engleski

EXPRESSION AND CHARACTERISATION OF RESPONSE REGULATOR CiaR VARIANTS WITH HIS- OR FLAG-TAGS IN Streptococcus pneumonaie TO IDENTIFY CiaR INTERACTION PARTNERS

In CiaRH two component regulatory system in Streptococcus pneumoniae, under certain conditions, CiaH histidine kinase is not needed for phosphorylation of CiaR response regulator. In that situation, acetyl phosphate AcP is probably alternative donor of phosphate for CiaR. There are some indications that AckA acetate kinase acts negatively on CiaR phosphorylation via AcP. The aim of this work is to detect interactions between CiaR and AckA in vivo. For easier purification, two tagged variants of CiaR (with His and Flag tag) were expressed. CiaR without tag was used as a control. Activity of CiaR variants was determined through measurement of β-galactosidase activity. After pull down of CiaR tagged variants, isolates were tested with Western blot for the presence of AckA. Cross- linking was also performed and CiaR-His variant was isolated. Isolates were tested with Western blot for the presence of AckA. AckA was not detected. Results of this work show that CiaR and AckA do not interact in vivo.

CiaRH two component system, Streptococcus pneumoniae, CiaR response regulator, AckA acetate kinase

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