使用微蒂特萝卜无线电标签进行多维欧大肠杆菌测量 (p) ppGpp,然后是薄层色谱
Summary
微酸度皿中放射性标记细菌培养物的生长有助于高通量采样,从而允许对核苷酸池丰度进行多种技术和生物复制检测,包括 (p) ppGpp。可以监测生理压力来源引起的生长转变以及压力恢复的影响。
Abstract
(p)ppGpp核苷酸作为细菌的全球调节器,对各种物理和营养压力作出反应。它有一个快速的开始,在几秒钟内,这导致积累的水平接近或超过的GTP池的水平。应力反转场合迅速消失(p)ppGpp,通常半寿命不到一分钟。(p)ppGpp的存在导致细胞基因表达和代谢的改变,以对抗压力的破坏性影响。革兰氏阴性和革兰氏阳性细菌有不同的反应机制,但都取决于(p)ppGpp浓度。在任何情况下,都需要同时监测许多放射性标记细菌培养物的时间间隔,在临界应力过渡期间,时间间隔可能从 10 秒到数小时不等。此协议解决了这一技术难题。该方法利用温度和摇摇控制微酸盐培养箱,允许平行监测生长(吸收)和均匀磷酸盐放射性标记培养物的快速采样,通过PEI纤维素上的薄层色谱。分析的多重技术和生物复制需要少量样品。复杂的增长过渡,如二次增长和快速(p)ppGpp周转率可以通过这种方法进行定量评估。
Introduction
(p)ppGpp第二信使是一个全球调节器,调节大量基因的表达,包括用于合成核糖体和氨基酸1、2的基因。虽然最初发现在大肠杆菌3,(p)ppGpp可以发现在革兰氏阳性和革兰氏阴性细菌,以及在植物叶绿体4,5。对于大肠杆菌和其他革兰氏阴性细菌,(p)ppGpp在6、7、8两个不同位点与RNA聚合酶直接相互作用。在革兰氏阳性,(p)ppGpp抑制GTP丰度,这是由CodY,一种GTP结合蛋白与基因特异性DNA识别主题,导致调控9,10。(p)ppGpp积累,以回应饥饿的不同营养和压力条件,导致缓慢生长和调整基因表达,使适应压力11,12。
ppGpp累积的净量反映了合成酶和水酶活动之间的平衡。在大肠杆菌RelA是一种强合成酶和SpoT是双功能,具有强水酶和弱合成酶,其中每一种可能以不同的方式调节在应力依赖的方式。强RelA合成酶被激活时,通币指定的带电tRNA绑定到核糖体A位点时,它未能跟上蛋白质合成13,14,15的需求。弱孢子(p)ppGpp合成酶被激活,而强(p)ppGpp水解酶被抑制,以响应其他应力条件并通过其他机制。在某些情况下,ACP或Rsd等蛋白质可以结合到SpoT,这也改变了水解和合成16、17之间的平衡。在革兰氏阳性物中,合成和水解反映了单一RelA SpoT同源(RSH)蛋白之间更复杂的平衡,具有很强的合成和水解活性,以及较小的水酶和/或合成酶12。
(p)ppGpp核苷酸首次被发现为不寻常的32P标记点,出现在薄层色谱图(TLC)的自动放射图上,在氨基酸饥饿3引起的严格反应。更详细的标签协议已经审查18。此处描述的协议(图1)是对这些协议的修改,允许监测微小板上多个样品的生长。这有利于对(p)ppGpp丰度变化的多重生物和技术估计,最初是为研究二代移位19而开发的。带有32P 和 TLC 检测的 (p) ppGpp 标签也允许测量 (p) ppGpp 降解率。已开发替代方法来确定(p)ppGpp水平,如质谱、HPLC20、荧光化学传感器21、22和GFP基因融合到受ppGpp23影响的启动子。24.荧光化学传感器目前用途有限,因为结合ppGpp后光谱偏移小,以及ppGpp和ppGpp21之间的问题。该方法在体外检测(p)ppGpp是有效的,但在细胞提取物中却不能。涉及HPLC的方法已经改进了20个,但需要昂贵的设备,并且不能很好地适应高通通。最后,GFP融合可以估计ppGpp依赖激活或抑制,但不测量ppGpp本身。虽然每种替代方法都是有价值的,但它们需要昂贵的设备或大量的动手时间,否则它们不能接受多种动力学采样和后续处理。通过本文所述的方法,96 个样品可在大约 20 分钟(每板 18 个样本)内应用于 6 个 TLC 板,TLC 开发可在几个小时内解决,在几个小时或一夜之间获得定量数据,具体取决于标签强度。
Protocol
Representative Results
Discussion
实现细胞近乎均匀的标签是该协议的关键步骤。因此,使用定义的介质(如 MOPS 或 Tris 介质)对于允许携带磷酸盐浓度和特定活动的变化至关重要。不能使用磷酸盐缓冲介质,如 M9 或介质 A。大多数未定义的介质含有可变量的磷酸盐,如LB、胰锥酮和卡萨米诺酸。磷酸盐同位素33P是一种较弱的发射体,可以替代32 P。 然而,32 P的更高能量排放大大提高了检测灵敏度。必须强?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国国家卫生研究院尤尼斯·肯尼迪·施莱佛国家儿童健康与人类发展研究所(Eunice Kennedy Shriver)内部研究计划的支持。
Materials
| (NH4)6(MO7)24 | Fisher Scientifics | A-674 | |
| Autoradiography film | Denville scientific inc. | E3218 | |
| CaCl2 | J.T.Baker | 1-1309 | |
| Chloramphenicol | RPI | C61000-25.0 | |
| CoCl2 | Fisher Scientifics | C-371 | |
| CuSO4 | J.T.Baker | 1843 | |
| FeSO4 | Fisher Scientifics | I-146 | |
| Formic acid | Fisher Biotech | BP1215-500 | |
| Glucose | Macron | 4912-12 | |
| H3BO4 | Macron | 2549-04 | |
| H3PO4 | J.T.Baker | 0260-02 | |
| K2SO4 | Sigma | P9458-250G | |
| KH2PO4 | Fisher Biotech | BP362-500 | |
| L-Valine | Sigma | V-6504 | |
| MgCl2 | Fisher Scientifics | FL-06-0303 | |
| Microplate reader Synergy HT | Biotek | Synergy HT | |
| MnCl2 | Sigma | M-9522 | |
| MOPS | Sigma | M1254-1KG | |
| Na2HPO4 | Mallinckrodt | 7892 | |
| NaCl | J.T.Baker | 3624-01 | |
| NaH2PO4 | Mallinckrodt | 7917 | |
| NH4Cl | Sigma | A0171-500G | |
| P-32 radionuclide, orthophosphoric acid in 1 mL water (5 mCi) | Perkin Elmer | NEX053005MC | |
| Storage phosphor screen | Kodak | So230 | |
| Thermomixer | Eppendorf | 5382000015 | |
| Thiamine | Sigma | T-4625 | |
| TLC PEI Cellulose F | Merk-Millipore | 1.05579.0001 | |
| Tricine | RPI | T2400-500.0 | |
| Typhoon 9400 imager | GE Healthcare | ||
| ZnSO4 | Fisher Scientifics | Z-68 |
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