Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ.

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.

The essential tension: opposed reactions in bacterial two-component regulatory systems.

In many different bacteria several sensory-response functions are controlled by systems of similar design. Most consist of two proteins, one of which regulates the phosphorylation of the other in response to an environmental stimulus. Regulation is achieved by balancing opposed phosphorylation and dephosphorylation reactions against each other. Remarkably, such a system can generate a signal whose strength is independent of the concentration of either component.

Mutations that affect separate functions of OmpR the phosphorylated regulator of porin transcription in Escherichia coli.

OmpR is a member of a family of bacterial transcriptional regulators whose activity is controlled by phosphorylation. It regulates the transcription of two genes, serving as an activator of ompC, and as both an activator and a repressor of ompF. A previously isolated collection of ompR mutations was analyzed for the effect of each on the expression of both genes simultaneously. The results of this analysis indicate that the activation, repression, and DNA binding functions of OmpR can be disrupted independently, and that mutations interfering with each of these functions cluster within the sequence of the OmpR protein. The nature of these mutations is discussed in terms of the mechanisms by which OmpR regulates transcription, and potentially similar mechanisms operating within closely related response regulators.

Direct simulation of yeast 2-microns circle plasmid amplification.

The 2-microns circle is a plasmid found in most strains of the yeast Saccharomyces cerevisiae at approximately 60-100 copies per cell. The plasmid possesses the novel capacity for replicative amplification induced by site-specific recombination. To address the question of whether the recombination model is adequate to account for observed rates of 2-microns circle amplification, we developed a direct computational simulation of the amplification system. Results of this simulation show that theoretically at least six copies per plasmid can be produced in each generation, and that previously unanticipated replication intermediates contribute largely to this degree of amplification.

Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.

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.

EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregulation of the porin genes.

Osmoregulation of the bacterial porin genes ompF and ompC is controlled by a two-component regulatory system. EnvZ, the sensor component of this system, is capable both of phosphorylating and dephosphorylating OmpR, the effector component. Mutations were isolated in envZ that abolish the expression of both porin genes. These mutants appear to have lost the kinase activity of EnvZ while retaining their phosphatase activity, so that in their presence OmpR is completely unphosphorylated. The behavior of these mutants in haploid, and in diploid with other envZ alleles, is consistent with a model in which EnvZ mediates osmoregulation by controlling the concentration of a single species. OmpR-P.