Abstract
The transcriptional factors, OmpR and EnvZ, are crucially involved in the osmotic regulation of ompF and ompC 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 ompR allele, ompR96A and ompR115S, whose phenotype is OmpF- OmpC- irrespective of the medium osmolarity, were characterized. These mutations result in amino acid conversions, Glu96 to Ala and Arg115 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.
Highlights
The transcriptional factors, OmpR and EnvZ, are phorylation of OmpR results in enhancement of its crucially involved in the osmotic regulation of o m p F DNA binding ability as to bothomthpeF and ompC promoter and o m p C expression in Escherichia coli
Two examples of a novel type of mutant o m p R allele, o m p R 9 6 A and ompR115S, whose phenotype is OmpF- OmpC- irrespective of the medium osmolarity, were characterized. These mutations result in amino acid conversions, G ~ touAla~ an~d Arg’’’ to Ser, respectively, withinthe phosphorylation domain of OmpR
It was revealed that these penedto isolatetwo independent ompR mutants,both of mutant OmpR represent a novel type in terms of the which exhibit an OmpF- OmpC- phenotype irrespective of mechanism opf hosphorylation-dependentactivation of the medium osmolarity
Summary
OF T H E ACTIVATORPROTEIN, OmpR,RESULTSIN A DEFECTINITSPHOSPHORYLATION-DEPENDENTDNABINDING*. Two examples of a novel type of mutant o m p R allele, o m p R 9 6 A and ompR115S, whose phenotype is OmpF- OmpC- irrespective of the medium osmolarity, were characterized These mutations result in amino acid conversions, G ~ touAla~ an~d Arg’’’ to Ser, respectively, withinthe phosphorylation domain of OmpR. Cells carrying the wild-type ompR gene displayed the normal osmoregulatory phenotype as expected (see pMANI 0 4 ) , while amounts of both the OmpF and ompC proteinsproduced in teins were prepared by extraction of cell envelopes with sodium N - lauroyl sarcosinate and analyzed by urea-SDS-polyacrylamide gel electrophoresis as described previously (18).
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