Biochemical characterization of the folate biosynthetic pathway of a rickettsial endosymbiont of Ixodes pacificus
Graduation Date
2015
Document Type
Thesis
Program
Other
Program
Thesis (M.S.)--Humboldt State University, Biology, 2015
Committee Chair Name
Jianmin Zhong
Committee Chair Affiliation
HSU Faculty or Staff
Keywords
Humboldt State University -- Theses -- Biology, Biochemical characterization, Folate biosynthesis pathway, Ixodes pacificus, Rickettsia, Lyme disease vector, Protein purification, Symbiosis
Abstract
Nonpathogenic bacterial endosymbionts have been shown to contribute to their arthropod hosts' fitness by supplying them with essential vitamins and amino acids. Little is known about the nutritional basis for the symbiotic relationship of endosymbionts in ticks. Recent metabolic reconstructions in our laboratory have shown that Rickettsia species phylotype G021 in Ixodes pacificus carries all five genes for de novo folate synthesis. Folates are one-carbon donors in purine biosynthesis, act as cofactors in the formation of methionine as well as in protein and DNA methylation reactions, and hence in epigenetic modifications. In this study, sequence annotations, recombinant protein expressions and purifications, and in vitro enzyme assays were used to study de novo folate biosynthesis of the Rickettsia species. The rickettsial folate genes (folA, folC, folE, folKP, and ptpS) were amplified by PCR. Sequencing and bioinformatic analyses have identified the putative promoter and ribosome binding site as well as the functional domain of the open reading frame for each folate gene. The folA and folKP gene loci are located in a multi-cistronic operon; BLAST searches confirmed that several species of Rickettsia and Wolbachia have the same folA-folKP gene organization. The amplified folate genes were digested and cloned into the pET-41a(+) expression vector, and transformed into the competent expression host BL21(DE3). SDS-PAGE results showed that all five recombinant rickettsial proteins were overexpressed in BL21(DE3) E. coli and that the FolA protein was overexpressed as a soluble recombinant protein, whereas FolC, FolE, FolKP, and PtpS proteins were overexpressed as insoluble proteins. Folate proteins were purified by either affinity column chromatography or batch method. In vitro enzyme assays were performed using purified proteins to assess the biochemical function of dihydrofolate reductase activity of the rickettsial FolA and GTP cyclohydrolase I activity of the FolE protein. The specific activity of FolA and FolE protein of phylotype G021 was calculated to be 16.1 U/mg and 7.9 U/mg, respectively. This study has shown that folate genes exist in the genome of Rickettsia species phylotype G021 of I. pacificus and are capable of producing functional enzymes for making folate, confirming the nutritional interactions of the symbiosis between Rickettsia species phylotype G021 and Ixodes pacificus.
Recommended Citation
Bodnar, Jimmy, "Biochemical characterization of the folate biosynthetic pathway of a rickettsial endosymbiont of Ixodes pacificus" (2015). Cal Poly Humboldt theses and projects. 2261.
https://digitalcommons.humboldt.edu/etd/2261
https://scholarworks.calstate.edu/concern/theses/dz010s563