title: Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression.
abstract: Effects of changes in intracellular ion concentrations on the interactions of Escherichia coli lac repressor with lac operator mutants and on the interactions of RNA polymerase with various promoters have been investigated in vivo. The intracellular ionic environment was reproducibly varied by changing the osmolality of the 4-morpholinepropanesulfonic acid minimal growth medium. As the osmolality of the growth medium is varied from 0.1 to 1.1 osmolal, the total intracellular concentration of K+ increases linearly from 0.23 +/- 0.03 to 0.93 +/- 0.05 molal and the total intracellular concentration of glutamate increases linearly from 0.03 +/- 0.01 to 0.26 +/- 0.02 molal. The sum of the changes in the total concentrations of these two ions appears sufficient to compensate for a given change in external osmolality, indicating that K+ and glutamate are the primary ionic osmolytes under these conditions and that these ions are free in the cytoplasm. In support of this, in vivo 39K NMR experiments as a function of external osmolality indicate that changes in the total cytoplasmic K+ concentration correspond to changes in the free cytoplasmic K+ concentration. Extents of interaction of lac repressor and RNA polymerase with their specific DNA sites were monitored by measuring the amounts of beta-galactosidase produced under the control of these sites. For both lac repressor and RNA polymerase, it was found that formation of functional protein-DNA complexes in vivo is only weakly (if at all) dependent on intracellular ion concentration. These results contrast strongly with those obtained on these systems in vitro, which showed that both the equilibria and kinetics of binding are extremely salt-dependent. We discuss several possible mechanisms by which E. coli may compensate for the potentially disruptive effects of these large changes in the intracellular ionic environment.
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007763:Lac Operon; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase; D011188:Potassium