Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB in Staphylococcus aureus
Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB in Staphylococcus aureus
复制标题
DOI：
10.1128/jb.00117-09
复制
发表时间：
2009-07-01
影响因子：
3.2
通讯作者：
Ohlsen, Knut
中科院分区：
文献类型：
--
作者：
Donat, Stefanie;Streker, Karin;Ohlsen, Knut
利用DNA微阵列技术和生物化学方法对金黄色葡萄球菌真核样丝氨酸/苏氨酸蛋白激酶PknB的功能进行了研究。转录谱显示PknB对编码参与嘌呤和嘧啶生物合成、细胞壁代谢、自溶和谷氨酰胺合成的蛋白质的基因的表达具有强烈的调节作用。使用髓鞘碱性蛋白作为替代底物证明了过表达和纯化的PknB激酶的功能活性。磷酸化以时间依赖性方式发生，Mn 2+作为优选的辅因子。此外，生物化学表征显示腺苷酸琥珀酸合成酶（PurA）活性的磷酸化调节。与未磷酸化的PurA相比，磷酸化的PurA的酶活性降低了1.8倍。PknB的缺失导致形成更大的细胞簇，并且pknB缺失菌株对细胞壁活性抗生素衣霉素的敏感性高32倍。本研究结果有力地表明PknB在S.金黄色。
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 Mn2+ 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.