Department of Biological Sciences, Humboldt State University, 1 Harpst Street, Arcata, CA, 95521, USA.
Ticks Tick Borne Dis. 2018 Mar;9(3):443-449. doi: 10.1016/j.ttbdis.2017.12.013. Epub 2017 Dec 24.
Although nonpathogenic bacterial endosymbionts have been shown to contribute to their arthropod host's 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. Our lab has previously reported that Rickettsia species phylotype G021 in Ixodes pacificus carries all five genes for de novo folate synthesis, and that these genes are monophyletic with homologs from other Rickettsia species. In this study, the rickettsial folate synthesis folA gene, coding for dihydrofolate reductase, was PCR amplified, cloned into an expression vector, and overexpressed in E. coli. Bioinformatic analysis identified that the FolA protein of phylotype G021 has the conserved DHFR domain, NADP binding sites, and substrate binding sites of bacterial dihydrofolate reductase. SDS-PAGE results showed that recombinant rickettsial FolA protein was overexpressed in BL21(DE3) E. coli in its soluble form. Affinity chromatography was used to purify the protein, and in vitro enzyme assays were performed to assess the biochemical activity of dihydrofolate reductase. The specific activity of recombinant FolA from phylotype G021 was determined to be 16.1 U/mg. This study has revealed that Rickettsia species phylotype G021 of I. pacificus is capable of producing a functional enzyme of the folate biosynthesis pathway, addressing the nutritional interactions behind the symbiosis between Rickettsia species phylotype G021 and its host.
尽管非致病性细菌内共生体通过为节肢动物宿主提供必需的维生素和氨基酸来提高其适应性，但对于蜱虫内共生体共生关系的营养基础知之甚少。我们的实验室之前曾报道过，太平洋硬蜱中的立克次体 G021 型携带从头合成叶酸所需的全部 5 个基因，这些基因与其他立克次体的同源物具有单系性。在这项研究中，我们扩增了立克次体叶酸合成 folA 基因（编码二氢叶酸还原酶），将其克隆到表达载体中，并在大肠杆菌中过表达。生物信息学分析表明，G021 型 FolA 蛋白具有保守的 DHFR 结构域、NADP 结合位点和细菌二氢叶酸还原酶的底物结合位点。SDS-PAGE 结果表明，重组立克次体 FolA 蛋白以可溶性形式在 BL21(DE3)大肠杆菌中过表达。亲和层析用于纯化该蛋白，并进行体外酶测定以评估二氢叶酸还原酶的生化活性。确定 G021 型立克次体的重组 FolA 的比活性为 16.1 U/mg。这项研究揭示了太平洋硬蜱中的立克次体 G021 型能够产生叶酸生物合成途径的功能酶，解决了 G021 型立克次体与其宿主共生关系背后的营养相互作用问题。
