Signal Transduction and Gene Regulation in Bacteria

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The E.coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signaling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and invitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) in TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF^-/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibting a wild-type phenotype of OmpF^+/OmpC^+, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and in vitro catlytic activities caused by EnvZ(P41S), through an intermolecular complementation. These results are best interpreted as meaning that EnvZ molecules function an an oligomer (most likely a dimer), and that an intimate intermolecular interaction between the membrane-spanning segments is crucial fro transmembrane signaling in response to and external osmotic stimulus.