Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes which entered the host cell and were retained as endosymbionts. Here, we model this earliest stage of the endosymbiotic theory of mitochondrial evolution by engineering endosymbiosis between two genetically tractable model organisms, Escherichia coli and Saccharomyces cerevisiae. In this model system, we engineered E. coli strains to survive in the yeast cytosol and provide ATP to a respiration-deficient yeast mutant. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. This readily manipulated chimeric system was stable for more than 40 doublings and should allow us to investigate various aspects of the endosymbiotic theory of mitochondrial evolution.

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae. An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast–E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host–endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.

Endosymbiotic theory suggests that mitochondria were once free-living prokaryotes which entered the host cell and were retained as endosymbionts (1⇓⇓–4). The earliest recognized instance of endosymbiosis, which dramatically shaped the emergence of present-day eukaryotic cells, occurred more than 1.5 billion years ago (5). Previous studies hypothesized that an alphaproteobacterium became established in an archaeal host cell as an endosymbiont and triggered the evolution of the mitochondrion, an organelle specialized for efficient energy production, particularly ATP synthesis (3, 6). However, a recent study suggests that mitochondria evolved from a proteobacterial lineage that branched off before the divergence of all sampled alphaproteobacteria (7). The recent discovery of the Asgard superphylum archaea, which encode in their genomes homologs of cytoskeletal proteins and vesicular trafficking machinery, indicates that the last archaeo–eukaryotic common ancestor could have already featured precursors to the endomembrane system of modern eukaryotes (8).

Significant details have emerged about the nature of the premitochondrial endosymbiont. Studies involving the reconstruction of its genome from endosymbiont genes retained in eukaryotes and genes found in present-day alphaproteobacteria point to a bacterium related to intracellular endosymbionts from the Rickettsiales order (class of Alphaproteobacteria), some of which are human and animal pathogens (9⇓–11). This premitochondrial endosymbiont likely possessed metabolic pathways that included glycolysis, the tricarboxylic acid cycle (TCA), the pentose phosphate pathway, and the fatty acid biosynthesis pathway. The reconstructed endosymbiont is also predicted to have an electron transport chain capable of functioning under low oxygen tension and an ADP/ATP translocase, the protein functionally homologous to the one used by many intracellular bacteria for ATP import from the host cytoplasm.

Herein, we attempted to experimentally recapitulate the early stages of mitochondrial evolution by generating Escherichia coli endosymbionts capable of providing ATP to host yeast cells deficient in ATP synthesis. Such an experimental system with easy-to-manipulate genomes may allow us to attempt evolution of a bacterial endosymbiont with a minimal genome in yeast that recapitulates key features of modern mitochondria.

Results

Experimental Approach. Our initial strategy involved engineering E. coli strains that are auxotrophic for an essential cofactor and that would depend on the host for this cofactor. We further engineered the bacteria to express a functional ADP/ATP translocase and GFP as a marker. As the host cells, we used Saccharomyces cerevisiae strains that had mitochondrial defects and were deficient in utilizing nonfermentable carbon sources (e.g., glycerol) for growth. Introduction of such engineered E. coli cells into the mutant S. cerevisiae cells followed by selection for yeast cells that can grow on a nonfermentable carbon source for multiple generations was expected to afford a stable yeast–E. coli chimera (Fig. 1). Fig. 1. Strategy to engineer S. cerevisiae–E. coli endosymbiont chimera. (A) Wild-type S. cerevisiae can grow on medium with glucose or glycerol due to ATP production by glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria. (B) Yeast cells with a defect in oxidative phosphorylation cannot utilize glycerol for ATP synthesis and cannot grow in the absence of glucose. Introduction of E. coli-expressing ADP/ATP translocase and SNARE proteins into such mutant yeast can restore yeast growth with glycerol as the sole carbon source. Growth of intracellular E. coli is dependent on thiamin diphosphate (vitamin B1) provided by yeast. ER, endoplasmic reticulum; G, Golgi apparatus; M, mitochondria; N, nucleus; V, vacuole.

Engineering an E. coli Strain Expressing Functional ADP/ATP Translocase. We first engineered an E. coli DH10B strain that had key features of an endosymbiont as outlined above. Because thiamin pyrophosphate is an essential bacterial cofactor, deletion of thiamin biosynthetic genes results in thiamin auxotrophy (12, 13). To create growth dependency of an experimental bacterial endosymbiont on the yeast host, the thiamin biosynthetic gene thiC was deleted and replaced with a gene cassette that coded for superfolder gfp and kanamycin resistance genes to afford the E. coli ΔthiC::gfp-kanR strain. We confirmed that the resulting E. coli strain required exogenous thiamin for growth (growth was enabled by the endogenous expression of TbpA, a thiamin transporter, in E. coli ΔthiC::gfp-kanR strain; see SI Appendix, Fig. S1), and that it expressed GFP (SI Appendix, Fig. S2 A and B). Next, we constructed an expression plasmid (pAM94) that encoded the ADP/ATP translocase gene from the intracellular bacterium Protochlamydia amoebophila strain UWE25, a symbiont of Acanthamoeba spp. (14), under control of a pBAD promoter. This construct was transformed into E. coli ΔthiC::gfp-kanR and the cellular activity of the ADP/ATP translocase was assayed. When translocase expression was induced by addition of 1 mM arabinose, and cells expressing the translocase were incubated with [γ-35S]ATP, we observed significant ATP uptake by the bacteria. In contrast, no ATP uptake was observed by the parent E. coli strain, which lacked the translocase-encoding plasmid (Fig. 2A and SI Appendix, Fig. S15A). To test release of ATP from the translocase-expressing E. coli cells, ADP, AMP, or potassium phosphate were separately added to the suspension of cells preloaded with [γ-35S]ATP. A significant, time-dependent drop in cellular radioactivity was observed on incubation with ADP, but not AMP or phosphate, indicating ADP-specific release of intracellular [γ-35S]ATP (Fig. 2A). Next, we analyzed the efflux of intracellular ATP on ADP stimulation during E. coli growth. Levels of extracellular ATP were quantified by a luciferase assay. We observed that cells expressing the ADP/ATP translocase released a significant amount of ATP on addition of ADP to the growth medium. For comparison, the media from E. coli lacking the plasmid pAM94 (expressing the ADP/ATP translocase) produced ∼50-fold lower luciferase signal with or without ADP addition (Fig. 2B and SI Appendix, Fig. S15B). Low levels of ATP were observed in the growth medium of cells expressing the ADP/ATP translocase even without addition of ADP, likely due to a small amount of cell lysis. These studies demonstrated that the E. coli ΔthiC::gfp-kanR cells expressing the translocase released ATP into the extracellular milieu in response to the presence of extracellular ADP. Fig. 2. Release of ATP by E. coli cells encoding ADP/ATP translocase. (A) Cellular [γ-35S]ATP uptake/release by E. coli cells expressing the UWE25 ADP/ATP translocase (pAM94 plasmid) in the presence of 1 mM arabinose. Cellular [γ-35S]ATP was released when E. coli cells expressing the ADP/ATP translocase were challenged with extracellular ADP (10 mM), but not with phosphate (Pi) or AMP (each at 10 mM). (B) Release of ATP into the growth medium by E. coli cells expressing the UWE25 ADP/ATP translocase (pAM94 plasmid) in presence of 20 µM ADP and 1 mM arabinose. The ATP concentration in the medium was determined by luciferase assay. Data bars show a mean of three technical replicates; error bars represent SE of the mean.

Introduction of Engineered E. coli Cells into Yeast. Next, we optimized the protocol for introducing bacteria into yeast cells. Previously, bacterial cells were fused with yeast to clone whole bacterial genomes in yeast (15, 16). Since we wanted to retain intact live bacteria within yeast cells, we decided to use an alternative protocol that was previously developed for introduction of mitochondria into yeast by polyethylene glycol (PEG)-induced fusion (17). To test the latter protocol, we first isolated mitochondria from the respiration-competent YPH500 S. cerevisiae strain, and used a ρ0 S. cerevisiae mutant as the recipient for isolated mitochondria. ρ0 strains completely lack mitochondrial DNA (mtDNA) and are unable to utilize nonfermentable carbon sources for growth. As reported previously, PEG-induced fusion of isolated, respiratory-competent mitochondria with the ρ0 yeast spheroplasts led to the formation of yeast cybrids (cytoplasmic hybrids), which were able to grow on a nonfermentable carbon source (minimal medium + 3% glycerol/0.1% glucose, selection medium I) due to the presence of heterologous, respiring mitochondria in cells (SI Appendix, Fig. S3). Next, we attempted to introduce the engineered E. coli cells (the ΔthiC::gfp-kanR strain expressing the UWE25 ADP/ATP translocase under control of pBAD promoter from the pAM94 plasmid; see SI Appendix, Fig. S4) into the ρ0 mutant by following the same protocol. Similar to fusion with purified mitochondria, we expected to select yeast cells harboring E. coli endosymbionts that could utilize glycerol for the synthesis of ATP and provide it to the S. cerevisiae ρ0 host. However, we did not observe formation of any yeast colonies after the yeast/E. coli fusion mixtures were plated on a selection medium (rich medium + 3% glycerol, 0.1% glucose medium, 1 mM arabinose; selection medium II). Mitochondria of ρ0 yeast cells lack both the electron transport chain and F 1 F 0 ATP synthase (Atp6, Atp8, and Atp9 subunits are encoded in mitochondrial DNA). As a result, the ρ0 yeast cells energize their inner mitochondrial membrane inefficiently by electrogenic exchange of mitochondrial ADP for cytosolic ATP (catalyzed by the mitochondrial ADP/ATP translocase). To sustain ADP/ATP exchange and maintain an energized inner mitochondrial membrane, intramitochondrial ATP in the ρ0 cells undergoes futile hydrolysis to ADP (18). We reasoned that such an unproductive consumption of ATP by ρ0 mitochondria can critically deplete cellular ATP in chimera cells and block their growth. We therefore turned to a yeast mutant with a more limited mitochondrial defect as a host, S. cerevisiae cox2-60 (NB97), which possesses mtDNA but has an insertion in the COX2 gene. As a result, the cox2-60 strain lacks Cox2 protein, does not assemble a functional cytochrome c oxidase complex and, similar to ρ0 mutants, it does not grow in media with a nonfermentable carbon source (19). However, F 1 F 0 ATP synthase expressed in mitochondria of the cox2-60 strain couples hydrolysis of intramitochondrial ATP to proton transport across the inner mitochondrial membrane resulting in more efficient generation of the inner membrane electrochemical potential. When S. cerevisiae cox2-60 spheroplasts were fused with the engineered E. coli cells containing the pAM94 plasmid, a few yeast colonies grew on the selection medium that had plated yeast/E. coli fusion mixtures, but not on plates with control mixtures that had omitted E. coli cells. However, most of the colonies were E. coli. To suppress E. coli (a thiamin auxotroph) growth, we also plated the fusion mixtures on a minimal selection medium, but did not observe formation of any colonies, neither yeast nor E. coli.

Imaging E. coli Endosymbionts Within Yeast Cells by Fluorescence Microscopy. To obtain further evidence for the presence of the E. coli endosymbionts in yeast cells, we imaged the yeast cells from a third round of replating with total internal reflection fluorescence (TIRF) microscopy to detect the expression of the bacteria-encoded GFP protein within the yeast cells. We observed the presence of compartmentalized GFP-positive signals in the fused yeast cells, but not in the parental control NB97 yeast cells (Fig. 4A). Similarly, cox2-60 S. cerevisiae–E. coli chimeras and control S. cerevisiae were imaged by fluorescence confocal microscopy. The S. cerevisiae cell wall was labeled with FITC-labeled Con A (a lectin which binds to the yeast cell wall) (27), and the E. coli endosymbionts were labeled with a EUB338-Cy3 FISH probe, which can detect bacterial rRNA (28). We detected FISH probe signals only in the yeast–E. coli chimera cells, but not in control cells (Fig. 4B), further confirming the presence of E. coli endosymbionts in yeast cells. Fig. 4. Imaging intracellular endosymbiont E. coli by fluorescent microscopy. (A) TIRF microscopic images of chimeric cells (Right) and control yeast cells (Left). Two representative cells of indicated chimera type are shown. All panels are merged images of TIRF (green) and differential interference contrast (grayscale). (Scale bar in the NB97 panel, 10 µm; scale bar in the NB97–E. coli ΔthiC panel, 5 µm.) (B) Confocal fluorescence microscopy images of control and chimeric yeast–E. coli cells. Yeast cell wall was stained with Con A-FITC (blue) and bacterial rRNA with EUB338-Cy3 probe (purple). Yellow arrowheads indicate examples of EUB338-positive yeast cells. (Scale bar in the Middle, 10 µm.)

Mimicking Mitochondrial Genome Reduction with Additional E. coli Auxotrophies. Having established the presence of E. coli endosymbionts in yeast cells, we next investigated if we could begin to replicate genome reduction events that occurred during mitochondrial evolution. As a first step, we investigated if a strain of E. coli auxotrophic for another key cofactor could generate endosymbionts within yeast. We deleted the NAD biosynthetic gene nadA by replacing it with the gfp-kanR gene cassette in E. coli DH10B (to afford E. coli ΔnadA::gfp-kanR) and confirmed that the resulting E. coli strain is a NAD auxotroph (growth in media with NAD+ enabled by E. coli pnuC transporter) (29). We then transformed this strain with the pAM136 plasmid (coding for the ADP/ATP translocase and three SNARE-like proteins), fused it with the S. cerevisiae cox2-60 spheroplasts, and plated the fusion mixtures on selection medium II. As before, most of the colonies that formed on the plates were yeast. We again confirmed the presence of E. coli and yeast genomes by PCR (SI Appendix, Fig. S8). These chimeras were also stable for four rounds of replating (>40 generations, Fig. 3A) and displayed a compartmentalized, E. coli-encoded GFP signal by TIRF microscopy (Fig. 4A) as well as bacterial RNA FISH signal by confocal fluorescence microscopy (Fig. 4B). We also assessed the fraction of S. cerevisiae cox2-60–E. coli ΔnadA::gfp-kanR chimeric cells present in the yeast culture grown on selection medium II. We plated a single cell suspension of one such culture on plates with nonselective (YPD + carbenicillin, allows growth of all yeast cells) and selective (selection medium II + carbenicillin, allows growth of chimeric cells only) media. We observed formation of a comparable number of colonies on both media (Fig. 3B), suggesting that most of yeast cells in the culture contained intracellular E. coli. This conclusion was further confirmed by detecting by PCR the E. coli gfp gene in 10 randomly selected yeast colonies from selection medium II (Fig. 3C). Next, we generated a thiamin/NAD double auxotroph of E. coli DH10B, transformed it with pAM136, and used these cells in fusions with yeast as before. Formation of yeast colonies was again observed on selection plates (selection medium II) and the chimeras could be replated for four rounds on the selection medium III in the presence of carbenicillin (Fig. 3A). Additionally, we observed the E. coli-encoded GFP signal in these chimeric yeast cells by TIRF microscopy (Fig. 4A). Further, we confirmed the presence of both genomes by PCR amplification of the MATa gene for yeast and gfp and kanR genes for E. coli (SI Appendix, Fig. S9). Finally, we transformed a serine auxotroph of E. coli (E. coli ΔserA::kanR) with pAM136 plasmid and fused it with the S. cerevisiae cox2-60 spheroplasts to select chimeras on selection medium II, which were further replated on selection medium III for three rounds of growth. The presence of E. coli and yeast genomes was demonstrated by PCR during each round of replating (SI Appendix, Fig. S10). These preliminary experiments suggest that it might be relatively straightforward to eliminate a significant fraction of the E. coli genome by complementing essential bacterial metabolites and other cellular building blocks with those from the yeast cytosol.