Novel type of linear mitochondrial genomes with dual flip-flop inversion system in apicomplexan parasites, Babesia microti and Babesia rodhaini

Abstract

Background: Mitochondrial (mt) genomes vary considerably in size, structure and gene content. The mt genomes of the phylum Apicomplexa, which includes important human pathogens such as the malaria parasite Plasmodium, also show marked diversity of structure. Plasmodium has a concatenated linear mt genome of the smallest size (6-kb); Babesia and Theileria have a linear monomeric mt genome (6.5-kb to 8.2-kb) with terminal inverted repeats; Eimeria, which is distantly related to Plasmodium and Babesia/Theileria, possesses a mt genome (6.2-kb) with a concatemeric form similar to that of Plasmodium; Cryptosporidium, the earliest branching lineage within the phylum Apicomplexa, has no mt genome. We are interested in the evolutionary origin of linear mt genomes of Babesia/Theileria, and have investigated mt genome structures in members of archaeopiroplasmid, a lineage branched off earlier from Babesia/Theileria.

Results: The complete mt genomes of archaeopiroplasmid parasites, Babesia microti and Babesia rodhaini, were sequenced. The mt genomes of B. microti (11.1-kb) and B. rodhaini (6.9-kb) possess two pairs of unique inverted repeats, IR-A and IR-B. Flip-flop inversions between two IR-As and between two IR-Bs appear to generate four distinct genome structures that are present at an equi-molar ratio. An individual parasite contained multiple mt genome structures, with 20 copies and 2 - 3 copies per haploid nuclear genome in B. microti and B. rodhaini, respectively.

Conclusion: We found a novel linear monomeric mt genome structure of B. microti and B. rhodhaini equipped with dual flip-flop inversion system, by which four distinct genome structures are readily generated. To our knowledge, this study is the first to report the presence of two pairs of distinct IR sequences within a monomeric linear mt genome. The present finding provides insight into further understanding of evolution of mt genome structure.

Figure 1

Four distinct mitochondrial (mt) genome structures in Babesia microti (A) and Babesia rodhaini (B). These mt genomes possess two pairs of inverted repeats, IR-A and IR-B. Genes shown above bold line are transcribed from left to right and those below from right to left. Light and dark gray blocks indicate fragments of small subunit (SSU) and large subunit (LSU) rRNA genes, respectively. Abbreviations: cox1, cytochrome c oxidase subunit 1 gene; cox3, cytochrome c oxidase subunit 3 gene; cob, cytochrome b gene. Black arrowheads, r1, r2 and r3 in the B. microti mt genome and r in the B. rodhaini mt genome, indicate short direct or inverted repeat sequences (see Additional file 2: Table S1 and Additional file 1: Figure S4).

Figure 2

Southern blot hybridization showing four distinct mitochondrial genome structures in Babesia microti (A) and Babesia rodhaini (B). (A) Genomic regions of probes Bm-1, Bm-2 and Bm-3 and restricted enzyme sites of DraI and Eco065I are shown in the lower map of the B. microti mt genome type-I (see Figure 1-A). Lanes 1, 4 and 7 for undigested DNA, lanes 2, 5 and 8 for DraI-digested DNA, and lanes 3, 6 and 9 for Eco0651I-digested DNA. (B) Genomic regions of probes Br-1, Br-2 and Br-3 and restriction enzyme sites of HindIII and XhoI are shown in the lower map of the B. rodhaini mt genome type-I (see Figure 1-B). Lanes 1, 4 and 7 for undigested DNA, lanes 2, 5 and 8 for DNA digested with HindIII, and lanes 3, 6 and 9 for DNA digested with XhoI (lanes 3, 6 and 9).

Figure 3

A postulated mechanism for inversions of Region A and Region B. Recombination between two IR-As (or two IR-Bs) produces an isomeric form characterized by a flip-flop of Region A (or Region B). Dual flip-flop inversions of IR-A and IR-B can generate four distinct genome structures.

Figure 4

The ML phylogenetic tree of mitochondrial protein coding genes, cox1 and cob, from 17 apicomplexans.Crypthecodinium cohnii was used as an outgroup. Concatenated amino acid sequences (696 sites) were used with 1,000 heuristic replicates under a Jones Taylor, and Thornton model [20] (α = 0.69) for constructing this tree. Numbers shown along nodes represent bootstrap values.