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The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme.

ABSTRACT: 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.

SUBMITTER: Bodnar JL

PROVIDER: S-EPMC5857425 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme.

Bodnar James L JL Fitch Sergio S Rosati Allison A Zhong Jianmin J

Ticks and tick-borne diseases 20171224 3

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. I ...[more]

PMID: 29284564

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Project description:A previously unrecognized Rickettsia species was isolated in 1976 from a pool of Ixodes pacificus ticks collected in 1967 from Tillamook County, Oregon, USA. The isolate produced low fever and mild scrotal oedema following intraperitoneal injection into male guinea pigs (Cavia porcellus). Subsequent serotyping characterized this isolate as distinct from recognized typhus and spotted fever group Rickettsia species; nonetheless, the isolate remained unevaluated by molecular techniques and was not identified to species level for the subsequent 30 years. Ixodes pacificus is the most frequently identified human-biting tick in the western United States, and as such, formal identification and characterization of this potentially pathogenic Rickettsia species is warranted. Whole-genome sequencing of the Tillamook isolate revealed a genome 1.43 Mbp in size with 32.4 mol% G+C content. Maximum-likelihood phylogeny of core proteins places it in the transitional group of Rickettsia basal to both Rickettsia felis and Rickettsia asembonensis. It is distinct from existing named species, with maximum average nucleotide identity of 95.1% to R. asembonensis and maximum digital DNA-DNA hybridization score similarity to R. felis at 80.1%. The closest similarity at the 16S rRNA gene (97.9%) and sca4 (97.5%/97.6% respectively) is to Candidatus 'Rickettsia senegalensis' and Rickettsia sp. cf9, both isolated from cat fleas (Ctenocephalides felis). We characterized growth at various temperatures and in multiple cell lines. The Tillamook isolate grows aerobically in Vero E6, RF/6A and DH82 cells, and growth is rapid at 28 °C and 32 °C. Using accepted genomic criteria, we propose the name Rickettsia tillamookensis sp. nov., with the type strain Tillamook 23. Strain Tillamook 23 is available from the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (WDCM 1093), Atlanta, GA, USA (CRIRC accession number RTI001T) and the Collection de Souches de l'Unité des Rickettsies (WDCM 875), Marseille, France (CSUR accession number R5043). Using accepted genomic criteria, we propose the name Rickettsia tillamookensis sp. nov., with the type strain Tillamook 23 (=CRIRC RTI001=R5043).

Dataset Information

The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme.

Publications

The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme.

Similar Datasets

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