EnvZ, an osmosensor histidine kinase in Eschericia coli, senses medium osmolarity and regulates the phosphorylation state of its own cognate response regulator, OmpR. OmpR functions as a transcription factor regulating the expression of the outer membrane porins OmpF and OmpC. EnvZ is composed of a periplasmic sensor domain, a transmembrane domain and a cytoplasmic signaling domain. The cytoplasmic domain contains linker and enzymatic subdomains A and B, which catalyze autophosphorylation, OmpR phosphoryl transfer and dephosphorylation. Therefore, the linker is considered to play an important role in transducing signals from the periplasm to the catalytic domains of EnvZ.

The EnvZ linker mutation F220D resulted in increasing the OmpR-P concentration when the cytoplasmic part was attached to the transmembrane domain. However, if the cytoplasmic EnvZ domain was cleaved from the transmembrane region, no phenotype for F220D was observed (mutated EnvZ behaved like the wild-type EnvZ), possibly due to loss of the native structure of the linker domain.

Therefore, to study the effect of linker mutations on the EnvZ structure in vitro, we created a chimeric cytoplasmic EnvZ containing fully folded archea A. fulgidus linker (Hulko et al, 2006, Cell 126, 929-940). Using site-specific mutagenesis, we introduced an I319D mutation (homologous to F220D) into the chimeric EnvZ and tested the biochemical properties of the recombinant protein. Phenotype of enzymatic activities of mutated HAB I319D was kinase+/phosphatase-; this phenotype is consistent with in vivo EnvZ linker mutation F220D.

This work allowed us to test whether the chimeric protein could be used to study the mechanism of signal transduction through the linker. Therefore, this approach using chimeric protein could be used to study the effect of linker mutations in enzymatic activities of EnvZ in vitro.