Nonpathogenic nutrient-providing bacterial endosymbionts have been shown to contribute to their hosts' fitness by supplying them with essential vitamins and amino acids. The metabolic basis for the symbiotic relationship of endosymbionts in ticks has been unknown. Recent metabolic reconstruction, PCR, and DNA sequencing results in our lab have shown that a Rickettsia species in Ixodes pacificus carries genes for the anabolism of cofactors and vitamins. The biosynthetic pathway of folate (vitamin B9) is of particular interest because previous bioinformatic metabolic reconstruction in our lab indicates that the pathway exists in its entirety in the genome of the Rickettsia species. The overall objective of the project is to study bacterial vitamin biosynthetic genes that are essentia for the symbiotic relationship with the tick host. We hypothesize that the endosymbiotic Rickettsia species synthesizes folate, and that the vitamin is utilized by the tick host. The proposed work has three major aspects: 1) Characterization of genes in the folate pathway of the Rickettsia species in Ixodes pacificus using PCR, cloning, and sequencing. 2) Characterization of gene expression patterns of genes in the folate pathway of the Rickettsia species in Ixodes pacificus using quantitative reverse transcriptase PCR (qRT-PCR). 3) Characterization of function of gene products in the folate biosynthetic pathway of the Rickettsia species in Ixodes pacificus by complementation and in vitro enzymatic assay. The research will increase our understanding of the metabolism of folate in ticks, as well as the metabolic basis of symbiotic interactions between the symbiotic Rickettsia species and Ixodes pacificus ticks. Describing the functions of the biosynthetic genes and defining the capacity of the vitamins contributed by the Rickettsia species in Ixodes pacificus, will provide important insights into molecular mechanisms of the endosymbiotic relationship in Ixodes pacificus. PUBLIC HEALTH RELEVANCE: Lyme borreliosis and anaplasmosis are the most common tick-borne diseases in the United States, and prevention of these diseases has become the focus of current tick research. Bacteria that live within eukaryotic cells have allowed their hosts to acquire metabolic capabilities otherwise unavailable. These bacterial partners contribute to their hosts' fitness by providing essential nutrients such as vitamins and amino acids. Arthropods that feed primarily or entirely on blood are virtually a breeding ground for these types of partnerships because vertebrate blood rarely contains sufficient quantities of essential B-vitamins and amino acids. Therefore, the diet of blood feeding arthropods is complemented with bacterially synthesized vitamins (2, 5, 76). The sole diet of most ticks is blood from vertebrates, but the sources of B-vitamins for Ixodes pacificus ticks are unknown. The recent sequencing of the Ixodes scapularis genome revealed genomic sequences of the tick's bacterial partner. Using freely available computer software, as well as computer algorithms written in our lab, we have discovered all six genes involved in the vitamin B9 biosynthetic pathway. The pathway is from a Rickettsia species in Ixodes pacificus, the West Coast relative of Ixodes scapularis. In this proposal, we hypothesize that the Rickettsia species, living within Ixodes pacificus cells, produces essential B9 vitamin that is not synthesized by Ixodes pacificus. We will determine if all genes in the B9 vitamin pathway of the Rickettsia species are actively transcribed in Ixodes pacificus ticks, and if these bacterial genes encode proteins that synthesize vitamin B9. The proposed studies will offer an ideal model system to study the impact of bacterial endosymbionts on the metabolism of ticks and more specifically, the effect of the Rickettsia endosymbiont on Ixodes pacificus fitness.