Identifying novel components of the riboflavin biosynthetic pathway in invasive bacteria

苜蓿中华根瘤菌属于a-变形杆菌的根瘤目，也包括动物和人类的病原体，如布鲁氏菌和巴通氏菌。虽然致病性布鲁氏菌和共生苜蓿链球菌感染的结果不同，但它们在建立和维持慢性细胞感染方面有很大的相似之处。与布鲁氏菌类似，布鲁氏菌入侵寄主细胞，改变寄主细胞内环境，侵染豆科植物的根细胞，触发共生器官的发育。在这些根瘤中，细菌还原大气中的二氮，并为寄主植物提供氨以供生长。这使得苜蓿链霉菌成为研究入侵细菌与真核宿主相互作用机制的良好模型。氧化还原辅助因子FMN和FAD及其前体核黄素(RF)是许多执行细胞生化反应的酶的活性所必需的。有一个强烈的共识，即RF的生物合成在细菌、真菌和植物中通过类似的途径进行。然而，对RF生物合成途径的分析表明，a-蛋白细菌使用不止一种类型的GTP环水解酶。我这个项目的长期目标是更好地了解黄素的产生和分泌，以及细菌中控制这些过程的因素。这项研究的目的是确定RF生物合成途径的新的结构和调节成分，并研究参与明显不同的RF产生和分泌途径模块的蛋白质之间的相互作用。假设α-蛋白细菌有经典的RF生物合成酶的替代品，这些酶是进行RF生物合成所必需的，以满足细菌的细胞内需求。我推测，入侵的α-蛋白细菌具有两个部分重叠的RF生物合成模块，一个用于满足细菌新陈代谢对黄素的内部需求，另一个用于产生分泌所需的黄素，这与细菌-植物的交流有关。这项研究的基本原理是，入侵细菌中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节组件的基本知识，包括入侵病原体和在农业上具有重要意义的共生体。这将为提高根瘤菌-豆科植物固氮共生效率提供新的途径。这一知识可以进一步扩展到其他能够合成RF的生命系统，并有望与开发针对人类病原体中核黄素生物合成酶的抗菌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.