Ticks and other arthropods often are hosts to nutrient provisioning bacterial
endosymbionts,
which contribute to their host’ s fitness by providing nutrients such
as vitamins. It has been detected, in our lab, that Ixodes pacificus is
host to Rickettsia species phylotype G021. This endosymbiont is
universally present and 100% maternally transmitted. To study the role
of phylotype G021 in I. pacificus, bioinformatic and molecular
approaches were carried out. Results from alignments of whole genome
sequence data of I. scapularis by MUMmer and reconstruction of metabolic
pathways by KEGG Pathway Database indicated that phylotype G021 has the
metabolic capability to de novo synthesize folate, commonly known as
vitamin B9. To examine the metabolic capability of the bacterium, genes
of the folate biosynthesis pathway of phylotype G021 were PCR amplified
using degenerative primers. BLAST searches identified that the folA
(encoding dihydrofolate reductase), folC, folE, folKP, ptpS genes
possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to
the corresponding genes of the Rickettsia endosymbiont of I. scapularis.
Phylogenetic tree constructions show that the folate genes of phylotype
G021 and homologous genes from various Rickettsia species are
monophyletic. To further test our hypothesis that phylotype G021 in I.
pacificus synthesizes folate de novo, the FolA recombinant protein was
successfully overexpressed in BL21(DE3) E. coli using IPTG. The
dihydrofolate reductase activity of the FolA protein of phylotype G021,
which was purified using a GST affinity column, was measured by the
decline of absorbance of NADPH at 340 nm in an in vitro enzymatic assay.
The specific activity of FolA of phylotype G021 was calculated to be
8.99 μmol/min/mg. This study has shown that folate genes exist in the
genome of Rickettsia species phylotype G021 and that this bacterium is
capable producing functional FolA protein for de novo synthesis of
folate in I. pacificus.
奈良先端科学技術大学院大学