苏云金杆菌中一个新的多效、双能转录因子——YvoA的调控网络解析

N-acetylglucosamine (GlcNAc) is a ubiquitous monosaccharide derivative of glucose and an essential molecule for all forms of life. GlcNAc is in addition a preferred carbon source for many microorganisms as it provides both carbon and nitrogen. Moreover, since complete hydrolysis of GlcNAc by the NagA (N-acetylglucosamine-6-phosphate deacetylase) and NagB (glucosamine-6-phosphate deaminase) enzymes generates acetate, ammonia (NH3), and Fru-6-P, GlcNAc utilization influences major biological processes, such as glycolysis, the tricarboxylic acid (TCA) cycle, respiration, nucleic acids, nitrogen, and fatty acid metabolism, as well as cell wall biosynthesis.. YvoA in Bacillus subtilis is a GntR/HutC transcription regulator implicated in the regulation of genes encoding GlcNAc degradative and biosynthetic enzymes from the N-acetylglucosamine-degrading pathway. It represses expression of the three GlcNAc-inducible genes nagA, nagB, and nagP by directly binding a 16-bp sequence identified within their upstream regions and has been renamed to NagR. However, we provide a compilation of in silico, in vitro, and in vivo evidence that defines YvoA from Bacillus thuringiensis as a novel pleiotropic regulatory protein. It controls genes of chitin，GlcNAc，tricarboxylic acid (TCA) cycle, respiration, nucleic acids metabolism as well as fatty acid metabolism. Moreover，YovA acts as dual regulator possessing both activator and repressor functions in B. thuringiensis. However, HutC/GntR regulators are almost exclusively repressor proteins. . This research is going to investigate the YvoA regulatory network in B. thuringiensis. First, we will utilize ChIP-nexus and RNA-seq to screen potential genes directly controlled by YvoA and the dre sites bind specifically by YvoA. The candidate genes will be identified via electrophoretic mobility shift assay(EMSA) and DNase I footprinting assay in vitro, qRT-PCR tests in vivo. Furthermore，bioinformatics tools(such as MEME, CisFinder, HOMER,PREDetector and BioProspector ) will be used to analysis if the potential dre sites exist in the upstream of candidate genes. Based upon the compilation of in silico, in vitro, and in vivo data, we will construct the regulatory network of YvoA. In addition, we will investigate the affinities of YvoA for different dre sites since YvoA proper function relies on its DNA binding affinities to different dre sequences. . Relative positions between dre sites and promoter may essential to YvoA acting as a dual regulator. This appears to hold true for several regulators implicated in both the transcription of anabolic and catabolic genes such as the E. coli FadR regulator and B. subtilis arginine repressor/activator AhrC. The FadR protein of Escherichia coli has been shown to play a dual role in transcription of the genes of bacterial fatty acid metabolism. The protein acts as a repressor of beta-oxidation and an activator of unsaturated fatty acid synthesis. When FadR binds downstream of the RNA polymerase-binding site within the promoter, it blocks the polymerase and acts as a repressor. Conversely, when bound upstream, it promotes binding of the RNA polymerase and acts as an activator. Location relationship of dre sites and promoter also will be determined.. Significance and impact of the research is not only conducive to construct the regulatory network of YvoA in B. thuringiensis and elucidate its regulation mechanism，but also help deepen our understanding of GntR/HutC transcription regulators.

枯草芽胞杆菌的YvoA蛋白是只能负调控N-乙酰葡萄糖胺(GlcNAc)代谢的转录因子。我们研究发现，苏云金芽胞杆菌（Bt）的YvoA却是一种多效、双能转录因子，既可作为激活子，又可作为阻遏子，调控的基因也远远超出GlcNAc代谢范围。.有鉴于此，本项目拟对Bt转录因子YvoA的调控网络进行解析：应用RNA-seq和ChIP-nexus筛选YvoA的调控基因和结合位点（dre位点）；应用生物软件分析（in silico），结合qRT-PCR，EMSA和ITC等体内（in vivo）、体外（in vitro）技术方法，从三方面验证YvoA直接调控的靶基因和dre位点，构建YvoA的调控网络，并进一步明确YvoA与不同dre位点的亲和力以及dre位点与启动子之间的位置关系。.本项目不仅可以明确YvoA的调控网络，有助于其调控机理的阐释，也将推进对整个GntR / HutC转录因子家族的深入了解。

NagR是一种属于GntR家族、HutC亚家族的转录因子，在枯草杆菌中仅负调控氨基糖代谢基因（nagA和nagB）及其转运基因（nagP）的表达。.我们从计算机预测（in silico）、EMSA的体外（in vitro）实验和nagR基因敲除后对体内基因表达量（in vivo）的影响三方面证实了NagR对基因表达的调控作用，解析了苏云金杆菌Bti75菌株中的NagR的调控网络，发现其不仅直接调控GlcNAc的摄入和代谢，还调控几丁质酶将几丁质降解为GlcNAc的这一过程，并影响氨基糖代谢、核糖代谢、脂肪酸代谢、磷酸转运系统和EMP途径等多种生物过程，其中19个基因受其直接调控，是一个多效应转录因子；并进一步证实Bt中NagR还是一个双功能转录因子，能够正调控pgi和BTF1_19025基因的表达。明确了NagR的双功能性由其结合位点的位置决定，结合位点在启动子中或下游表现为抑制作用，结合位点在启动子上游表现为促进作用。此外，我们还应用DNA足迹法（DNA-foot printing）技术进一步明确了NagR结合位点的共有序列，应用和微量热泳动技术（MST）测定了NagR与其特异结合序列的亲和力。.另外，我们还发现了一个新的HutC亚家族的转录因子，它直接或间接调控核苷转运蛋白、ABC转运系统、Opp转运系统、离子转运、色素和毒素合成等生物进程，我们将它并名为NupR。NagR和NupR两者间不存在相互调控作用。.本研究的意义不仅局限于作为生物农药的苏云金芽胞杆菌本身，对于严重威胁人类健康的炭疽芽胞杆菌和机会致病菌如蜡状芽胞杆菌等的研究也有重要的借鉴意义，对于更深入地了解GntR/HutC转录因子家族的功能也有积极地推动作用。