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.

Large Linear Plasmids of Borrelia Species That Cause Relapsing Fever

“…Only 5 to 10% of the deduced ORFs of the 3 large plasmids defined here had identifiable homologs to proteins from organisms outside the Borrelia genus (see Tables S2 and S3 in the supplemental material). These ORFs encode products involved with nucleotide biosynthesis (thyX, nrdE, nrdF, and nrdI) (38,50) and putative bacterial plasmid replication and partitioning (paralogous family 32 [PFam32], PFam49, PFam50, and PFam57) (16,43). The remaining protein sequences of ORFs were found to be homologous to proteins found in LD Borrelia species as well as in RF species.…”

“…Sanger sequencing of shotgun libraries of 2-to 3-kb fragments of genomic DNA was carried out as described previously (31,38). For the present study, pyrosequencing using 454 FLX technology (Roche, Branford, CT) also was performed following the manufacturer's recommended protocols.…”

Helicobacter pylori Relies Primarily on the Purine Salvage Pathway for Purine Nucleotide Biosynthesis

“…The existence of other bacteria lacking de novo synthesis pathways points out that, given the right microenvironment, purine salvage can be sufficient within a human host (36,55). As many as 10 years ago, loss of the purine de novo nucleotide biosynthesis pathway was thought to represent a relatively rare evolutionary event.…”

“…Similarly, the purine salvage pathway has also been investigated in numerous organisms (42,55). PRTases, the enzymes capable of joining salvaged purine bases with PRPP in order to generate purine nucleotides, have also been studied in numerous pathogens (17,18,38,39).…”

“…These nucleotides can then be fed into the nucleotide biosynthesis pathway, where they can then be used to generate GMP or AMP. Additionally, based on the established pathway, enzymes such as GMP reductase (GuaC) and adenine/adenosine deaminases (Add/Ade) allow the cycling of nucleotides by creating bypasses in the pathway, thereby allowing for various purine bases to be used for generating both GMP and AMP, instead of only one or the other (46,55).…”