Evolution of Mitochondria Reconstructed from the Energy Metabolism of Living Bacteria

The ancestors of mitochondria, or proto-mitochondria, played a crucial role in the evolution of eukaryotic cells and derived from symbiotic α-proteobacteria which merged with other microorganisms - the basis of the widely accepted endosymbiotic theory. However, the identity and relatives of proto-mitochondria remain elusive. Here we show that methylotrophic α-proteobacteria could be the closest living models for mitochondrial ancestors. We reached this conclusion after reconstructing the possible evolutionary pathways of the bioenergy systems of proto-mitochondria with a genomic survey of extant α-proteobacteria. Results obtained with complementary molecular and genetic analyses of diverse bioenergetic proteins converge in indicating the pathway stemming from methylotrophic bacteria as the most probable route of mitochondrial evolution. Contrary to other α-proteobacteria, methylotrophs show transition forms for the bioenergetic systems analysed. Our approach of focusing on these bioenergetic systems overcomes the phylogenetic impasse that has previously complicated the search for mitochondrial ancestors. Moreover, our results provide a new perspective for experimentally re-evolving mitochondria from extant bacteria and in the future produce synthetic mitochondria.

cytochrome and the frequent association with PQQ-dependent dehydrogenases; its richness in mono-and diheme c cytochromes suggests possible evolutionary relationships with the operon of cbb 3 oxidases. Finally, type a-III characteristically contains a doublet of ctaE/COX3 proteins (Figs. 3A and S4) and is widely distributed among α-proteobacteria (Table S2). Although COX operons of type a are present also in β-and γproteobacteria (Tables S2-S4), COX operon type b is present only in α-proteobacteria, showing no fusion among COX subunits and more variation in the contiguity of the gene sequence than in the gene sequence itself. Indeed, while Beijerinckia or Tistrella have a continous gene sequence, Rhodobacterales and other taxa present different fragmentations of the operon in separate gene clusters (Figs. 3A and S4A). Of note, previous phylogenetic studies on aa 3 cytochrome c oxidases predominantly used the proteins belonging to COX operon type b [6,7,13,47,64].
With regard to the possible evolution of COX operons, we hypothesize that an early duplication of COX4 might have been crucial in the progressive breaking apart of the operon core sequence and the major structural transition of COX3 from the 5-helices to the 7-helices form (Fig. S3), presumably by fusion of a two-helices COX4-like protein. This large molecular change has enhanced COX3 binding to specific lipids, in particular PG1 and PG2 [60] (see alsoTable S4). The resulting dense lipo-protein packing could modulate the entry of oxygen in the catalytic centre [60], thereby reducing the affinity for the oxygen substrate. This molecular change affecting the affinity for oxygen appears to correspond to the juncture between COX operons type a-II lacking a separate COX4 subunit and the transition a-b type (Figs. 3 and S3). We surmise that such a junction might have been concurrent with the separation of the β-and γ-lineages from primordial α-proteobacteria ( Fig. 3C), an event which occurred around the time when oxygen levels strongly increased on the planet [31].  (Table S1B). The asterisk* labels the same subset as in Fig. 1B (main text), but with fewer representative taxa. Underlined organisms are symbionts or pathogens. Each of the six bioenergetic systems presented in Fig. 1 was identified from its catalytic protein subunits and was considered functionally absent when one or more of these subunits were not found in their completeness, as indicated by the profile of their conserved domains (cf. [41]). The functional absence of a given system is represented by an empty square as in Fig. 1B In bold black are the residues that are identical in the aligned position of at least two COX4 sequences, or are positive substitutions [83] across at least three aligned COX4 sequences; they are additionally yellowhighlighted when identical between at least one bacterial COX4 and one mtDNA-encoded protein (cf. A). In bold dark blue are the residues that are positive substitutions between bacterial COX4 and mtDNA-encoded proteins, while those in bold light blue are identical or positive substitutions among the aligned mtDNAcoded proteins. This colour labelling enhances the limited similarity between the sequences shown.
Hypothetical steps in the evolution of COX operons are indicated.   7A). The C terminus of some sequences is truncated at the residue indicated by the numeral before the slash. Key residues for the iron-sulfur cluster, including Y165 influencing its redox potential [71], are in bold. highlighted in pale blue with some conserved residues in gray.

Supporting Information Tables
These tables contain large datasets that do no fit the recommended format of the text and are therefore presented as pasted images; they are also available as Excel files, if requested.   Table S2.
A Table S1a BIONERGETIC SYSTEMS NOTES Genome* Photosynthetic 1. bc1 4. aa3 2. bd -types 5. cbb3 3. bo Nitrogen (N) metabolism (see also     We constructed a matrix of 11 independent characters (indicated concisely on top of the columns) that could differentiate the gene sequence of COX subunits in the mitochondria of some protists from the gene sequence of bacterial COX operons (Table S2). The cumulative phenetic analysis indicate that COX operon type a-II of methylotrophs and Tistrella (highlighted) share the largest number of characters with COX gene clusters of protist mitochondria (F. Comandatore and C. Bandi, unpublished).