Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis inBorreliaspp

Abstract

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.Spirochetes are a coherent group of bacteria with a unique morphology and cell structure (38). The point of divergence of spirochetes from other bacterial groups was deep in time, and the topology of spirochetes' branching from other phyla, such as proteobacteria and cyanobacteria, remains unresolved. Phylogenetic inference has been advanced with the availability of genome sequences of pathogenic spirochete species from the following three genera: Borrelia, including Borrelia burgdorferi (13,19) and B. garinii (23), two agents of Lyme borreliosis (LB); Treponema, including Treponema pallidum (20), the agent of syphilis, and Treponema denticola (51), a mouth organism associated with periodontal disease; and two serovars of Leptospira interrogans (36,44). Leptospires have free-living capacity and, not surprisingly, larger chromosomes, at ϳ4.5 Mb, than the ϳ1-Mb chromosomes of the obligately parasitic organisms T. pallidum (20) and B. burgdorferi (18). But the leptospires' branching from the common ancestor of Treponema and Borrelia appears to be nearly as ancient as the spirochetes' origin itself, and for the most part, leptospire sequences have been of limited use for further defining evolution within the spirochete clade.The several Borrelia species can be divided into two major groups on the basis of DNA sequence and biological differences (2). One group includes all the agen...

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.…”

“…3B), the GC skew around the operons for outer surface proteins A and B (bba15 and bba16) and for decorin-binding proteins A and B (bba24 and bba25) is opposite to that of the surrounding sequence that is common to RF and LD species. This is an indication that these operons, which are unique to LD species, were likely acquired horizontally in the LD clade after the descent from an ancestral taxon, as appears to be the case for other operons (50,61).…”

“…kb 36 to 75 of B. hermsii lp174 (Fig. 2C), is just left or upstream of a functional pyrimidine biosynthesis locus containing thyX (bha078), which encodes a thymidylate synthase (50), and the ribonucleotide reductase subunit cluster nrdFEI (bha075 to -077) (38,50). The majority of the coding sequences in this region were on the minus strand (Fig.…”

Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria

“…Heterologous complementation tests have shown that E. coli thyA can be functionally replaced by thyX genes from a variety of sources (9,11,20,29). To investigate whether the reciprocal nonorthologous replacement is possible, we constructed transferable plasmids carrying either the R. sphaeroides thyA gene alone (pDL14) or the R. sphaeroides thyA and folA genes (pDL12) (Fig.…”

“…Previous genetic studies on thyX either were performed using poorly defined genetic backgrounds (7) or were based upon multicopy heterologous complementation systems using either bacterial (9,12,20,29) or viral (10,11) thyX genes.…”