Purine salvage pathways among Borrelia species

Abstract

Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.

FIG. 1.

Graphical representation of the ORFs in B. hermsii. (A) Chromosomal genes hpt, purA and purB; (B) plasmid-encoded genes nrdI, nrdE, and nrdF. In B. hermsii hpt, purA, and purB are located between orthologous genes BB0421 and BB0422 in B. burgdorferi. The ERGO bioinformatics suite (Integrated Genomics, Chicago, IL) (34) was used to produce these genetic maps. The scale bars indicate size in base pairs.

FIG. 2.

Southern blot analysis for the presence of purB and nrdF among Borrelia species. (A) Ethidium bromide-stained agarose gel of EcoRI-digested genomic DNA. (B) Hybridization pattern with the purB probe. (C) Hybridization pattern with the nrdF probe. Molecular size standards are shown on the left.

FIG. 3.

PRT activities of B. burgdorferi and B. hermsii cell lysates with different purines as substrate. Activity was measured in femtomoles of purine base converted to ribonucleotide per milligram of lysate per minute. Error bars indicate standard deviations.

FIG. 4.

[³H]hypoxanthine incorporation into nucleic acids. The values are based on two measurements from each of three independent assays. The counts per minute per microgram of nucleic acid are represented by the mean and one standard deviation.

FIG. 5.

Southern blot analysis for the adenine deaminase gene (ade) in B. hermsii. (A) Agarose gel with genomic DNA digested with EcoRI and stained with ethidium bromide. (B) Hybridization with the probe containing the sequence present in both the intact gene and pseudogene. (C) Hybridization with the probe with the sequence present only in the intact gene. Isolate designations are indicated above each lane. Molecular size standards are shown on the left.

FIG. 6.

Schematic representation of purine metabolism pathways identified in B. burgdorferi, B. hermsii, and B. turicatae. Solid arrows represent enzymatic steps associated with their corresponding gene designation identified in Table 3. Genes present only in B. hermsii and B. turicatae are in a box, while those present in all three species are not enclosed. The dashed arrow represents a pathway with no gene yet identified. This figure was modified from reference .