BIOCHEMICAL CHARACTERIZATION OF THE FOLATE BIOSYNTHETIC PATHWAY IN A RICKETTSIAL ENDOSYMBIONT OF IXODES PACIFICUS
Sergio Fitch, Jianmin Zhong.
Humboldt State University, Arcata, CA.
Ixodes pacificus ticks have been identified as the paramount vector for Lyme disease and anaplasmosis in North America. Recently, a novel Rickettsia species classified as phylotype G021 has been identified as living within the tissue of I. pacificus. Its genome contains the 6 genes (folA, folC, folE, folK, folP, and ptpS) required for de novo folate biosynthesis. Since I. pacificus cannot produce folate nor obtain it from blood, it was hypothesized it obtains this essential nutrient from the Rickettsia endosymbiont. To address this hypothesis, recombinant protein expression of these enzymes was used to study de novo folate biosynthesis. The 6 rickettsial folate genes were PCR amplified and cloned into a pET-41a(+) expression vector. The clones were transformed into competent non-expression NovaBlue E. coli, and later into a competent high-expression BL-21 E. coli strain. The clones were verified by digestion, and the correct open reading frame for each gene was verified by sequencing. FolA, FolC, FolE, and PtpS have been overexpressed and optimized by different incubation temperatures, induction time, and IPTG concentrations. The amount of soluble and insoluble portions of the overexpressed proteins were determined and analyzed by SDS-PAGE. We found that the genes were successfully amplified and cloned into the expression vector in the correct reading frame and 4 of them have been successfully expressed for protein analysis. This study has shown that folate genes exist in the genome of a novel rickettsial endosymbiont of I. pacificus and may be capable of producing functional enzymes, providing fitness advantages to the host.