The Role of Calcium in Bacteria: a Proteomic and Genomic Approach

钙在细菌中的作用：蛋白质组学和基因组学方法

基本信息

批准号：
7282238
负责人：
DELFINA C DOMINGUEZ
金额：
$ 15.76万
依托单位：
UNIVERSITY OF TEXAS EL PASO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7282238
关键词：
Antibiotic A23187 Antibodies Bacteria Binding Binding Proteins Biochemical Biological Assay Calcium Calmodulin Cell Differentiation process Cells Cellular biology Characteristics Chelating Agents Condition Data Development EEF1A1 gene Electrophoresis Escherichia coli Eukaryota Eukaryotic Cell Event Gene Expression Genes Genetic Transcription Genomics Goals Growth Heating Homeostasis Ionophores Ions Lead Maps Mass Spectrum Analysis Measures Microarray Analysis Molecular Weight Nature Organism Other Finding Peptide Elongation Factor Tu Personal Communication Personal Satisfaction Pertussis Physiology Play Process Prokaryotic Cells Protein Sequence Analysis Proteins Proteomics Publishing Purpose Radioactive Regulation Regulon Research Role Shock Signal Transduction Stress Verapamil Work base biological adaptation to stress cross reactivity design human EEF1A1 protein inhibitor/antagonist liquid chromatography mass spectrometry microorganism novel therapeutics pathogenic bacteria protein expression research study response

项目摘要

The importance of calcium (Ca2+) as a cell regulator is well established in eukaryotes. However, the role of Ca2+ in prokaryotes still remains elusive. Recent experimental work has suggested that Ca2+ ions may play a regulatory role in prokaryotic organisms. An essential step toward an increased understanding of the role of Ca2+ is the identification and characterization of Ca2+ binding proteins (CaBPs). In eukaryotes, CaBPs are involved in the regulation of multiple cellular events including: cell differentiation, gene expression, transport mechanisms and others. Our preliminary data indicates that several CaBPs are present in bacteria (E. coli, B. subtilis and B. pertussis). These proteins share similar biochemical characteristics with eukaryotic CaBPs, including calmodulin (CaM). In an effort to isolate and sequence these proteins we analyzed crude cell lysates by 2D-electrophoresis followed by mass spectrometry. Most of the proteins found with CaBP characteristics are associated with stress responses. Based on these initial findings and other published data the purpose of this application is: 1) to map changes in protein expression as a function of cytosolic Ca2+ levels during both optimal growth conditions and during induced stress responses (heat/cold/pH shock). 2) to study the role of Ca2+ ions in transcription and establish the nature of the calcium regulon. The long-term goal of this research is to illuminate the role of Ca2+ in bacteria. We hypothesize that CaBPs play an important role in Ca2+ homeostasis and that Ca2+ ions are involved in the regulation of several intracellular processes in bacterial cells. In particular, we hypothesize that the intracellular concentration of Ca2+ ions are involved in the regulation of stress response proteins with associated changes in gene expression. The following specific aims were developed: 1) To characterize further the previously identified CaBPs and to identify new CaBPs that are involved in the bacterial response to changes in calcium levels 2) To identify and characterize the adaptive mechanisms utilized by cells to maintain calcium homeostasis 3) To determine the role of calcium in stress-related proteomic changes 4) To investigate the specific genomic changes that occur in bacteria in response to changing calcium levels. This study will lead to better understanding of basic cell biology and physiology of both pathogenic and environmental microorganisms and may lead to development of new therapeutics for the control of pathogenic bacteria.

钙 (Ca2+) 作为细胞调节剂的重要性在真核生物中已得到充分证实。然而，角色原核生物中的 Ca2+ 仍然难以捉摸。最近的实验工作表明 Ca2+ 离子可能发挥作用在原核生物中起调节作用。加深对角色理解的重要一步 Ca2+ 的鉴定和表征是 Ca2+ 结合蛋白 (CaBP) 的鉴定和表征。在真核生物中，CaBP 参与多种细胞事件的调节，包括：细胞分化、基因表达、运输机制及其他。我们的初步数据表明细菌中存在多种 CaBP （大肠杆菌、枯草芽孢杆菌和百日咳芽孢杆菌）。这些蛋白质与真核生物具有相似的生化特征 CaBP，包括钙调蛋白 (CaM)。为了分离和测序这些蛋白质，我们分析了粗蛋白通过二维电泳和质谱分析细胞裂解物。大多数与 CaBP 一起发现的蛋白质特征与应激反应相关。基于这些初步发现和其他已发表的数据本应用的目的是：1) 绘制蛋白质表达随胞质 Ca2+ 函数的变化在最佳生长条件和诱导应激反应（热/冷/pH 冲击）期间的水平。 2）到研究 Ca2+ 离子在转录中的作用并确定钙调节子的性质。长期来看这项研究的目的是阐明 Ca2+ 在细菌中的作用。我们假设 CaBPs 发挥着 Ca2+离子在Ca2+稳态中发挥重要作用，并且参与多种细胞内的调节细菌细胞中的过程。特别是，我们假设细胞内 Ca2+ 离子浓度为参与应激反应蛋白的调节以及相关的基因表达变化。这制定了以下具体目标： 1) 进一步描述先前确定的 CaBP 的特征并识别参与细菌对钙水平变化的反应的新 CaBP 2) 识别并表征细胞用来维持钙稳态的适应性机制 3) 确定钙在应激相关蛋白质组变化中的作用 4) 研究特定的基因组变化细菌响应钙水平的变化而发生。这项研究将有助于更好地理解基础知识病原微生物和环境微生物的细胞生物学和生理学，可能导致开发控制病原菌的新疗法。