Mitochondrial Genome of Palpitomonas bilix: Derived Genome Structure and Ancestral System for Cytochrome c Maturation

Abstract

We here reported the mitochondrial (mt) genome of one of the heterotrophic microeukaryotes related to cryptophytes, Palpitomonas bilix The P. bilix mt genome was found to be a linear molecule composed of "single copy region" (∼16 kb) and repeat regions (∼30 kb) arranged in an inverse manner at both ends of the genome. Linear mt genomes with large inverted repeats are known for three distantly related eukaryotes (including P. bilix), suggesting that this particular mt genome structure has emerged at least three times in the eukaryotic tree of life. The P. bilix mt genome contains 47 protein-coding genes including ccmA, ccmB, ccmC, and ccmF, which encode protein subunits involved in the system for cytochrome c maturation inherited from a bacterium (System I). We present data indicating that the phylogenetic relatives of P. bilix, namely, cryptophytes, goniomonads, and kathablepharids, utilize an alternative system for cytochrome c maturation, which has most likely emerged during the evolution of eukaryotes (System III). To explain the distribution of Systems I and III in P. bilix and its phylogenetic relatives, two scenarios are possible: (i) System I was replaced by System III on the branch leading to the common ancestor of cryptophytes, goniomonads, and kathablepharids, and (ii) the two systems co-existed in their common ancestor, and lost differentially among the four descendants.

Keywords: Cryptista; cytochrome c maturation; inverted repeats; mitochondrial genome.

Fig. 1.—

Palpitomonas bilix mitochondrial genome. (A) Putative mitochondrial (mt) genome structure. Inverted repeats are shown in blue, and the central portion of the mt genome (single copy region) is shown in yellow. The DIG probe was designed to hybridize atp1 gene region in inverted repeats (open arrowheads). According to the monomeric linear form of the P. bilix mt genome, XbaI digestion is anticipated to produce ∼44 kb- and ∼32 kb-fragments. Likewise, ∼5.3 kb- and ∼9.6 kb-fragments are likely to be detected by Southern blot analyses following EcoRI and SalI digestion, respectively. (B) Gene order. Protein-coding genes and ribosomal RNA genes are shown by boxes, and their putative functions are color-coded (see the inset). Transfer RNA and transfer-messenger RNA genes are shown by lines.

Fig. 2.—

Southern blot analyses of the Palpitomonas bilix mitochondrial genome. A DIG DNA probe set within atp1 gene region (fig. 1) was used in all the Southern blot analyses in this study. (A) Analysis of the DNA sample separated by pulsed-field gel electrophoresis (PFGE). The DNA samples with and without XbaI digestion were run in lanes 1 and 2, respectively. The sizes of the detected DNA fragments were found to be consistent with the predicted monomeric linear form of the mitochondrial (mt) genome (fig. 1A). Note that the precise sizes of the two DNA fragments were difficult to estimate from the PFGE image. (B). Analysis of the DNA samples digested by EcoRI and SalI. Both restriction enzymes generated the DNA fragments, the sizes of which were found to be similar to those expected from the mt genome contig reconstructed from Illumina pair-end short reads (fig. 1A).

Fig. 3.—

Venn diagram to compare the gene content among the mitochondrial genomes of Palpitomonas bilix and two cryptophytes (Rhodomonas salina and Hemiselmis andersenii). Note that tatA, rps1, and atp4 are not annotated in the GenBank file of the R. salina mitochondrial (mt) genome (GenBank accession no. NC_002572), but listed in the genome map (fig. 1) in Hauth et al. (2005). Only functionally assignable and vertically inherited protein-coding genes are included in this diagram. Functionally unassignable genes in the three mt genomes are indicated in parentheses.

Fig. 4.—

Scenarios for the evolution of cytochrome c maturation system in Palpitomonas bilix, kathablepharids, goniomonads, and cryptophytes. The phylogenetic relationship amongst the four lineages/species is based on Yabuki et al. (2014). (A) Scenario 1 assuming no co-existence of Systems I and III. Under this assumption, the distribution of Systems I and III can be predicted as follows. Although no nuclear genome is available for Palpitomonas bilix, we can conclude that this organism lacks a HCCS gene for System III, as its mitochondrial (mt) genome contains ccm genes for System I. Likewise, although no mt genome data is available for goniomonads or kathablepharids, we can conclude that the two lineages lack ccm genes for System I, as HCCS genes were identified in their transcriptomic data. By combining both nuclear and mt genome data of cryptophytes, this lineage most probably possesses a single system for cytochrome c maturation (i.e., System III). For each of the four lineages/species, the presence/absence (+/−) of ccm genes and a HCCS gene is indicated. This scenario assumes (i) the ancestral cell used the bacterial system (i.e., System I; designated as “sI”), and (ii) the switch from System I to System III (designated as “sIII”) occurred in the common ancestor of kathablepharids, goniomonads, and cryptophytes. (B) Scenario 2. This scenario assumes (i) the ancestral cell possessed the two evolutionarily distinct systems, and (ii) System I or System III was differentially lost on the branch leading to P. bilix and that leading to the common ancestor of kathablepharids, goniomonads, and cryptophytes. If the co-existence of Systems I and III is possible in a single cell, some uncertainties about the distribution of Systems I and III remain (represented by question marks). As no nuclear genome is available for P. bilix, it is uncertain whether a HCCS gene (System III) is absent in this organism. Likewise, no complete mt genome is available for kathablepharids or goniomonads, and the presence of ccm genes for System I in either (or both) lineages remains unclear.