Stringent response and bmp expression in B.burgdorferi

描述（由申请人提供）：营养压力通常是致病菌如伯氏疏螺旋体在其宿主生长过程中遇到的。这种刺激触发了细菌的全局调控反应，即严格反应，该反应由警报(p)ppGpp介导，并调节许多基因的表达。在之前的资助期内，我们发现编码旁系膜bmp脂蛋白BmpD、BmpC、BmpA和BmpB的伯氏疏螺旋体bmp染色体基因簇存在于所有的伯氏疏螺旋体中，并形成两个复杂的转录单位，转录成可变的单反式和多反式信息，以产生不同的mRNA和基因产物浓度。我们现在的初步证据表明，伯氏疏螺旋体具有(p)ppGpp生成所需的转录活性relA/spoT基因，并且(p)ppGpp的存在与不同环境条件下bmp簇基因的表达调节有关。在还原和浓缩的伯氏疏螺旋体基因组背景下，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序列对染色体基因调控的理想模型。该项目的假设是bmp和其他伯氏疏螺旋体基因的表达在反式中受到(p)ppGpp直接和间接通过sigma-54和sigma-S的严格响应的调节，在顺式中受到其5个上游启动子区的DNA序列的调节。该项目的具体目的是：1)通过RelA/ spot介导的严格反应来表征伯氏疏螺旋体在BSKH培养基、与蜱细胞共培养以及大鼠腹膜培养champers和蜱中生长过程中bmp表达的调控；2)通过体外和体内对伯氏疏螺旋体的严格反应，确定sigma-S和sigma-54在调节bmp表达中的作用；3)鉴定rpsL、bmpD、bmpC、bmpA和sigma-S基因5个上游DNA序列的推定调控蛋白及其DNA结合位点。提出的一系列分子、功能和遗传实验将使我们能够确定bmp基因调控的全局因素、分层调控网络和顺式作用DNA序列，并将对伯氏疏螺旋体的基因表达和调控进行研究，从描述性范式转向由机制和因果解释框架的范式。

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.