Genetic fusions as experimental tools.
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Cloning, Molecular↗
Biomedical subjects
J M Slauch
Publications and source records attributed to J M Slauch.
cis-acting ompF mutations that result in OmpR-dependent constitutive expression.
OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.
Bacterial Outer Membrane Proteins↗
Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase.
We have isolated mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that specifically affect transcriptional control by OmpR and EnvZ, the two-component regulatory system that controls porin gene expression in Escherichia coli. Characterization of these mutations and a previously isolated rpoA allele suggests that both positive and negative regulation of porin gene transcription involves a direct interaction between OmpR and RNA polymerase through the alpha subunit. Several of the rpoA mutations cluster in the carboxy-terminal portion of the alpha protein, further suggesting that it is this domain of alpha that is involved in interaction with OmpR and perhaps other transcriptional regulators as well.
Alleles↗
Signal transduction in bacteria: kinases that control gene expression.
A new paradigm, termed two-component regulatory systems, is emerging from the study of signal transduction in bacteria. A simple example of such a system is provided by the Omp regulon of Escherichia coli. This regulon, which controls the expression of the major outer membrane porin proteins OmpF and OmpC in response to changes in osmolarity, includes the inner membrane protein EnvZ (a receptor kinase) and the DNA-binding protein OmpR (a transcriptional activator). Although we do not know what "ligand" is sensed in the Omp system, we can trace the signal transduction pathway from the receptor at the cell surface directly to regulatory sequences within the DNA. Perhaps signal transduction in bacteria can serve as a simple archetype for understanding certain functions performed by receptor kinases and phosphorylated DNA-binding proteins in higher organisms.
Bacterial Outer Membrane Proteins↗
Genetic analysis of the switch that controls porin gene expression in Escherichia coli K-12.
The two-component regulatory system, OmpR and EnvZ, in Escherichia coli controls the differential expression of ompF and ompC in response to medium osmolarity. Previous studies suggest that EnvZ functions as a membrane sensor relaying information to the DNA-binding protein, OmpR, which in turn activates expression of the appropriate promoter. A strategy has been devised to isolate and characterize a collection of missense mutations in ompR that alter, but do not abolish protein function. Mutants were isolated using strains that contain the ompR and envZ genes in separate chromosomal locations yet maintain the production of both regulatory proteins at physiological levels. Such an arrangement facilitates ompR diploid analysis and tests of epistasis with known envZ mutations. The data obtained indicate that OmpR works in both a positive and negative fashion to control the transcription of ompF and this result forms the basis of a model for porin regulation that explains the switch from OmpF to OmpC production in response to increasing medium osmolarity.
Bacterial Outer Membrane Proteins↗
EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12.
The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.
Bacterial Outer Membrane Proteins↗
Differences in regulatory sequences of naturally occurring JC virus variants.
The regulatory region was sequenced for DNAs representative of seven independent isolates of JC virus, the probable agent of progressive multifocal leukoencephalopathy. The isolates included an oncogenic variant (MAD-4), an antigenic variant (MAD-11), and two different isolates derived from the urine (MAD-7) and from the brain (MAD-8) of the same patient. The representative DNAs were molecularly cloned directly from diseased brain tissue and from human fetal glial cells infected with the corresponding isolated viruses. The regulatory sequences of these DNAs were compared with those of the prototype isolate, MAD-1, sequenced previously (R. J. Frisque, J. Virol. 46:170-176, 1983). We found that the regulatory region of JC viral DNA is highly variable due to complex alterations of the previously described 98-base-pair repeat of MAD-1 DNA. On the basis of these alterations, there are two general types of JC virus. There were no consistent alterations in regulatory sequences which could distinguish brain tissue DNAs from tissue culture DNAs. Furthermore, for each isolate except MAD-1 (R. J. Frisque, J. Virol. 46:170-176, 1983), the regulatory regions of brain tissue and tissue culture DNAs were not identical. The arrangement, sequence, or both of potential regulatory elements (TATA sequence, GGGXGGPuPu, tandem repeats) of JC viral DNAs are sufficiently different from those in other viral and eucaryotic systems that they may effect the unique properties of this slow virus.
Animals↗