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首页> 美国卫生研究院文献>Infection and Immunity >Stress-Induced Membrane Association of the Streptococcus mutans GTP-Binding Protein an Essential G Protein and Investigation of Its Physiological Role by Utilizing an Antisense RNA Strategy
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Stress-Induced Membrane Association of the Streptococcus mutans GTP-Binding Protein an Essential G Protein and Investigation of Its Physiological Role by Utilizing an Antisense RNA Strategy

机译：应力诱导变形链球菌GTP结合蛋白一种必需的G蛋白的膜关联并通过利用反义RNA策略研究其生理作用。

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SGP (for Streptococcus GTP-binding protein) is a Streptococcus mutans essential GTPase which has significant sequence identity to the previously identified Escherichia coli Era protein and to numerous other prokaryotic GTPase proteins of unknown function. Recent studies in our laboratory have addressed the possible role of SGP in the stress response of the oral pathogen S. mutans. Here we report that during growth in the early stationary phase, and in response to elevated temperatures or acidic pH, the distribution of SGP between the cytoplasm and the membranes of S. mutans cells varies. Immunoblot analysis of soluble and membrane protein fractions collected from the mid-log and early stationary growth phases of bacterial populations grown at normal temperature (37°C) and at the elevated temperature of 43°C, or at acidic pH, demonstrated that the total amount of SGP increased with the age of the bacterial culture, elevated temperature, or acidic pH. Furthermore, it was established that a substantial amount of SGP is associated with the membrane fraction under stress conditions. In order to investigate the physiological role of SGP, we constructed an S. mutans strain capable of chromosomal sgp antisense RNA expression, which interferes with the normal information processing of the sgp gene. Utilizing this strain, we determined conditions whereby the streptococcal cells can be depleted of SGP, thus avoiding the problem of constructing a conditional lethal system. From the results of measurements of the nucleotide pools extracted from the antisense strain and its isogenic counterpart, we propose that one of the physiological roles of SGP is regulation and modulation of the GTP/GDP ratio under different growth conditions. Moreover, we observed that in SGP-depleted cells the levels of glucan-binding protein A (GbpA) substantially increased, suggesting that GbpA may have stress response-related physiological functions. Finally, the potential applications of the antisense RNA approach that we employed are discussed.
机译：SGP（针对链球菌GTP结合蛋白）是变形链球菌必不可少的GTPase，它与先前鉴定的大肠杆菌时代蛋白以及功能未知的许多其他原核GTPase蛋白具有显着的序列同一性。我们实验室中的最新研究已经探讨了SGP在口腔病原体变形链球菌的应激反应中的可能作用。在这里，我们报告说，在早期静止期的生长过程中，以及对升高的温度或酸性pH的响应，SGP在变形链球菌细胞质和细胞膜之间的分布有所不同。从正常温度（37°C）和高温（43°C）或酸性pH下生长的细菌种群的对数中期和静止早期生长阶段收集的可溶性和膜蛋白组分的免疫印迹分析表明，总的SGP的量随细菌培养物的年龄，高温或酸性pH的增加而增加。此外，已经确定在应力条件下大量SGP与膜部分相关。为了调查SGP的生理作用，我们构建了一种变形链球菌菌株，该菌株能够进行染色体sgp反义RNA表达，从而干扰sgp基因的正常信息处理。利用该菌株，我们确定了链球菌细胞可以耗尽SGP的条件，从而避免了构建条件致死系统的问题。从反义菌株及其等基因对应物提取的核苷酸库的测量结果，我们建议SGP的生理作用之一是在不同生长条件下调节和调节GTP / GDP比率。此外，我们观察到，在SGP耗尽的细胞中，葡聚糖结合蛋白A（GbpA）的水平显着增加，表明GbpA可能具有与应激反应相关的生理功能。最后，讨论了我们采用的反义RNA方法的潜在应用。

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