Stringent response and bmp expression in B.burgdorferi

描述（由申请方提供）：病原菌（如伯氏疏螺旋体）在其宿主中生长期间通常会遇到营养应激。这种刺激触发细菌的全局调节反应，即严格反应，其由alarmon（p）ppGpp介导并调节许多基因的表达。在上一个融资期，我们发现B。在所有B中存在编码旁系同源膜Bmp脂蛋白BmpD、BmpC、BmpA和BmpB的burgdorferi bmp染色体基因簇。Burgdorferi sensu lato的基因组，并且它形成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以产生不同的mRNA和基因产物浓度。我们的初步证据表明B。burgdorferi具有产生（p）ppGpp所需的转录活性relA/spoT基因，并且（p）ppGpp的存在与bmp簇基因在不同环境条件下的表达调节有关。在B的简化和浓缩基因组背景下BMP基因簇的遗传、转录和生物学特征。burgdorferi是研究（p）ppGpp和sigma因子等反式调节因子以及顺式DNA序列对染色体基因调控的理想模型。该项目基础假设是bmp和其他B的表达。通过（p）ppGpp直接和通过σ-54和σ-S间接的严格响应，以及通过其5个上游启动子区域的DNA序列，来反式调节burgdorferi基因。本项目的具体目标是：1）表征B生长过程中RelA/SpoT介导的严格反应对BMP表达的调控。在BSKH培养基中、与蜱细胞共培养中以及在大鼠腹膜培养箱和蜱中检测到伯氏螺旋体; 2）通过B中的严格反应确定σ-S和σ-54在调节BMP表达中的作用。3）鉴定与rpsL、bmpD、bmpC、bmpA和sigma-S基因的5个上游DNA序列结合的推定调控蛋白及其DNA结合位点。所提出的一系列分子、功能和遗传实验将使我们能够确定涉及BMP基因调控的全局因素、分级调控网络和顺式作用DNA序列，并将研究B中的基因表达和调节。burgdorferi从一个描述性的范式框架的机械和因果解释。

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.