The theory of endosymbiosis is a theory that was developed principally by an American biologist called Lynn Margulis in the 1960s. At its most simple level, the theory suggests that the modern Eukaryotic cell evolved from symbiotic associations with prokaryotic ancestors. Free-living bacteria and photosynthetic cyanobacteria became incorporated inside larger nucleated prokaryotic cells, where they developed into the forerunners of the mitochondria and chloroplasts seen in modern eukaryotes. Margulis postulates that these events have occurred on several occasions, producing various lineages of both heterotrophic and phototrophic organisms, from which ancestors of animals, plants and fungi have evolved. Evidence for the theory is relatively strong, particularly the finding that mitochondria and chloroplasts have circular DNA similar in form to that of bacterial DNA, and that they contain prokaryotic type ribosomes. The double membrane, and mitochondrial specific transcription and translation machinery all point to this conclusion.
Discussion of the Theory & Examination of Supporting Evidence
Looking at the phylogenetic tree of life (Figure 1) it is widely recognised that from the root it split in two separate directions. Bacteria in one direction and what would eventually diverge into the separate domains know as Archaea and Eukarya in the other direction.
The exact time of origin of eukaryotes is not pinpointed in the fossil record. Prokaryotes were definitely first seen 3.5 x 10 9 years ago, and the first undoubted eukaryotes (which would have been multicellular algae) were seen 0.9-0.8 x 10 9 years ago. The size of cells in microfossils remained constant from their earliest appearance until about 1.6 x 10 9 years ago. After this point, the size of some of the cells began to increase (1.4-1.2 x 10 9 years ago). This is interpreted by some as the approximate time protoeukaryotes or eukaryotes first developed.
The evolutionary origins of eukaryotes can be grouped into two main categories of theories; autogenous theories and symbiotic theories. In autogenous theories it is suggested that all structures and functions of eukaryotes evolved gradually from a single stock of prokaryotes. One common feature in this type of theory is the proposed infolding of regions of the cell membrane forming internal vesicles, which subsequently evolved into the various organelles.
In symbiotic theories it is thought that certain eukaryotic organelles evolved from prokaryotic organisms, which entered into symbiosis with an ancestor of eukaryotic cells, "the protoeukaryote".
The theory is known as the endosymbiotic theory, literally meaning "inside symbiont" or "internal symbiont". The theory suggests that a stable residence was established by aerobic bacteria inside the cytoplasm of a primitive eukaryotic-like cell, providing the cell with energy in return for a protected environment and an easily obtainable source of nutrients. This symbiotic relationship created what was to be the forerunner of the mitochondrion in the modern eukaryotic cell. Similarly a primitive eukayrote would have gained photosynthetic properties after the endosymbiotic uptake of an oxygen producing phototroph, the forerunner of the modern chloroplast.
Evidence suggests that mitochondria probably arose from a major group of Bacteria called the Proteobacteria, specifically from relatives like the Agrobacterium, Rhizobium and the rickettsias. Like mitochondria, the latter organisms are capable of an intracellular existence either within plants or animals.
Although Margulis and her colleagues were working on the endosymbiotic theory in the 1960s, the theory had been partially anticipated by early scholars & naturalists. Ivan E. Walin (1883-1969) was an anatomist at the University of Colorado and one of Margulis' predecessors. In his book he argued "tissues, organs, organisms and even species originated by the establishment of long term symbiogenesis" (1). Wallin did not specifically use the word symbiogenesis, but his basic principle was the same. He wrote of eukaryotic symbiosis with bacteria, a process he called "the establishment of microsymbiotic complexes" or "symbonticism". He believed that mitochondria were of bacterial origin as they were "indistinguishable by sight"(2) from bacteria. After intense criticism at the time he stopped researching his idea, only for it to be proved at least partially correct after his death.
The outline of Margulis' Serial Endosymbiosis Theory (SET), is that a sulphur and heat loving kind of bacterium called a fermenting "archaebacterium" (or "thermoacidophil"), merged with a swimming bacterium. The complete SET phylogeny is shown in figure 2. Together the two integrated components of the merger became "nucleocytoplasm"(1), the basic substance of the precursors of plant, animal and fungal cells.
Precursor cells, similar to these are then thought to have serially engulfed different free-living bacteria over time in separate endosymbiotic events.
This type of transient engulfment of prokaryotic cells by larger cells is not uncommon in the microbial world. In regard to mitochondria, such transient relations became permanent as the bacterial cell lost DNA, making it incapable of independent living, and the host cell became dependent on the ATP generated by its tenant. This is generally referred to as "middle ground" SET, as this part of the theory has a considerable amount of supporting evidence, and is widely accepted.
As well as suggesting that mitochondria and chloroplasts are endosymbiants of bacterial origin, Margulis' proposed a version of SET that was taken by many as "extreme". In this "extreme" version, as well as chloroplasts, mitochondria and the nucleocytoplasm descending from bacteria, Margulis postulates that cilia, sperm tails and other appendages arose from fusion with a swimming bacterium. She theorises that this fusion was occurred 2000 million years ago, before the engulfment of mitochondria and chloroplasts. There is however little hard evidence to support this extension of the theory
Mitochondria are among the larger organelles in the cell, each being about the size of an E. coli bacterium. Most eukaryotic cells contain mitochondria, which collectively occupy about 25% of the volume of the cytoplasm. They are large enough to be seen under a light microscope, but a detailed examination can only be carried out using an electron microscope. They have a smooth outer membrane, while the inner membrane has many invaginations called cristae. The membranes form two compartments, the intermembrane space between the two membranes and the matrix or central compartment. The outer membrane contains a mitochondrial porin. This is a transmembrane channel protein and is very similar in structure to bacterial porins.
With the exception of vacuoles, chloroplasts are the largest and most characteristic organelles of plants and algae, and can vary in size and shape. As well as the double membrane that encompasses a chloroplast, this organelle also contains an extensive internal system of interconnected membrane-limited sacs called thylakoids. These thylakoids, which are embedded in the matrix, are flattened to make disks, which often form stacks called grana. Thylakoid membranes contain green pigments (chlorophylls) and other pigments that absorb light, as well as enzymes that generate ATP during photosynthesis.
Most DNA in eukaryotes is found in the nucleus, however, around the beginning of the 20 th century, two plant scientists working independently (H. De Vries & C. Correns) found chloroplasts to have their own genes, rediscovering Mendel's 'factors' or genes. Further research has shown small amounts of DNA, arranged in a covalently closed circular form, to be present in the mitochondria of plants, animals and fungi and in the chloroplasts of plants. As well as being circular in nature, it has a single start site for DNA replication, and its genes are arranged in operons, which are sequences of functionally related genes under common control. This circular DNA is a typical trait of prokaryotes and enforces the theory of a previously free-living lifestyle, before beginning a symbiotic residence within eukaryotic cells many hundreds of thousands of years ago. However, if these organelles were indeed free living at one time, they would have had similar amounts of DNA to modern bacteria. This expected quantity of DNA is significantly greater than the amount found in modern mitochondria and chloroplasts.
The genomes of mitochondria range broadly in size across species. The mitochondrial genome of the protist Plasmodium falciparum has fewer than 6 kbp, while human mitochondrial DNA consists of 16,569 bp and encodes 13 respiratory-chain proteins. However, mitochondria also contain many proteins encoded by nuclear DNA.
The low DNA content of mitochondria and chloroplasts is accounted for in the theory, by assuming a substantial loss and transfer to the nucleus of genetic information. The movement of DNA between different genomes within eukaryotic cells is well documented. During the examination of eukaryotic cells, it has been shown that they contain genes from the Bacterial and Archaea domains. Chromosomes store the eukaryotic genome in the nucleus. Ribosomal RNA gene sequencing and phylogeny techniques can be used to show that mitochondria and chloroplasts are highly related ancestors of specific Bacteria. A particularly relevant example is subunit 9 of the F0 component in mitochondrial ATPase, which is coded by mitochondrial DNA in yeast Saccharomyces cerevisiae, but by nuclear DNA in the filamentous fungus Nurospora (Borst and Grivel 1978). The ancestral condition is not known, although the two fungi are phylogenically close, suggesting that the transposition occurred relatively recently.
The DNA retained in modern chloroplasts and mitochondria encodes proteins essential for organellar function as well as ribosomal RNAs required for their translation and tRNAs. Eukaryotic cells can therefore be said to have multiple genetic systems. There is a primary DNA system in the nucleus and secondary systems in the mitochondria and chloroplasts with their own DNA. The proteins encoded for by mitochondrial or chloroplast DNA are synthesised on ribosomes within the organelles.
With the rapid accumulation of sequence data for mitochondrial and bacterial genomes, it is possible to speculate on the origin of the "original" mitochondrion. The most mitochondrial-like bacterial genome is that of Rickettsia prowazekii, the cause of louse borne typhus. This organism has a genome of more than a million bp in size and contains 834 protein encoding genes. Sequence data suggest that all extant mitochondria are derived from an ancestor of R. prowazekii as the result of a single endosymbiotic event.
The evidence that modern mitochondria result from a single event comes from examination of the most bacteria-like mitochondrial genome, that of Reclinomonas americana. It has a genome consisting of 97 genes, 62 of which specify proteins. Yet this genome encodes less than 2% of the protein-coding genes in the bacterium E. coli. It seems unlikely that mitochondrial genomes resulting from several endosymbiotic events could have been independently reduced to the same set of genes found in R. americana. The evidence suggests that chloroplasts in higher plants and green algae are derived from a single endosymbiotic event, whereas those in red and brown algae arose from at least one additional event.
The presence of DNA in the mitochondria cannot be accounted for in most autogenous theories.
However, Raff and Mahler (1975) suggested that when part of the protoeukaryote's cell membrane infolded, forming the mitochondrial membranes, the resultant vesicle incorporated a plasmid bearing the minimum information necessary for sustained functioning.
The growth and division of mitochondria and chloroplasts are not linked with nuclear division. New organelles form by division of pre-existing organelles, while the organelles grow by the incorporation of cellular proteins and lipids. Both of these processes occur continuously during the interphase period of the cell cycle, showing quite a high degree of semi-autonomy.
This 'separateness' is echoed by the fact that even modern eukaryotic microorganisms are known that have no mitochondria or chloroplasts, which suggests a time before mitochondria and chloroplasts were included in eukaryotic cells. Microsporidia and diplomonads lack mitochondria but contain a membrane-enclosed nucleus. These cells are probably similar to those that first accepted the pre-organelle bacteria many hundreds of generations ago.
Both mitochondria and chloroplasts contain ribosomes, which are clearly of the prokaryotic type of 70 Svedberg Units, rather than eukaryotic ribosomes, which are 80 Svedberg Units in overall size. Ribosomes from these organelles show ribosomal RNA sequences characteristic of specific Bacteria. However, more convincing evidence of their joint ancestry, is the fact that their function is inhibited by the same antibiotics that affect ribosomal function in free-living Bacteria. Most antibiotics that specifically affect Bacteria have no effect on organisms in the archaeal or eukarayal domains.
In the presence of oxygen, pyruvate formed in glycolysis is transported into mitochondria, where it is oxidised by O 2 to CO 2 in a series of oxidative phosphorylation reactions collectively called cellular respiration. These reactions generate an estimated 28 additional ATP molecules per glucose molecule, outstripping the yield of anaerobic ATP by far. This clear energetic benefit to the cell is arguably one of the reasons why these cells prevailed. In bacteria both photosynthesis and oxidative phosphorylation occur on the plasma membrane.
The molecular mechanisms that form ATP in mitochondria and chloroplasts are very similar. Chloroplasts and mitochondria have other features in common: both often migrate from place to place within cells and both mitochondria and chloroplasts contain similar types of electron transport proteins and use an F-class ATPase to synthesise ATP. Remarkably gram-negative bacteria also exhibit these characteristics. Striking evidence for the endosymbiotic theory of an ancient evolutionary relationship can be found in the many proteins of similar sequences shared by mitochondria, chloroplasts and bacteria including some of the proteins involved in membrane translocation.
Endocytosis of a bacterium by an ancestral eukaryotic cell would create an organelle with a double membrane. According to this theory the inner mitochondrial membrane would be derived from the original bacterial membrane, and the outer membrane would be derived from the eukaryotic plasma membrane.
Recent research by Martin and Müller (1998) into the origin of the mitochondrion has led to a new theory of endosymbiosis called the "hydrogen hypothesis." In the current picture of the origin of the eukaryotic cell, the mitochondrion was a "lucky accident" (Vogel 1998). The ancestral host cell simply engulfed the mitochondrion ancestor, did not fully ingest it, and an even more successful cell resulted. According to the hydrogen hypothesis, however, the first eukaryotic cell did not form simply by accident. Instead, it was the result of a purposeful union between an archaebacterial host cell, a methanogen that consumed hydrogen and carbon dioxide to produce methane, and a future mitochondrion symbiont that made hydrogen and carbon dioxide as waste products of anaerobic metabolism. Thus, although the symbiont was probably capable of aerobic respiration, the symbiosis began as a result of the products of anaerobic metabolism. The host's dependence upon hydrogen produced by the symbiont is identified as the selective principle that consolidated the common ancestor of eukaryotic cells (Martin and Müller 1998).
The hydrogen hypothesis has some important implications that contradict the current view of the relationship between eukaryotes and archaebacteria. In the current view, the eukaryotes branched off from the archaebacteria long before the archaebacteria had divided into their present-day groups. The hydrogen hypothesis implies that the first eukaryotes appeared much later in the evolutionary picture, meaning they are more closely tied to the archaebacteria. In order for the hydrogen hypothesis to be confirmed, an analysis of the complete sequences of eukaryotic and archaebacterial genomes must be completed (Vogel 1998).
Another recent explanation of the origin of eukaryotes called the "syntrophic hypothesis" was presented by López-García and Moreira (1998). Though they were independently proposed, the syntrophic hypothesis is complementary in several aspects to the hydrogen hypothesis. Both hypotheses agree in the suggestion of an anaerobic metabolism for the origin of mitochondrial symbiosis. The theories are also similar in some metabolic details of the symbiosis and molecular features of archaea (López-Garcia and Moreira 1998). The major difference between the two hypotheses is in the nature of the original bacterial partnership. As previously stated, in the hydrogen hypothesis, the original symbiosis is thought to have taken place between a methanogenic archaebacterium and a eubacterial ancestor to the mitochondrion. In the syntrophic hypothesis, the original symbiosis is conceived to have taken place between a methanogenic archaebacterium and an ancestral sulfate-respiring delta-proteobacterium. The former provided the central genetic material and nucleic acid metabolism while the latter provided most metabolic characteristics (López-Garcia and Moreira 1998). Mitochondria are thought to have derived from a later, independent symbiotic event. As with the hydrogen hypothesis, further genetic sequencing analyses are necessary in order for the claims of the syntrophic hypothesis to be upheld.
Conclusion & Summary
It is generally agreed by the scientific community that eukaryotic cells originated from some prokaryote-like ancestor. There is mounting evidence which supports the theory that "the modern, organelle containing eukaryotic cell evolved in steps through the stable incorporation of chemo-organotrophic and phototrophic symbionts from the domain Bacteria". Mitochondria and chloroplasts reproduce through division of pre-existing organelles and are not linked to nuclear division. Eukaryotic genomes contain bacterial genes, which are thought to have been transferred from the bacteria-like organelles at some point in the past. The organelles have double membranes, which is explained by the engulfment process, they are structurally very similar to bacteria and contain their own ribosomes.
The evidence all seems to support the "middle ground" SET, which is now widely accepted. However, Margulis' "extreme" SET is still quite controversial and has little supporting evidence.