB, they were both osmoregulated under S. typhi or E. coli ompB. Thus, the lack of osmoregulation of OmpC expression in S. typhi is determined in part by the ompB operon, as well as by other unknown transacting elements present in S. typhi.
Journal of Biological Chemistry, 1991
The transcriptional factors, OmpR and EnvZ, are crucially involved in the osmotic regulation of o m p F and o m p C expression in Escherichia coli. The DNA binding ability of the positive regulator, OmpR, is modulated through its phosphorylation and dephosphorylation mediated by EnvZ in response to the medium osmolarity. In this study, two examples of a novel type of mutant o m p R allele, ompR96A and ompR115S, whose phenotype is OmpF-OmpC-irrespective of the medium osmolarity, were characterized. These mutations result in amino acid conversions, G~u~~ to Ala and Arg''' to Ser, respectively, within the phosphorylation domain of OmpR. Nevertheless, these mutant proteins were capable of undergoing phosphorylation and dephosphorylation normally, just like wild-type OmpR. However, the phosphorylation-dependent enhancement of their in vitro DNA binding ability was found to be severely affected. It was thus revealed that these mutant OmpR represent a novel type in terms of the mechanism of phosphorylation-dependent activation of the function of OmpR, i.e. those are normally phosphorylated but not activated to bind to the cognate promoter DNAs. In this respect, it was further suggested that OmpR oligomerization may be involved in the mechanism underlying the phosphorylation-dependent enhancement of the DNA binding ability of OmpR. The mutant proteins characterized in this study seem to be defective in this particular oligomerization process observed in vitro. Expression of the Escherichia coli outer membrane proteins, OmpF and OmpC, is regulated in response to the medium osmolarity. The transcriptional factors, OmpR and EnvZ, are crucially involved in the osmotic regulation of ompF and ompC expression, namely OmpR is the actual activator, which binds to both the ompF and ompC promoter DNAs, and EnvZ is a transmembrane osmotic sensor, which exhibits both OmpR phosphorylation and dephosphorylation abilities (see Refs. 1-4 for reviews). Recently, it was revealed that phosphotransfer between these two regulatory components, originally observed i n vitro, appears to play a crucial role in signal transduction and the consequent osmotic regulation i n vivo (see Refs. 3 and 4 for review). In this respect, we previously addressed the main question of what the biochemical consequence of OmpR phosphorylation is and demonstrated i n vitro that the phos-* This work was supported by grants from the Ministry of Education, Science and Culture of Japan.
Journal of Bacteriology
Expression of the Escherichia coli OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in Salmonella typhi is not influenced by osmolarity, while OmpF is regulated as in E. coli. To better understand the lack of osmoregulation of OmpC expression in S. typhi, we compared the expression of the ompC gene in S. typhi and E. coli, using ompC-lacZ fusions and outer membrane protein (OMP) electrophoretic profiles. S. typhi ompC expression levels in S. typhi were similar at low and high osmolarity along the growth curve, whereas osmoregulation of E. coli ompC in E. coli was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in S. typhi, while expression of both was regulated by osmolarity in E. coli. Complementation experiments with either the S. typhi or E. coli ompB operon in an S. typhi ⌬ompB strain carrying the ompC-lacZ fusions showed that both S. typhi and E. coli ompC were not regulated by osmolarity when they were under the control of S. typhi ompB. Interestingly, in the same strain, both genes were osmoregulated under E. coli ompB. Surprisingly, in E. coli ⌬ompB, they were both osmoregulated under S. typhi or E. coli ompB. Thus, the lack of osmoregulation of OmpC expression in S. typhi is determined in part by the ompB operon, as well as by other unknown transacting elements present in S. typhi.
Journal of bacteriology, 1999
Expression of the Escherichia coli OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in Salmonella typhi is not influenced by osmolarity, while OmpF is regulated as in E. coli. To better understand the lack of osmoregulation of OmpC expression in S. typhi, we compared the expression of the ompC gene in S. typhi and E. coli, using ompC-lacZ fusions and outer membrane protein (OMP) electrophoretic profiles. S. typhi ompC expression levels in S. typhi were similar at low and high osmolarity along the growth curve, whereas osmoregulation of E. coli ompC in E. coli was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in S. typhi, while expression of both was regulated by osmolarity in E. coli. Complementation experiments with either the S. typhi or E. coli ompB operon in an S. typhi DeltaompB strain carrying the ompC-lacZ fusions showed that both S. typh...
Study the Expression of ompf Gene in Esherichia coli Mutants
Indian Journal of Pharmaceutical Sciences, 2013
The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5’ end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of o...
Journal of Bacteriology, 1990
Integration host factor (IHF) of Escherichia coli is a DNA-binding protein involved in gene expression and other cellular functions in E. coli and some of its bacteriophages and plasmids. We report here that IHF is a direct negative effector of the ompC operon of E. coli. IHF binds to ompC DNA and protects a region of 35 base pairs located upstream from the ompC promoters. The addition of IHF to a purified in vitro transcription system inhibited transcription from two of the three ompC promoters. In vivo experiments suggest that the in vitro results are physiologically relevant. IHF mutants show increased expression of OmpC. In addition, the OmpC- phenotype of certain strains is completely suppressed by a mutation in IHF.