Molecular evolution before the ancestors of the bacterial and archaeal domains and before the Last Universal Common Ancestor Funded through the NASA Exobiology and NSF Assembling the Tree of Life Programs Origins 2014, Nara, Japan, July 6-11, 2014 J. Peter Gogarten University of Connecticut Dept. of Molecular and Cell Biol. Collaborators: Dr. Greg Fournier (UConn/MIT) Dr. Cheryl Andam (UConn/Harvard)

Outline: MuralatNASAAmesResearchCenter Gene duplica,ons and deep molecular phylogenies Proper,es of the Last Universal Common Ancestor(s) The history of the transla,on machinery during the expansion of the gene,c code The ribosomal tree of life and inferred op,mal growth temperature Tree shape, the ar,fact of apparent lonely ancestors Indica,ons for early ex,nc,on events due to increased environmental temperature Phylogene,c evidence for LUCAs compatriots

Catalytic subunits Non catalytic subunits speciation gene duplication time

ATPase / ATPsynthase ATP binding Subunits

N C V-proteolipid N CN C A-proteolipids Halobacterium Methanococcus c A B A A B B c N C F-proteolipid V-type ATPase A-type ATPase F-type ATPase Methanopyrus

? mesophilicthermophilic Archaea Eukarya Bacteria endosymbionts 1 2 3 4 5 A B C D E 12 proteolipid Ds / 6 cataly,c SU = 2 H+(Na+) / ATP 12 proteolipid Ds / 3 cataly,c SU = 4H+(Na+) / ATP 6 proteolipid Ds / 3 cataly,c SU = 2H+(Na+) / ATP 12 proteolipid Ds / 3 cataly,c SU = 4H+(Na+) / ATP 12 proteolipid Ds / 3 cataly,c SU = 4H+(Na+) / ATP Reversible Enzyme Reversible Enzyme Dedicated Ion Pump Dedicated Ion Pump Reversible Enzyme

C. R. Woese and G. E. Fox (1977) J. Mol. Evol. 10, 1-6: Eucaryotes did arise from procaryotes, but only in the sense that the procaryo6c is an organiza6onal, not a phylogene6c dis6nc6on. In analogous fashion procaryotes arose from simpler en66es. The la

According to Woese and Fox According to V/F/A-ATPases From: GOGARTEN J.P., OLENDZENSKI L., (1999) The Progenote, Encyclopedia of Molecular Biology, Thomas Creighton, ed., John Wiley and Sons, NY (submieed version at gogarten.uconn.edu)

In R.P. Mortlock: (ed), The Evolu,on of Metabolic Func,on , CRC Press,1992

Organisms represented by the root of the universal evolu,onary tree were most likely complex cells with a sophis,cated protein transla,on system and a DNA genome encoding hundreds of genes.

Outline: MuralatNASAAmesResearchCenter Gene duplica,ons and deep molecular phylogenies Proper,es of the Last Universal Common Ancestor(s) The history of the transla:on machinery during the expansion of the gene:c code The ribosomal tree of life and inferred op,mal growth temperature Tree shape, the ar,fact of apparent lonely ancestors Indica,ons for early ex,nc,on events due to increased environmental temperature Phylogene,c evidence for LUCAs compatriots

A Radical Proposal by Eugene Koonin : Anthropic Chemical Evolution (The Logic of Chance FT Press 2012) Modern cosmologies postulate parallel worlds, for example assuming an eternal inflation period, resulting in an infinite number of universes (Villinkin, 2007). Given an infinite number of universes, even unlikely events are bound to happen in some universes (and because we are made from two biopolymers, we are in one of the universes where this rare event occurred). Koonin suggests that the assembly of the translation machinery is a candidate for such an unlikely event. Finding exceedingly rare events in evolution would argue for a Multi World Cosmology.

These hypotheses can be tested by examining the composi,on of reconstructed ancestor sequences Do synthetase paralogs retain evidence of pre-LUCA evolu,onary events?

Hypothesis Testing 1-2: neofunctionalization 3: subfunctionalization 4: takeover (parafunctionalization) Probability density graph of all positions with X+Y plurality consensus in ancestral reconstruction of cognate paralog ancestor.

Results Majority of high-probabilitiy positions are resolved for Ile or Val Supports both amino acids are specifically encoded at the time of the paralog ancestor, Parafunctionalization Large number of nondiscriminating positions between Ile and Val would support subfunctionalization However, these positions are all low-probability, and match with the control simulation, so probably artifact of poorly conserved positions.

"RNA world" (single biopolymer world) Replica,on Machinery

"RNA world" (single biopolymer world) Replica,on Machinery Rise of protein as second biopolymer tRNAs, "RNA" Ribosome, RNA based tRNA charging mechanisms

"RNA world" (single biopolymer world) Replica,on Machinery Rise of protein as second biopolymer tRNAs, "RNA" Ribosome, RNA based tRNA charging mechanisms Expansion of the gene,c code to include Isoleucine and Valine

"RNA world" (single biopolymer world) Replica,on Machinery Rise of protein as second biopolymer tRNAs, "RNA" Ribosome, RNA based tRNA charging mechanisms Expansion of the gene,c code to include Isoleucine and Valine Takeover of charging mechanism by proteins (inven,on of aminoacyl tRNA synthetases) 1IVS.pdb valRS + tRNAval

"RNA world" (single biopolymer world) Replica,on Machinery Rise of protein as second biopolymer tRNAs, "RNA" Ribosome, RNA based tRNA charging mechanisms Expansion of the gene,c code to include Isoleucine and Valine Takeover of charging mechanism by proteins (inven,on of aminoacyl tRNA synthetases) Expansion of the gene,c code to include Tryptophan 1IVS.pdb valRS + tRNAval

Conclusions 1st part Extrapolation of ATPsynthase structure suggests that LUCA was able to use transmembrane ion gradients to synthesize ATP. LUCA was not a progenote The expansion of the genetic code did not parallel the divergence of aaRSs; rather aaRS acquired specificity in cells that were already able to charge tRNAs with their cognate aa through other means (likely exception tryptophan).

Outline: MuralatNASAAmesResearchCenter Gene duplica,ons and deep molecular phylogenies Proper,es of the Last Universal Common Ancestor(s) The history of the transla,on machinery during the expansion of the gene,c code The ribosomal tree of life and inferred op:mal growth temperature Tree shape, the ar,fact of apparent lonely ancestors Indica,ons for early ex,nc,on events due to increased environmental temperature Phylogene,c evidence for LUCAs compatriots

Evolution of the Ribosome Core of ribosome consists of RNA + subset of ribosomal proteins universally conserved in all life (~29 proteins) (Harris et al., 2003) Likely coevolved with genetic code within an RNA world (Wolf & Koonin, 2007)

Compositional Stratigraphy We perform a compositional analysis of ribosomal proteins and ATPase subunits in bacterial and archaeal lineages, using conserved positions that came and remained under purifying selection before and up to the most recent common ancestor. An observable shift in amino acid usage at these conserved positions likely provides an untapped window into the history of protein sequence space, allowing events of genetic code expansion to be identified. Fournier GP, Gogarten JP. 2007. Signature of a primitive genetic code in ancient protein lineages. J Mol Evol. 65(4):425-436

Roo,ng the Ribosomal Tree of Life using an Echo from the Early Expansion of the Gene,c Code (Fournier and Gogarten, MBE 2010)

Fig. 3. The classical SSUrRNA distance tree, presented as rooted in the bacterial branch. Bold lines indicate extreme hyperthermophiles. From Steeer (1996).

LUCA (located on the bacterial branch) was less thermophilic than the ancestor of the bacterial and archaeal domains Boussau, B, Blanquart, S, Necsulea, A, Lartillot, N and Gouy, M (2008). Parallel adaptations to high temperatures in the Archaean eon. Nature 456(7224): 942-945 Reconstruction of ancestral protein and rRNA sequences Based on IVYWREL and rRNA stem G+C content LUCA was less thermophilic than the domain ancestors. Galtier, N, Tourasse, N and Gouy, M (1999). A nonhyperthermophilic common ancestor to extant life forms. Science 283(5399): 220-221.8 rRNA 60C!80C IVYWREL (corrected for GC content) 20C!70C

Outline: MuralatNASAAmesResearchCenter Gene duplica,ons and deep molecular phylogenies Proper,es of the Last Universal Common Ancestor(s) The history of the transla,on machinery during the expansion of the gene,c code The ribosomal tree of life and inferred op,mal growth temperature Tree shape, the ar:fact of apparent lonely ancestors Indica:ons for early ex:nc:on events due to increased environmental temperature Phylogene,c evidence for LUCAs compatriots

Tree, Web, or Coral of Life? Charles Darwin painted by George Richmond in the late 1830 Page B26 from Charles Darwins (1809-1882) notebook (1837/38) The tree of life should perhaps be called the coral of life, base of branches dead

The Coral of Life (Darwin) ZHAXYBAYEVAandGOGARTEN(2004): Cladogenesis,CoalescenceandtheEvolutionoftheThreeDomainsofLife. TrendsinGenetics20(4):182-187

The Coral of Life (Darwin) ZHAXYBAYEVAandGOGARTEN(2004): Cladogenesis,CoalescenceandtheEvolutionoftheThreeDomainsofLife. TrendsinGenetics20(4):182-187

Coalescence the process of tracing lineages backwards in ,me to their common ancestors. Every two extant lineages coalesce to their most recent common ancestor. Eventually, all lineages coalesce to the cenancestor. t/2 (Kingman, 1982) Illustra,on is from J. Felsenstein, Inferring Phylogenies, Sinauer, 2003

EXTANT LINEAGES FOR THE SIMULATIONS OF 50 LINEAGES

Bacterial 16SrRNA based phylogeny (from P. D. Schloss and J. Handelsman, Microbiology and Molecular Biology Reviews, December 2004.) The devia,on from the long branches at the base paeern could be due to under sampling an actual radia,on due to an inven,on that was not transferred following a mass ex,nc,on

Near frustra,on of early life From: Gogarten-Boekels M, Hilario E, Gogarten JP. Orig Life Evol Biosph. 1995 Jun;25(1-3): 251-64. The eects of heavy meteorite bombardment on the early evolu:on the emergence of the three domains of life.

From: hep://www.origin-life.gr.jp/3603/3603055/3603055.html Alterna,ve: tail of early heavy bombardment Nicolle Zellners talk on Tuesday See Marchi et al. Nature 2014 for a recent update.

Outline: MuralatNASAAmesResearchCenter Gene duplica,ons and deep molecular phylogenies Proper,es of the Last Universal Common Ancestor(s) The history of the transla,on machinery during the expansion of the gene,c code The ribosomal tree of life and inferred op,mal growth temperature Tree shape, the ar,fact of apparent lonely ancestors Indica,ons for early ex,nc,on events due to increased environmental temperature Phylogene:c evidence for LUCAs compatriots

Molecular Phylogenies " Lonely Ancestors From: hep://itol.embl.de/ iTol The interac,ve Tree of Life Ciccarelli et al, Science. 2006 311 :1283-7 Tree topology averaged over many genes (mainly ribosomal proteins). No re,cula,ons. Branches do not reect ,me. Only extant organisms and their lucky ancestors are includes Noteworthy:

The Coral of Life (Darwin) ZHAXYBAYEVAandGOGARTEN(2004): Cladogenesis,CoalescenceandtheEvolutionoftheThreeDomainsofLife. TrendsinGenetics20(4):182-187

The Coral of Life (Darwin) ZHAXYBAYEVAandGOGARTEN(2004): Cladogenesis,CoalescenceandtheEvolutionoftheThreeDomainsofLife. TrendsinGenetics20(4):182-187

Molecular phylogenies of aaRSs reveal other lineages that coexisted with LUCA and/or the domain ancestors and transferred some of their genes into extant lineages.

Pyrrolysine (Pyl) # 22nd genetically encoded amino acid to be discovered # Uses dedicated aminoacyl-tRNA synthetase (PylS) and a UAG-recognizing tRNA. # Found only within Methanosarcinae, Desulfitobacterium hafniense and a single marine worm symbiont delta-proteobacteria. # Used exclusively at the catalytic site of three enzymes responsible for the initial step of methylotrophic methanogenesis from methylamines. MtmB structure with Pyl residue in catalytic core (Hao et al., 2002) Synthesized from Pro and Lys Contains a peptide bond in the side chain

Class II aaRS Phylogeny LUCA -nodes

Horizontal Gene Transfer Pyl evolved and had a pervasive biological role in an ancient sister group to the MRCA. Transfer of cassette encoding methyltransferases and pyrrolysine system, selected for by the transfer of the methyltransferase genes. Subsequent extinction of the entire donor lineage Genetic Life Raft

Ancient origin of the divergent forms of leucyl-tRNA synthetases in the Halobacteriales Cheryl P Andam, Timothy J Harlow, R Thane Papke and J Peter Gogarten BMC Evolu6onary Biology 12:85 leucyl-tRNA synthetase (class I) phylogeny

Homeoalleles Variants that have the same general function, but can have distinct characteristics. Gene pool contains different homeoalleles, but individual strains and species usually contain only one of the alleles. Can be brought together temporarily in a lineage through HGT Andam, Williams, Gogarten 2010 PNAS

Andam and Gogarten 2011

Phylogeny of selected class II amino acyl tRNA synthetases

Andam and Gogarten 2011 Distribu:on of rare SerRS in Archaea

thrRS and serRS phylogeny Eukaryotes Euryachaeota Crenarchaeota Bacteria Alignment with PRANK and SAT, tree with phyml (WAG, gamma +I)

Conclusion 2nd part Tree shape and amino acid composition of ancestral sequences suggest a bottleneck due to increased environmental temperature at the base of the bacterial and archaeal domains. Studies of horizontal gene transfers of aaRSs suggest that more than two lineages passed through this bottleneck.

References Andam CP, Gogarten JP. 2011. Biased gene transfer in microbial evolu,on. Nat. Rev. Microbiol. 9:543555. Andam CP, Harlow TJ, Papke RT, Gogarten JP. 2012. Ancient origin of the divergent forms of leucyl-tRNA synthetases in the Halobacteriales. BMC Evol. Biol. 12:85. Andam CP, Williams D, Gogarten JP. 2010. Biased gene transfer mimics paeerns created through shared ancestry. Proc. Natl. Acad. Sci. U. S. A. 107:1067910684. Boussau B, Blanquart S, Necsulea A, Lar,llot N, Gouy M. 2008. Parallel adapta,ons to high temperatures in the Archaean eon. Nature 456:942945. Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Bork P. 2006. Toward automa,c reconstruc,on of a highly resolved tree of life. Science 311:12831287. Delaye L, Becerra A, Lazcano A. 2005. The last common ancestor: whats in a name? Orig Life Evol Biosph 35:537554. Felsenstein J. 2003. Inferring Phylogenies. Sinauer, Sunderland, MA Fournier GP, Andam CP, Alm EJ, Gogarten JP. 2011. Molecular evolu,on of aminoacyl tRNA synthetase proteins in the early history of life. Orig. Life Evol. Biosph. 41:621632. Fournier GP, Gogarten JP. 2007. Signature of a primi,ve gene,c code in ancient protein lineages. J. Mol. Evol. 65:425436. Fournier GP, Gogarten JP. 2010. Roo,ng the ribosomal tree of life. Mol. Biol. Evol. 27:17921801. Fournier GP, Huang J, Gogarten JP. 2009. Horizontal gene transfer from ex,nct and extant lineages: biological innova,on and the coral of life. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364:22292239.

References (con,nued) Gal,er N, Tourasse N, Gouy M. 1999. A nonhyperthermophilic common ancestor to extant life forms. Science 283:220221. Gogarten JP, Kibak H, Dierich P, et al. 1989. Evolu,on of the vacuolar H+-ATPase: implica,ons for the origin of eukaryotes. Proc Natl Acad Sci U S A 86:66616665. Gogarten JP, Taiz L. 1992. Evolu,on of proton pumping ATPases: Roo,ng the tree of life. Photosynth. Res. 33:137146. Gogarten-Boekels M, Hilario E, Gogarten JP. 1995. The eects of heavy meteorite bombardment on the early evolu,on--the emergence of the three domains of life. Orig Life Evol Biosph 25:251264. Goldman AD, Bernhard TM, Dolzhenko E, Landweber LF. 2013. LUCApedia: a database for the study of ancient life. Nucleic Acids Res. 41:D107982. Hao B, Gong W, Ferguson TK, James CM, Krzycki JA, Chan MK. 2002. A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296:14621466. Harris JK, Kelley ST, Spiegelman GB, Pace NR. 2003. The gene,c core of the universal ancestor. Genome Res 13:407412. Kim KM, Caetano-Anolls G. 2011. The proteomic complexity and rise of the primordial ancestor of diversied life. BMC Evol. Biol. 11:140. Kingman JFC. 1982. The coalescent. Stoch. Process. Their Appl. 13:235248. Koeberl C. 2006. Impact Processes on the Early Earth. Elements 2:211216. Koonin E V. 2011. The Logic of Chance: The Nature and Origin of Biological Evolu,on. FT Press; 1 edi,on

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