Regulation of Stationary Phase in Escherichia coli

大肠杆菌固定相的调节

• 批准号: 8681463
• 负责人: Thomas J. Silhavy
• 金额: $ 30.93万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8681463
• 关键词: 3' Untranslated Regions; Bacteria; Bacteria sigma factor KatF protein; Bacterial Infections; Biochemical; Bioinformatics; Carbon; Cell Survival; Cells; DNA-Directed RNA Polymerase; Data; Ensure; Escherichia coli; Face; Funding; Genes; Genetic; Genetic Transcription; Genotype; Holoenzymes; Mass Spectrum Analysis; Messenger RNA; Mutation; Nitrogen; Noise; Nutrient; Organism; Orphan; Pattern; Peptide Hydrolases; Phase; Phosphorylation; Phosphotransferases; Physiological; Physiology; Polyadenylation; Polyadenylation Pathway; Polynucleotide Adenylyltransferase; Population; Production; Prophages; Protein Dephosphorylation; Proteins; Proteobacteria; Proteolysis; Regulation; Reporting; Sigma Factor; Signal Transduction; Source; Starvation; Stress; Testing; Update; biological adaptation to stress; combat; degradosome; genetic analysis; genetic regulatory protein; insight; mRNA Stability; novel; research study; response; sensor; small molecule; transcription factor

DESCRIPTION (provided by applicant): The alternate sigma factor RpoS is the master regulator of stationary phase and the general stress response in Escherichia coli and many other proteobacteria. In rapidly growing cells, RpoS is made but it is degraded by the ClpX/P protease. When starved for a particular nutrient, RpoS synthesis increases, degradation ceases, activity is increased, or some combination of these effects occurs. Two novel regulatory proteins are the focus of this proposal, SprE (RssB) and Crl. SprE is an orphan response regulator that functions as an adaptor to direct RpoS to the ClpX/P protease in growing cells. When carbon sources are depleted, this degradation stops. We know that SprE phosphorylation/dephosphorylation is not involved in starvation signaling. Genetic analysis suggests that starvation is sensed as a decrease in ATP levels, and biochemical data suggest that this decrease is sensed by ClpX and RpoS itself. Genetic analysis has further revealed a second function for SprE in polyadenylation and the control of mRNA stability, and it suggests that phosphorylation of SprE is important for this activity. We will identify mutations that separate the two functions of SprE and identify the relevant small molecule or kinase. Using mass spectrometry we have discovered that SprE controls the association of Poly(A) polymerase and Hfq with the mRNA degradosome and data from microarrays suggest that SprE functions to silence foreign genes. We will probe the physiological significance of these novel activities. We have shown that Crl facilitates the association of RpoS with core RNA polymerase, and we know that this activity is especially important under nitrogen starvation conditions. Under these conditions crl transcription increases 25 fold. However, there is no significant corresponding change in Crl levels. We demonstrate that this change in transcription allows a switch from noisy to more uniform Crl production and we will probe the physiological significance of these expression patterns under both conditions. Nutrient starvation is the most common stress that bacteria face and RpoS is arguably the most important global regulatory protein in E. coli. A better understanding of stationary phase physiology may provide insights into how to combat bacterial infections.

描述（由申请人提供）：替代Sigma因子RPOS是固定相的主调节剂和大肠杆菌和许多其他蛋白质细菌中的一般应力反应。在快速生长的细胞中，制造了RPO，但被CLPX/P蛋白酶降解。当饿死特定营养素时，RPOS合成的增加，降解停止，活性增加或某种效果的组合发生。该提案的重点是两个新型的调节蛋白，即SPRE（RSSB）和CRL。 SPRE是一种孤儿响应调节剂，可作为将RPO引导至生长细胞中CLPX/P蛋白酶的适配器。当碳源耗尽时，这种降解就会停止。我们知道，SPRE磷酸化/去磷酸化不涉及饥饿信号。遗传分析表明，将饥饿视为ATP水平的降低，并且生化数据表明，CLPX和RPOS本身会感知这种降低。遗传分析进一步揭示了SPRE在多腺苷酸化和mRNA稳定性的控制中的第二个功能，这表明SPRE的磷酸化对于这种活性很重要。我们将确定将SPRE的两个函数分开并确定相关的小分子或激酶的突变。使用质谱法，我们发现SPRE控制了聚（A）聚合酶和HFQ与mRNA降解体的关联，而微阵列的数据表明，SPRE功能可以使外源基因保持沉默。我们将探究这些新活动的生理意义。我们已经表明，CRL促进RPO与核心RNA聚合酶的关联，并且我们知道在氮饥饿条件下，这种活性尤为重要。在这些条件下，CRL转录增加了25倍。但是，CRL水平没有显着的相应变化。我们证明，这种转录的这种变化使从嘈杂到更均匀的CRL产生，我们将在两种条件下探测这些表达模式的生理意义。营养饥饿是细菌面部和RPO的最常见压力，可以说是大肠杆菌中最重要的全球调节蛋白。对固定相生理的更好理解可以提供有关如何对抗细菌感染的见解。

项目成果

RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

RpoS 蛋白水解通过不需要 SprE (RssB) 反应调节器磷酸化位点的机制进行调节。

• DOI: 10.1128/jb.186.21.7403-7410.2004
• 发表时间: 2004
• 期刊: Journal of bacteriology.
• 作者: Peterson,CelesteN;Ruiz,Natividad;Silhavy,ThomasJ
• 通讯作者: Silhavy,ThomasJ

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions.

• DOI: 10.1016/j.jasms.2010.02.004
• 发表时间: 2010-06
• 期刊: Journal of the American Society for Mass Spectrometry
• 影响因子: 3.2
• 作者: Carabetta VJ;Li T;Shakya A;Greco TM;Cristea IM
• 通讯作者: Cristea IM

Transcriptional occlusion caused by overlapping promoters.

由重叠启动子引起的转录封闭。

• DOI: 10.1073/pnas.1323413111
• 发表时间: 2014
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Zafar,MAmmar;Carabetta,ValerieJ;Mandel,MarkJ;Silhavy,ThomasJ
• 通讯作者: Silhavy,ThomasJ