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EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in Escherichia coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K+ P−) and the OmpR-P phosphatase-dominant state (K− P+). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.

We have isolated mutations in rpoA, the gene encoding the a 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 a subunit. Several of the rpoA mutations cluster in the carboxy-terminal portion of the a protein, further suggesting that it is this domain of a that is involved in interaction with OmpR and perhaps other transcriptional regulators as well.Several different mechanisms have been proposed to explain how regulatory proteins function to catalyze transcription initiation (1). A common feature of many of these models is a direct physical interaction between the activator protein and the transcription apparatus. Evidence supporting such an interaction has been presented for several prokaryotic regulatory proteins. CRP, the cyclic AMP (cAMP) receptor protein, is an activator of many genes and operons and controls expression of lac, for example, by facilitating the binding of RNA polymerase (23). cI, A repressor, regulates its own expression by enhancing open complex formation at PRM (17,34). In this case as well, binding studies suggest a direct interaction (18). NR1 (NtrC) is the effector of the two-component regulatory system that stimulates expression of the genes of the nitrogen regulon. It activates the glnAp2 promoter, for example, via a mechanism that involves DNA looping (40). A complete understanding of transcriptional regulation requires a more detailed description of these activator-RNA polymerase interactions. In the case of cI, genetic studies suggest a region of the repressor molecule that is involved in contacting RNA polymerase(6). However, in all cases, little is known about the oppos-ing contact region of the polymerase. We have sought to address this question in our studies of the two-component system that regulates porin gene expression in Escherichia coli.The two-component regulatory system, OmpR and EnvZ, controls the differential expression of the porin genes, ompF and ompC, in response to medium osmolarity. EnvZ is a receptor kinase that functions as a sensor of osmolarity and communicates this information to the effector, OmpR (an NR1 homolog), by a mechanism involving phosphorylation and dephosphorylation. OmpR-P controls transcription initiation by binding to sites upstream of the appropriate promoters (19). We have shown previously that OmpR works in a positive fashion at the ompC promoter and both positively and negatively at the ompF promoter (37, 38). In media of low osmolarity, levels of OmpR-P are low and * Corresponding author. ompF expression is activated. In media of high osmolarity, levels of OmpR-P increase, ompC expression is activated, and ompF expression is repressed (11). Mutations in envZ that result in increas...

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