title: Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae.
abstract: The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.
authors.affiliation: Antimicrobials and Host Defense, GlaxoSmithKline Pharmaceuticals, Collegeville, PA, USA.
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Clausen
authors.forename: Weonhye
authors.initials: W
authors.lastname: Bae
authors.forename: John
authors.initials: J
authors.lastname: Throup
authors.forename: Martin K R
authors.initials: MK
authors.lastname: Burnham
authors.forename: Martin
authors.initials: M
authors.lastname: Rosenberg
authors.forename: Nicola G
authors.initials: NG
authors.lastname: Wallis
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D000071677:Histidine Kinase; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011994:Recombinant Proteins; D015398:Signal Transduction; D013211:Staphylococcus aureus / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; C119939:YycF protein, Bacteria; D011494:Protein Kinases; D000071677:Histidine Kinase