Stringent response and bmp expression in B.burgdorferi

伯氏疏螺旋体的严格反应和 bmp 表达

• 批准号: 6542927
• 负责人: Felipe Cardenas Cabello
• 金额: $ 38万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542927
• 关键词: Borrelia; Escherichia coli; Lyme disease; bacteria infection mechanism; bacterial DNA; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; binding sites; enzyme activity; gel mobility shift assay; gene expression; genetic regulation; laboratory mouse; laboratory rabbit; laboratory rat; mass spectrometry; membrane proteins; mixed tissue /cell culture; protein localization; transfection

DESCRIPTION (provided by applicant): Nutritional stress is commonly encountered by pathogenic bacteria such as Borrelia burgdorferi during growth in their hosts. This stimulus triggers a bacterial global regulatory response, the stringent response, which is mediated by the alarmon (p)ppGpp and modulates expression of many genes. In the previous funding period, we found that the B. burgdorferi bmp chromosomal gene cluster encoding the paralogous membrane Bmp lipoproteins BmpD, BmpC, BmpA and BmpB was present in all B. burgdorferi sensu lato and that it formed two complex transcriptional units transcribed into variable monocistronic and polycistronic messages to generate different mRNA and gene product concentrations. Our preliminary evidence now indicates that B. burgdorferi has a transcriptionally active relA/spoT gene needed for generation of (p)ppGpp and the presence of (p)ppGpp is associated with modulation of expression of bmp cluster genes under different environmental conditions. The genetic, transcriptional, and biological characteristics of the bmp gene cluster in the context of the reduced and condensed genome of B. burgdorferi makes it an ideal model to study chromosomal gene regulation by trans regulators such as (p)ppGpp and sigma factors and by cis DNA sequences. The hypothesis underlying this project is that expression of bmp and other B. burgdorferi genes is modulated in trans by the stringent response both directly by (p)ppGpp and indirectly through sigma-54 and sigma-S and in cis by the DNA sequences of their 5 upstream promoter regions. The specific aims of this project are: 1) characterize regulation of bmp expression by the RelA/SpoT-mediated stringent response during growth of B. burgdorferi in BSKH media, in co-culture with tick cells, and in rat peritoneal culture champers and ticks; 2) ascertain the roles of sigma-S and sigma-54 in modulating bmp expression by the stringent response in B. burgdorferi in vitro and in vivo; and 3) identify putative regulatory proteins binding to 5 upstream DNA sequences of the rpsL, bmpD, bmpC, bmpA and sigma-S genes and their DNA binding sites. The proposed array of molecular, functional and genetic experiments will permit us to identify global factors, hierarchical regulatory networks and cis acting DNA sequences involved in the regulation of bmp genes, and will take studies of gene expression and modulation in B. burgdorferi from a descriptive paradigm to a paradigm framed by mechanistic and causal interpretations.

描述(申请人提供)：伯氏疏螺旋体等病原菌在宿主生长过程中经常遇到营养压力。这种刺激触发了细菌的全球调控反应，即严格反应，该反应由Alarmon(P)ppGpp介导，调节许多基因的表达。在之前的资助期间，我们发现在所有的伯氏杆菌中都存在编码BmpD、BmpC、Bmpa和BmpB的伯氏杆菌BMP染色体基因簇，它形成了两个复杂的转录单位，转录成可变的单顺反子和多顺反子信息，产生不同的mRNA和基因产物浓度。我们的初步证据表明，伯氏杆菌具有转录活性的relA/Spot基因，是产生(P)ppGpp所必需的，并且(P)ppGpp的存在与不同环境条件下BMP簇基因的表达调控有关。BMP基因簇的遗传、转录和生物学特性使其成为研究(P)ppGpp和sigma因子等反式调控因子以及顺式DNA序列调控染色体基因的理想模型。该项目的假设是，骨形态发生蛋白和其他伯氏杆菌基因的表达在反式调控下，受到(P)ppGpp直接和间接通过sigma-54和sigma-S的严格反应的调控，在顺式调控下，受其5个上游启动子区域的DNA序列的调控。本项目的具体目标是：1)研究在伯氏杆菌生长过程中，RelA/Spot介导的严格反应对骨形态发生蛋白表达的调控；3)确定Sigma-S和Sigma-54在伯氏杆菌体内外的严格反应中调节BMP表达的作用；3)鉴定与rpsL、bmpD、bmpC、bmpa和sigma-S基因的5个上游DNA序列及其DNA结合位点相结合的可能的调控蛋白。拟议的一系列分子、功能和遗传学实验将使我们能够确定参与BMP基因调控的全球因子、层级调控网络和顺式作用DNA序列，并将把对伯氏杆菌基因表达和调控的研究从描述性范式转向机械和因果解释框架的范式。