Identifying novel components of the riboflavin biosynthetic pathway in invasive bacteria

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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.