Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB in Staphylococcus aureus.
The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn(2+) as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.
Universität Würzburg, Institut für Molekulare Infektionsbiologie, Würzburg, Germany.
Stefanie
S
Donat
Karin
K
Streker
Tanja
T
Schirmeister
Sonja
S
Rakette
Thilo
T
Stehle
Manuel
M
Liebeke
Michael
M
Lalk
Knut
K
Ohlsen
D000265:Adenylosuccinate Synthase / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008345:Manganese / Q000378:metabolism; D018625:Microscopy, Atomic Force; D009154:Mutation; D004676:Myelin Basic Protein / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004676:Myelin Basic Protein; D014415:Tunicamycin; D008345:Manganese; D017346:Protein Serine-Threonine Kinases; D000265:Adenylosuccinate Synthase