Stringent response and bmp expression in B. burgdorferi

描述(申请人提供)：在以前的研究中，我们已经证明编码邻近BMP膜脂蛋白的伯氏杆菌BMP染色体基因簇存在于所有伯氏杆菌中。这个簇形成了两个复杂的转录单位，它们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。伯氏疏螺旋体的营养胁迫触发了调控许多基因表达的严格反应，并由Alarmon(P)ppGpp介导。我们的工作表明，伯氏杆菌有一个转录活性基因REL/BBU，这是产生(P)ppGpp所必需的，并且(P)ppGpp的存在与不同环境条件下BMP和其他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，BMP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的严格反应的调节，在顺式中则受到其5‘上游启动子区域DNA序列的调控。本项目的具体目标是：1)利用细菌遗传学、基因芯片和蛋白质组学方法，研究REL/BBU表达的调控以及REL/BBU介导的骨形态发生蛋白在伯氏杆菌生长过程中的严格调控；(2)利用细菌遗传学、基因芯片和蛋白质组学方法，研究sRel/BBU和Sigma 54在调节骨形态发生蛋白表达中的作用；3)鉴定可能与rpsL、bmpD、bmpC、bmpa和sigma S基因5‘上游DNA序列及其DNA结合位点相结合的调控蛋白。拟议的实验将使我们能够识别参与BMP基因调控的全球因子、分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。

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.