James A Lake

The ring of life provides evidence for a genome fusion origin of eukaryotes

Genomes hold within them the record of the evolution of life on Earth. But genome fusions and horizontal gene transfer seem to have obscured sufficiently the gene sequence record such that it is difficult to reconstruct the phylogenetic tree of life. Here we determine the general outline of the tree using complete genome data from representative prokaryotes and eukaryotes and a new genome analysis method that makes it possible to reconstruct ancient genome fusions and phylogenetic trees. Our analyses indicate that the eukaryotic genome resulted from a fusion of two diverse prokaryotic genomes, and therefore at the deepest levels linking prokaryotes and eukaryotes, the tree of life is actually a ring of life. One fusion partner branches from deep within an ancient photosynthetic clade, and the other is related to the archaeal prokaryotes. The eubacterial organism is either a proteobacterium, or a member of a larger photosynthetic clade that includes the Cyanobacteria and the Proteobacteria.

The transorientation hypothesis for codon recognition during protein synthesis

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5′-stacked and the 3′-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon–anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.

Rings Reconcile Genotypic and Phenotypic Evolution within the Proteobacteria

Although prokaryotes are usually classified using molecular phylogenies instead of phenotypes after the advent of gene sequencing, neither of these methods is satisfactory because the phenotypes cannot explain the molecular trees and the trees do not fit the phenotypes. This scientific crisis still exists and the profound disconnection between these two pillars of evolutionary biology—genotypes and phenotypes—grows larger. We use rings and a genomic form of goods thinking to resolve this conundrum (McInerney JO, Cummins C, Haggerty L. 2011. Goods thinking vs. tree thinking. Mobile Genet Elements. 1:304–308; Nelson-Sathi S, et al. 2015. Origins of major archaeal clades correspond to gene acquisitions from bacteria. Nature 517:77–80). The Proteobacteria is the most speciose prokaryotic phylum known. It is an ideal phylogenetic model for reconstructing Earth’s evolutionary history. It contains diverse free living, pathogenic, photosynthetic, sulfur metabolizing, and symbiotic species. Due to its large number of species (Whitman WB, Coleman DC, Wiebe WJ. 1998. Prokaryotes: the unseen majority. Proc Nat Acad Sci U S A. 95:6578–6583) it was initially expected to provide strong phylogenetic support for a proteobacterial tree of life. But despite its many species, sequence-based tree analyses are unable to resolve its topology. Here we develop new rooted ring analyses and study proteobacterial evolution. Using protein family data and new genome-based outgroup rooting procedures, we reconstruct the complex evolutionary history of the proteobacterial rings (combinations of tree-like divergences and endosymbiotic-like convergences). We identify and map the origins of major gene flows within the rooted proteobacterial rings (P < 3.6 × 10−6) and find that the evolution of the “Alpha-,” “Beta-,” and “Gammaproteobacteria” is represented by a unique set of rings. Using new techniques presented here we also root these rings using outgroups. We also map the independent flows of genes involved in DNA-, RNA-, ATP-, and membrane- related processes within the Proteobacteria and thereby demonstrate that these large gene flows are consistent with endosymbioses (P < 3.6 × 10−9). Our analyses illustrate what it means to find that a gene is present, or absent, within a gene flow, and thereby clarify the origin of the apparent conflicts between genotypes and phenotypes. Here we identify the gene flows that introduced photosynthesis into the Alpha-, Beta-, and Gammaproteobacteria from the common ancestor of the Actinobacteria and the Firmicutes. Our results also explain why rooted rings, unlike trees, are consistent with the observed genotypic and phenotypic relationships observed among the various proteobacterial classes. We find that ring phylogenies can explain the genotypes and the phenotypes of biological processes within large and complex groups like the Proteobacteria.

Evidence from 18S ribosomal DNA that the lophophorates are protostome animals

The suspension-feeding metazoan subkingdom Lophophorata exhibits characteristics of both deuterostomes and protostomes. Because the morphology and embryology of lophophorates are phylogenetically ambiguous, their origin is a major unsolved problem of metazoan phylogenetics. The complete 18S ribosomal DNA sequences of all three lophophorate phyla were obtained and analyzed to clarify the phylogenetic relationships of this subkingdom. Sequence analyses show that lophophorates are protostomes closely related to mollusks and annelids. This conclusion deviates from the commonly held view of deuterostome affinity.

Origin of the eukaryotic nucleus determined by rate-invariant analysis of rRNA sequences

The origin of the eukaryotic nucleus is difficult to reconstruct. Eukaryotic organelles (chloroplast, mitochondrion) are eii bacterial1,2 endosymbionts3,but the source of nuclear genes has been obscured by multiple nucleotide substitutions. Using evolutionary parsimony4, a newly developed rate-invariant treeing algorithm, the eukaryotic ribosomal rRNA genes are shown to have evolved from the eocytes5, a group of extremely thermophilic, sulphur-metabolizing, anucleate cells. The deepest bifurcation yet found separates the reconstructed tree into two taxonomic divisions. These are a proto-eukaryotic group (karyotes) and an essentially bacterial one (parkaryotes). Within the precision of the rooting procedure, the tree is not consistent with either the prokaryotic–eukaryotic or the archaebacterial–eubacterial–eukaryotic groupings. It implies that the last common ancestor of extant life, and the early ancestors of eukaryotes, probably lacked nuclei, metabolized sulphur and lived at near-boiling temperatures.

Ribosome structure determined by electron microscopy of Escherichia coli small subunits, large subunits and monomeric ribosomes.

Abstract Unique, three-dimensional structures have been determined for Escherichia coli small subunits, large Subunits and monomeric ribosomes by electron microscopy of ribosomes and subunits and of antibody-labeled ribosomes and subunits. Small subunits orient on the carbon substrate with their long axes parallel to the plane of the carbon. In these projections the subunit is divided into a onethird and a two-thirds portion by a region of accumulated negative stain similar to that observed in eukaryotic small subunits. Four characteristic views, or projections, are readily recognized and correspond to orientations of approximately −40 °, 0 °, +50 ° and +110 ° about the long axis of the subunit. Three of these have been described (Lake et al., 1974a; Lake & Kahan, 1975). The two most distinctive views are a quasi-symmetric view (0 °) that is characterized by an approximate line of mirror symmetry that coincides with the long axis of the subunit, and an asymmetric view (110 °) that is characterized by a concave and a convex subunit boundary. In the asymmetric projection, a platform or ledge is viewed “face-on”. The platform is attached to the lower two-thirds of the subunit just below the one-third/two-thirds partition. It is separated from the upper one-third of the subunit at the level of the partition and above the partition it forms a cleft approximately 30 to 40 A wide, which has been suggested as the site of the codon-anticodon interaction (Lake & Kahan, 1975). Four characteristic views are presented for the large subunit. The most prominent of these, the quasi-symmetric view (θ = 90 °, φ = 0 °), is distinguished by a central protuberance located on a line of approximate mirror symmetry. The central protuberance is surrounded by projecting features inclined at about 50 ° on both sides of it. The smaller of these projections is rod-like, about 40 A wide and approximately 100 A long. The feature projecting from the other side of the central protuberance is shorter, more blunt and wider than the rod-like appendage. In another view approximately orthogonal to the quasi-symmetric projection, the asymmetric projection (θ = 10 °, φ = 90 °), the subunit profile is distinguished by a convex lower edge and an upper boundary which is indented by a notch. The subunit is separated, in projection, by the notch into two unequal regions. The smaller region comprises about 20% of the total projected density and consists of the central protuberance and the rod-like appendage. The profiles observed in fields of monomeric 70 S ribosomes result from superpositions of the 30 S and 50 S profiles. Two major views are observed, an overlap and a non-overlap view, corresponding to whether or not the profile of the small subunit overlaps that of the large subunit in the 70 S profile. The small subunit is oriented in the monomeric ribosome so that the platform is in contact with the large subunit. The central protuberance of the large subunit overlaps part of the upper one-third of the small subunit in the overlap view of 70 S ribosomes, although in three dimensions they are probably separated by 30 to 50 A. A region of the small subunit comprising the platform, the cleft and part of the upper one-third, suggested to be the approximate binding site of IF3 and IF2 (Lake & Kalian, 1975), is located at the interface between the large and small subunits, in a region of the small subunit that is close to, but probably not in physical contact with, the large subunit.

Ribosomal proteins L7L12 localized at a single region of the large subunit by immune electron microscopy

Ribosomal proteins L7L12 have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the L7L12 stalk. The L7L12 stalk is approximately 100 A long, about 40 A wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the L7L12 stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface. Anti-L7L12 antibody binding to the stalk was shown to be solely dependent upon the presence of L7L12 by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of L7L12 were unable to bind anti-L7L12 antibodies and that re-incorporation of L7L12 restored the ability of L7L12-depleted cores to react with anti-L7L12 antibodies. Anti-L7L12 antibodies pre-absorbed with L7L12 did not react with 50 S subunits. Anti-L7L12 antibodies used in these experiments reacted only with the L7L12 stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, L7L12-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the L7L12 stalk from more than 95% of the subunits, and that re-incorporation of L7L12 into depleted cores restores the L7L12 stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-L7L12 immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that L7L12 are located only on the L7L12 stalk.

Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences.

A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsimony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciculata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic species Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tarentolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phylogenetic tree is consistent with our cladistic analysis of the biological evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assuming that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 +/- 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukaryotic parasites have an evolutionary history that extends back to the origin of their insect host.

Evidence for an early prokaryotic endosymbiosis.

Endosymbioses have dramatically altered eukaryotic life, but are thought to have negligibly affected prokaryotic evolution. Here, by analysing the flows of protein families, I present evidence that the double-membrane, Gram-negative prokaryotes were formed as the result of a symbiosis between an ancient actinobacterium and an ancient clostridium. The resulting taxon has been extraordinarily successful, and has profoundly altered the evolution of life by providing endosymbionts necessary for the emergence of eukaryotes and by generating Earth’s oxygen atmosphere. Their double-membrane architecture and the observed genome flows into them suggest a common evolutionary mechanism for their origin: an endosymbiosis between a clostridium and actinobacterium.

Tracing origins with molecular sequences: metazoan and eukaryotic beginnings

Milestones in the evolution of the eukaryotic cell are being discovered through the analysis of molecular sequences. As sequence data become increasingly plentiful, our ability to reconstruct the most distant evolutionary branchings of evolutionary trees is limited only by the mathematics of phylogenetic reconstruction. Analysis of ribosomal RNAs agrees with traditional analyses of morphological and developmental characters that all multicellular animals probably arose from a common ancestor, but highlights one of the major limitations of the various mathematical algorithms used. Refined methods of sequence analysis also suggest a previously unsuspected sister relationship between the eukaryotic nucleus and eocytes, a group of extremely thermophilic, sulfur-metabolizing bacteria, that questions the classical eukaryote/prokaryote division.