He Kuang, Dragnea Vladimira, Bauer Carl E
Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA.
Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA
mBio. 2015 May 5;6(3):e00546-15. doi: 10.1128/mBio.00546-15.
Rhodospirillum centenum forms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che3 signaling cascade. The Che3 cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3 and CheR3, two CheW3 linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3 cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3 does not serve as a phosphor donor for CheY3; instead, CheA3 inhibits a CheS3→CheY3 two-component system by phosphorylating an inhibitory receiver domain of CheS3. In this study, we show that in addition to phosphorylation by CheA3, the phosphorylation state of CheS3 is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occur in vivo upon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicated in vitro, the phosphorylation level of CheS3 is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3 phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3.
Upon starvation, Rhodospirillum centenum undergoes a developmental process that forms metabolically dormant cysts, which withstand desiccation and nutritional limitation. This study explores the role of the cellular energy state as measured by the ratio of ATP to ADP as an important regulator of cyst formation in Rhodospirillum centenum. We show that R. centenum cells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When this in vivo level of energy starvation is simulated in vitro, CheS3 phosphorylation is reduced by 75%. This profound reduction in CheS3 autophosphorylation is contrasted with a much lower 25% decrease in CheA3 phosphorylation in response to a similar downward shift in ATP/(ATP + ADP). We argue that even though adenylate energy affects all ATP-dependent enzymes to an extent, the enhanced inhibition of CheS3 activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.
在诸如干燥或营养饥饿等不利生长条件下，百脉根红螺菌会形成代谢休眠的孢囊。孢囊的发育受到严格调控，涉及一种名为Che3信号级联的抑制孢囊形成的趋化样信号转导途径。Che3级联由一个甲基化学感受器（MCP3）、受体甲基化/去甲基化蛋白CheB3和CheR3、两个CheW3连接蛋白、一个CheA3-CheY杂合组氨酸激酶以及一个单结构域应答调节因子CheY3组成。除了类似Che的组分外，Che3级联还包含第二个杂合组氨酸激酶CheS3。最近的生化和遗传学研究表明，CheA3并非CheY3的磷酸供体；相反，CheA3通过磷酸化CheS3的抑制性接收结构域来抑制CheS3→CheY3双组分系统。在本研究中，我们表明，除了CheA3的磷酸化作用外，CheS3的磷酸化状态还受细胞能量水平的调节，细胞能量水平通过ATP/(ATP + ADP)的摩尔比来量化。营养物质下调导致孢囊形成时，体内细胞能量显示下降35%。当在体外复制这种能量下降时，CheS3的磷酸化水平降低约75%。最后，我们还表明，ADP介导的CheS3磷酸化减少是ADP增强CheS3自身去磷酸化的结果。
饥饿时，百脉根红螺菌会经历一个发育过程，形成代谢休眠的孢囊，这些孢囊能耐受干燥和营养限制。本研究探讨了以ATP与ADP的比率衡量的细胞能量状态作为百脉根红螺菌孢囊形成重要调节因子的作用。我们表明，使用ATP/(ATP + ADP)作为衡量指标时，百脉根红螺菌细胞在孢囊形成过程中ATP显著减少。当在体外模拟这种体内能量饥饿水平时，CheS3磷酸化降低75%。CheS3自身磷酸化的这种显著降低与在ATP/(ATP + ADP)类似下降时CheA3磷酸化仅降低25%形成对比。我们认为，尽管腺苷酸能量在一定程度上会影响所有依赖ATP的酶，但响应ATP/(ATP + ADP)比率降低时CheS3活性增强的抑制作用可能作为调节孢囊发育的重要输入信号发挥作用。
