Identifying novel components of the riboflavin biosynthetic pathway in invasive bacteria

苜蓿中华根瘤菌属于α-变形菌的根瘤菌目，其还包括动物和人类病原体如布鲁氏菌和巴尔通体。致病性布鲁氏菌和共生性S.苜蓿是不同的，有一个显着的相似之处，他们如何建立和维持慢性细胞感染。类似于布鲁氏菌，其侵入宿主细胞并改变宿主细胞内环境，S。苜蓿侵入豆科寄主植物的根细胞并引发共生器官的发育。在这些结核中，细菌减少大气中的二氮，并为宿主植物的生长提供氨。这使得S。苜蓿根瘤菌是研究入侵细菌与真核宿主相互作用机制的理想模型。氧化还原辅因子FMN和FAD及其前体核黄素（RF）是进行细胞生化反应的许多酶的活性所必需的。有一个强烈的共识，即RF生物合成通过类似的途径在细菌，真菌和植物中进行。然而，RF生物合成途径的分析表明，α-变形菌使用一种以上类型的GTP环化水解酶。我的计划的长期目标是更好地了解黄素的生产和分泌以及控制细菌中这些过程的因素。这里提出的研究的目标是确定RF生物合成途径的新的结构和调控成分，并研究RF生产和分泌的明显不同的途径模块中所涉及的蛋白质之间的相互作用。该假说是α-变形菌具有经典RF生物合成酶的替代物，所述经典RF生物合成酶是进行RF生物合成以满足细菌细胞内需求所需的。我推测，侵入性α-proteobacteria有两个部分重叠的RF生物合成模块，一个是为了满足内部需要的黄素在细菌代谢和其他生产黄素分泌，这是参与细菌-植物通信。这项研究的基本原理是，侵入性细菌中RF生物合成途径的组织与广泛假设的不同，拟议的研究将为RF生物合成的组织提供新的见解。该项目的预期成果是对细菌中RF生物合成网络的结构和调控成分的基本知识，包括入侵病原体和农业上重要的共生体。这将为提高根瘤菌-豆科植物共生固氮效率提供新的途径。这一知识可以进一步扩展到能够合成RF的其他生命系统，并且预计与开发靶向人类病原体中核黄素生物合成酶的抗微生物化合物的尝试高度相关。

Sinorhizobium meliloti belongs to the Rhizobiales order of a-proteobacteria that also includes animal and human pathogens like Brucella and Bartonella. While the outcomes of infection by pathogenic Brucella and symbiotic S. meliloti are different, there is a significant similarity in how they establish and maintain chronic cellular infections. Similar to Brucella spp, which invade host cells and modify the host intracellular environment, S. meliloti invades root cells of legume host plants and triggers development of symbiotic organs. In these nodules, the bacteria reduce atmospheric di-nitrogen and provide ammonia to the host plant for growth. This makes S. meliloti an excellent model to study the mechanism of interaction between invasive bacteria and eukaryotic hosts. The redox cofactors FMN and FAD and their precursor riboflavin (RF) are required for activity of many enzymes that carry out the cell's biochemical reactions. There is a strong consensus that RF biosynthesis proceeds through similar pathways in bacteria, fungi, and plants. However, analysis of the RF biosynthetic pathway showed that a-proteobacteria uses more than one type of GTP cyclohydrolase. The long-term objective of my program is to better understand the production and secretion of flavins and the factors controlling these processes in bacteria. The objectives of the research proposed here are to identify the novel structural and regulatory components of the RF biosynthetic pathway, and to study the interactions between the proteins involved in the apparently distinct pathway modules of RF production and secretion. The hypothesis is that alpha-proteobacteria have alternatives to the classical RF biosynthetic enzymes that are required to carry out RF biosynthesis for the intracellular needs of the bacteria. I speculate that invasive alpha-proteobacteria have two partly overlapping modules for RF biosynthesis, one to satisfy the internal need for flavins in bacterial metabolism and the other to produce flavins for secretion, which is involved in bacteria-plant communication. The rationale for this research is that the organization of the RF biosynthesis pathway in invasive bacteria is different from what has been broadly assumed, and the proposed research will provide novel insights into the organization of RF biosynthesis. The expected outcome of this project is the fundamental knowledge of structural and regulatory components of the RF biosynthetic network in bacteria, including invasive pathogens and agriculturally important symbionts. This will provide new approaches to the improvement of the efficiency of rhizobium-legume nitrogen fixing symbiosis. This knowledge can be further extended to other living systems able to synthesize RF and it is expected to be highly relevant to attempts to develop antimicrobial compounds targeting the riboflavin biosynthetic enzymes in human pathogens.