Stringent response and bmp expression in B. burgdorferi

描述（由申请人提供）：在以前的研究中，我们已经表明，B。编码旁系同源Bmp膜脂蛋白的burgdorferi bmp染色体基因簇存在于所有B中。广义上的伯多禄该簇形成两个复杂的转录单位，其被转录成可变的单顺反子和多顺反子信息以产生不同的bmp mRNA和基因产物浓度。伯氏疏螺旋体中的营养应激触发调节许多基因表达的严格应答，并且由alarmon（p）ppGpp介导。我们的工作现在表明，B。burgdorferi具有产生（p）ppGpp所需的转录活性rel/Bbu基因，并且（p）ppGpp的存在与bmp和其它基因在不同环境条件下的表达调节有关。bmp基因簇的遗传、转录和生物学特性使其成为研究染色体基因调控的理想模型，所述染色体基因调控既通过反式调节因子如（p）ppGpp和sigma因子，也通过顺式DNA序列。该项目的基本假设是bmp和其它B burgdorferi基因的表达通过（p）ppGpp直接和通过σ 54和σ S间接的严格应答反式调节，并且通过它们的5'上游启动子区的DNA序列顺式调节。本研究的具体目的是：1）研究B生长过程中rel/Bbu表达的调控以及Rel/Bbu介导的bmp表达的严格反应。在BSKH培养基中，在与蜱细胞共培养中，以及在大鼠腹膜培养室和蜱中，确定sigma S和sigma 54在通过严格响应和它们在B中的分级相互作用调节BMP表达中的作用。使用细菌遗传学方法、DNA微阵列和蛋白质组学在体外和体内鉴定伯氏螺旋体;和3）鉴定与rpsL、bmpD、bmpC、bmpA和sigma S基因的5'上游DNA序列结合的推定调节蛋白及其DNA结合位点。这些实验将使我们能够确定bmp基因调控的全局因素、层次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): In previous studies we have shown that the B. burgdorferi bmp chromosomal gene cluster encoding the paralogous Bmp membrane lipoproteins is present in all B. burgdorferi sensu lato. This cluster forms two complex transcriptional units that are transcribed into variable monocistronic and polycistronic messages to generate different bmp mRNA and gene product concentrations. Nutritional stress in Borrelia burgdorferi triggers the stringent response that modulates expression of many genes and is mediated by the alarmon (p)ppGpp. Our work now indicates that B. burgdorferi has a transcriptionally active rel/Bbu, gene needed for generation of (p)ppGpp and that the presence of (p)ppGpp is associated with the modulation of expression of bmp and other genes under different environmental conditions. The genetic, transcriptional, and biological characteristics of the bmp gene cluster makes it an ideal model to study chromosomal gene regulation both 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 the regulation of rel/Bbu expression and the Rel/Bbu-mediated stringent response on bmp expression during growth of B. burgdorferi in BSKH media, in co-culture with tick cells, and in rat peritoneal culture chambers and ticks; 2) ascertain the roles of sigma S and sigma 54 in modulating bmp expression by the stringent response and their hierarchical interactions in B. burgdorferi in vitro and in vivo using bacterial genetics methods, DNA microarrays and proteomics; 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 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.