Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage

Abstract

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5'-diphosphates to deoxynucleosides-5'-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2'-deoxyribosyltransferase (DRTase, encoded by bb0426), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues.

Fig. 1

Nucleotide incorporation by B. burgdorferi and B. hermsii using a variety of [³H]-labelled ribonucleotide and deoxyribonucleotide pairs as substrates. Error bars represent the standard deviation of triplicate samples, normalized to either DNA or RNA yield. Femtomoles of [³H]-labelled nucleotide incorporated were calculated from the counts per min (cpm) and the specific activity of the labelled substrate.

Fig. 3

Purification and characterization of BB0426_opt. A. Commassie-stained SDS-PAGE gel with (1) E. coli lysate expressing BB0426_opt and eluted fraction from a T7 affinity column. B. HPLC chromatograms showing the enzymatic activity of BB0426_opt. In this assay, deoxyinosine (dIno) served as the deoxyribosyl donor and adenine (Ade) the deoxyribosyl acceptor: deoxyadenosine (dAdo) and hypoxanthine (Hyp) are the products of the reaction.

Fig. 2

Amino acid alignment of the 2′-deoxyribosyltransferase of Lactobacillus species, Ureaplasma and B. burgdorferi BB0426. A. Alignment of Lactobacillus helveticus DRTase I active site residues (indicated by shaded boxes) and the catalytic residue unique to DRTase II (asterisk) (Anand et al., 2004) with the sequences of BB0426 and some other members of Pfam 05014 (Finn et al., 2006). B. Spatial arrangement of amino acid residues involved in deoxynucleoside binding with an dAdo molecule shown in bold (Anand et al., 2004).

Fig. 4

[³H]-Hyp conversion and incorporation into nucleic acids. A. [³H]-Hyp conversion and incorporation into the RNA and DNA of strains B31A3 68-1, ΔguaAB, ΔguaAB/pBSV2G (vector control) and ΔguaAB/pBSV2G guaAB (complement). Femtomoles of [³H]-Hyp incorporated were calculated from the counts per minute (cpm) and the specific activity of the labelled substrate. Error bars represent the standard deviations of triplicate samples. B. HPLC analyses of the incorporation of [³H] into individual deoxynucleosides of DNA isolated from [³H]-Hyp labelled B. burgdorferi cells. The left side of the z-axis, [³H]-Hyp was determined to be incorporated primarily as dGuo. The right of the z-axis shows the per cent composition of unlabelled DNA as a control to verify that DNA digestion went to completion.

Fig. 5

Characterization of guaA::kan and ΔguaAB mutant strains. A. Diagram of gene organization for wild-type guaAB, as well as the insertion points of antibiotic-resistance cassettes for the two mutant strain constructs (guaA::kan and ΔguaAB). The positions of the primers used for PCR are identified by number and correspond to those listed in Table 3. The location of GATase and PP-ATPase catalytic domains in guaA are indicated by rectangular boxes. B. Agarose gel containing PCR products using genomic DNA from the specified B. burgdorferi strains and primer sets corresponding to the target gene of interest. Standards in base pairs are shown on the left. Predicted product size (in base pairs) for each primer pair is indicated in the inset table.

Fig. 6

Proposed purine salvage pathway for B. burgdorferi. This diagram represents a proposed pathway for purine salvage based upon the experimental data presented in this report and genome data annotated in ERGO (Fraser et al., 1997; Overbeek et al., 2003). The deoxyribosyltransferase, BB0426, is shaded to highlight its pivotal role in this pathway. Genes assigned putative activities for non-specific substrates are designated with an asterisk. These assignments were based on activities reported for homologous genes in the BRENDA database (Chang et al., 2009).