Recombinant Cytosolic Truncations of Histidine Kinases Retain Function for Targeted In Vitro Investigations

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Histidine kinases are crucial for bacterial two-component systems (TCSs), regulating resistance and virulence.
• Their membrane-bound nature hinders isolation and in vitro studies, limiting research on signal transduction.
• Current research often focuses on response regulators, neglecting histidine kinases.

Purpose of the Study:

• To develop a straightforward method for generating soluble cytosolic truncation mutants of histidine kinases.
• To ensure these mutants retain essential autophosphorylation and phosphotransfer functions.
• To validate the utility of these mutants for in vitro biochemical techniques and drug discovery.

Main Methods:

• Generation of cytosolic truncation mutants of histidine kinases.
• Biochemical assays to confirm autophosphorylation and phosphotransfer capabilities.
• Production of functional mutants from diverse bacterial species including *Escherichia coli* and *Klebsiella pneumoniae*.

Main Results:

• Successfully generated functional cytosolic truncation mutants of histidine kinases.
• Demonstrated that these mutants retain autophosphorylation and phosphotransfer activities.
• Confirmed the applicability of this method across different bacterial species and histidine kinases (PhoQ, BasS, PmrB).

Conclusions:

• Soluble histidine kinase mutants are viable tools for in vitro studies.
• This approach overcomes solubility issues, facilitating research into bacterial signal transduction.
• The developed method supports inhibitor screening and broadens the understanding of TCSs for drug discovery.