RUI: Gamma-Butyrolactone Autoregulatory Signaling In Thaxtomin- And Naphthoquinone Polyketide-Producing Streptomyces

革兰氏阳性丝状链霉菌产生？-丁内酯（GBL）分子以生长速率和细胞密度依赖性方式调节参与次级代谢和形态发生的基因的表达。 GBL信号转导涉及GBL生物合成所需的GBL自调节因子和GBL结合蛋白（GBLBP），一种具有GBL结合结构域的TetR型转录调节因子。 几种链霉菌产生次级代谢物植物毒素，thaxtomins，这是有效的宿主定植和感染植物所必需的。 许多革兰氏阴性原核生物的宿主-微生物相互作用活性依赖于N-酰基高丝氨酸内酯（AHSL）衍生物，其具有与GBL相当的结构相似性。 由于thaxtomin是宿主-微生物相互作用所需的有效次级代谢物植物毒素，因此设想GBL化合物在革兰氏阳性链霉菌调节thaxtomin基因表达中的作用类似于AHSL在革兰氏阴性生物体中调节基因表达中的作用。 本计画将利用遗传学、生理学及生化学的方法，来阐明GBL信号在产生thaxtomin的生物体中所扮演的角色。 GBL自动调节子和GBLBP突变体将使用破坏和替换技术产生，并使用生长和形态发育的生理学研究进行分析。将对thaxtomins和其他代谢产物的产生进行定量测定。 将对野生型和突变衍生物中的自调节因子和thaxtomin结构基因进行转录图谱和生长速率依赖性转录分析。 GBLBP的生化分析将包括电泳迁移率变化和DNA酶足迹分析与DNA调控元件。 随着本科生的广泛参与，这项研究有望推进对链霉菌GBL自动调节因子介导的群体感应活动的有限理解，特别是关于thaxtomin依赖的宿主-微生物相互作用。

Gram-positive filamentous streptomycetes produce ?-butyrolactone (GBL) molecules in a growth rate- and cell density-dependent manner that regulate expression of genes involved in secondary metabolism and morphogenesis. GBL signaling involves the GBL autoregulator, required for GBL biosynthesis, and the GBL binding protein (GBLBP), a TetR-type transcriptional regulator with a GBL binding domain. Several streptomycetes produce secondary metabolite phytotoxins, thaxtomins, which are required for efficient host colonization and infection of plants. The elaboration of host-microbe interaction activities by many gram-negative prokaryotes relies on N-acylhomoserine lactone (AHSL) derivatives, which bear an appreciable structural similarity to GBLs. Since thaxtomins are potent secondary metabolite phytotoxins required for host-microbe interaction, a role for GBL compounds in the regulation of thaxtomin gene expression by gram-positive streptomycetes analogous to that of AHSLs in regulating gene expression in gram-negative organisms is envisioned. This project will employ genetic, physiological and biochemical methods to elucidate the role(s) played by GBL signaling in the biology of thaxtomin-producing organisms. GBL autoregulator and GBLBP mutants will be generated using disruption and replacement techniques and analyzed using physiological studies of growth and morphological development. Quantitative assays of production of thaxtomins and other metabolites will be carried out. Transcript mapping and growth-rate dependent transcriptional analysis of autoregulator and thaxtomin structural genes in wild type and mutant derivatives will be performed. Biochemical analysis of GBLBP will include electrophoretic mobility shift and DNase footprinting assays with DNA regulatory elements. With extensive undergraduate student involvement, the research is expected to advance the limited understanding of streptomycete GBL autoregulator-mediated quorum-sensing activities, particularly with regard to thaxtomin-dependent host-microbe interactions.