E. coli regulates envelope protein composition in response to its environment. Expression of a particular subset of envelope protein genes is affected by three different kinds of manipulations: alteration in medium osmolarity, treatment with the local anesthetic procaine and missense mutations in the regulatory gene envZ. The affected genes are ompF and ompC, specifying outer membrane porin proteins and the genes for two periplasmic proteins, alkaline phosphatase (phoA) and the maltose binding protein (malE). Our hypothesis is that expression of these genes is regulated by a single, environmentally responsive system that is sensitive to osmolarity and procaine and dependent on envZ. This proposal is designed to test this hypothesis and, in doing so, extend our knowledge of the scope and mechanism of osmoregulation. To establish the relationship between osmoregulation and procaine inhibition of gene expression, genes known to be procaine sensitive will be tested for response to osmolarity. These include malE and the mal porin gene, lamB. Additional pho regulon genes will be examined for both properties, especially the PhoE phorin. Osmoregulation of phoE and lamB would support the hypothesis that outer membrane permeability is the cellular property under control by this system. To identify other genes in to osmotic stimulon, cells will be labeled with radioactive amino acids during adaptation to altered osmolarity. The regulatory mechanism will be examined at the genetic and biochemical levels. To determine whether envZ is an osmoregulator gene controlling pho and mal expression, envZ missense and amber mutants will be examined for response to osmolarity, then mutants that are procaine resistant for phoA expression will be isolated and mapped. The structural requirements for procaine control of transcription will be determined by testing a range of drug analogs. The cellular site of the osmosensor and procaine target will be examined first in whole cell experiments and then in an in vitro coupled transcription-translation system. This analysis should ultimately lead to a detailed molecular description of osmoregulation and the interaction between procaine and its receptor in E. coli. These studies would aid in understanding mechanisms for coping with environmental stress and the pharmacologic properties of local anesthetics in eucaryotes.
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