Not much happening here atm so I thought I'd share something from a new paper I found interesting.

Experimental evidence for the thermophilicity of ancestral life
Satoshi Akanumaa, Yoshiki Nakajimaa, Shin-ichi Yokoboria, Mitsuo Kimuraa, Naoki Nemotoa, Tomoko Maseb, Ken-ichi Miyazonob, Masaru Tanokurab, and Akihiko Yamagishia

Abstract
Theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. Here we provide experimental support for the existence of a thermophilic universal common ancestor. We present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (NDK), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of Archaea and of Bacteria. These enzymes display extreme thermal stabilities, suggesting thermophilic ancestries for Archaea and Bacteria. The results are robust to the uncertainties associated with the sequence predictions and to the tree topologies used to infer the ancestral sequences. Moreover, mutagenesis experiments suggest that the universal ancestor also possessed a very thermostable NDK. Because, as we show, the stability of an NDK is directly related to the environmental temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished at a very high temperature.

http://www.pnas.org/content/early/2013/06/12/1308215110.abstract

Going over the interesting parts in the main text.

Elucidation of the origin and early evolution of life is funda-mental to our understanding of ancient living systems and their environment(s). One debate about the last universal com-mon ancestor, which has been denoted“LUCA,”“LCA,” or “senancestor,”and which we call “Commonote”(1), is its environmental temperature. In a well-referenced phylogenetic tree containing small-subunit rRNA sequences, those of hyperthermophilic archaea and bacteria are found at the deepest and shortest branches (2), and therefore it has been proposed that the common ancestors of Archaea and Bacteria were hyperthermophilic (3, 4). Given the apparent hyperthermophilic ancestry for both lineages, Occam’s razor suggests that the Commonote was a thermophilic organism. However, although some theoretical studies that focused on the environmental temperature of the Commonote support the thermophilic common-ancestry hypothesis (5–7), other theoretical studies have concluded that the universal ancestor was not (hyper)thermophilic (8–10). Therefore, the available theoretical studies disagree among them-selves. More seriously, these theoretical studies have not been tested empirically.

Information concerning the properties of ancient proteins is embedded in the sequences of their descendants, so the ances-tral sequence of a protein can be inferred by comparing a large number of extant homologous sequences (11–14). A powerful method for experimentally studying the properties of ancient life resurrects ancestral protein sequences, thereby allowing characterization oftheir features (15–23). Such resurrections have been used to understand the evolution of ethanol production and consumption in yeast (16), the evolutionary trajectory of changes in ligand specificity of hormone receptors (17, 18, 20), and the evolution of the increased complexity of vacu-olar H+-ATPases (22). This empirical technique has provided new ways to elucidate the environmental temperatures experienced by ancient bacteria (15, 19). For the study reported here, we chose archaeal and bacterial ancestral nucleoside diphosphate kinase (NDK) sequences as the resurrection targets. NDK cata-lyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. The ancestral NDK may have arisen early, because most extant organisms contain at least one gene that encodes NDK. The sequences of extant NDKs are relatively well conserved, allowing inference of the ancestral sequence with confidence. In addition, a number of 3D structures are available for the NDK family of proteins isolated from a wide variety of organisms, including bacteria, archaea, and eukar-yotes. Therefore, NDK is an ideal model for studying the physical characteristics of ancient proteins. By resurrecting ancestral NDK sequences and characterizing their properties, which should reflect the ancestor’s characteristics and its environment, we experimentally estimated the environmental temperature of the Commonote that would have existed about 3,800 million
years ago (24).

So, they use ancestral sequence reconstruction, focusing on a set of universally conserved protein enzymes in extant Archaea and Bactieria, Nucleoside Diphosphate Kinase (NDK), that function as an indispensible part of extant cell's metabolism. Given their central role and the fact of their universal conservation, the authors infer that these extant enzymes must have been inherited from a common ancestor, the protein sequence of which they then attempt to reconstruct.

I have to note as extremely interesting that this ancestral sequence they come up with is estimated by their molecular clocks to be 400 million years older than the oldest known geological evidence of life. Not sure how much emphasis to put on this, we know molecular clocks become increasingly uncertain the further back in time we extrapolate.

Results and Discussion
Ancestral Sequence Reconstruction Using a Small NDK Sequence Set. The first step in the reconstruction of an ancestral sequence is to prepare a multiple amino acid sequence alignment, using the sequences of a given protein from extant species, which then is used to build a phylogenetic tree (12). Two methods, the maximum-likelihood (ML) method (25) and the Bayesian method (26), have been used commonly for tree building and reconstructing the ancestral sequence. In the ML method, the likelihood of each type of amino acid at each position in the sequence associated with the deepest node of the tree is computed using a statistical model of evolution. The ancestral sequence is defined as the set of residues in which each residue has the greatest likelihood of existing at its associated position. The Bayesian method integrates uncertainties associated with the tree topology, branch lengths, and substitution models into the ancestral se-quence calculation, whereas only the most likely estimate of the tree and substitution models are assumed with the ML method.

So, using the gene sequences that code for proteins in extant life and using known methods from population genetics and probability theory, the authors are able to reconstruct the most likely common ancestral sequence.

They splice the new genes into E Coli which then faithfully expresses the resurrected proteins. They then go on to test the stability of the proteins by heating them in a dilution and see when they break apart.

The genes encoding the inferred ancestral proteins were PCR constructed, and the encoded proteins were expressed individually in Escherichia coli and purified. CD spectral changes at 222 nm as a function of temperature were acquired to assess the thermostability of the proteins at pH 6.0 and 7.6 (Table 1 and Fig. S3C–F).

They use a whole host of different phylogenetic methods to produce a library of genes which they subsequently express in E Coli, the results of which can be seen here in Table 1.

Afu NDK and Tth NDK are thermostable proteins taken from bacteria T. thermophilus and archaea A. fulgidus which both live near hydrothermal vents. They discover that there's an issue with their reconstruction methods used to construct the proteins Arc1, Arc2, Bac1 and Bac2, ...

The ancestral sequences predicted by CODEML are designated Arc1 (archaeal) and Bac1 (bacterial); those predicted by nhPhylobayes are designated Arc2 and Bac2 (Fig. S2). These reconstructed proteins are extremely thermally stable (Table 1and Fig. S3AandB), a property that is compatible with the proposal that the ancestors of bacteria and archaea were thermophiles. However, the 10 sequences used to build the trees seemed not to represent NDK sequence space accurately. Therefore, the thermal stability of the reconstructed proteins may be an artifact of the reconstruction methods (29).

... so they change to a different method to reconstruct the sequences Arc3, Arc4, Bac3 and Bac4. The remaining sequences(Bac3sec, Arc3sec etc.) are control sequences derived from additional measures against artifacts of their phylogenetic reconstruction methods, which basically just confirm that yes, they're pretty damn sure these anceint proteins really were very thermostable.

Ancestral Sequences Inferred from204 NDK Sequences. The accuracy of a predicted ancestral sequence depends on sequence sampling, which can be improved by including as many extant sequences as possible in the reconstruction. Since we performed the aforementioned experiment, improvements in computational power have allowed the use of a larger dataset. Therefore we built two ML phylogenic trees from 204 extant NDK sequences. [The Bayesian approach was not used for this tree-building exercise because Thornton and colleagues (30) recently reported that the ML phylogenetic algorithm accurately reconstructs ancestral sequences.]
One tree was built without constraints (Fig. 1A and Fig. S4A), and one was built with the constraint that Archaea and Bacteria each represent a monophyletic group (Fig. 1Band Fig. S4B). For both trees, the major phyla are well grouped into their own monophyletic groups. However, the relationship among the phyla is slightly different for the two trees. In the tree built without constraint, certain archaeal sequences (those of Desulfurococcales and Thermoplasmatales) are paraphyletic and are found among the bacterial sequences, positionings that are inappropriate for a nearly universal phylogenetic tree. In the constrained tree, the Desulfurococcales and Thermo-plasmatales sequences also are paraphyletic, but they are positioned near the root of the Archaea domain. Because the difference in the likelihood values of the two trees is not substantial, the sequences at the deepest archaeal and bacterial nodes were inferred from both trees. The resulting ancestral sequences are named, using the nomenclature given above, as Arc3 (nonconstrained) and Arc4 (constrained) and Bac3 (nonconstrained), and Bac4 (constrained) (Fig. S2). The amino acid sequences of Arc3 and Arc4 are very similar (131 of 139 residues are identical), and the sequences of Bac3 and Bac4 also are very similar (129 residues are identical).
The genes encoding the inferred ancestral proteins were PCR constructed, and the encoded proteins were expressed individually in Escherichia coli and purified. CD spectral changes at 222 nm as a function of temperature were acquired to assess the thermostability of the proteins at pH 6.0 and 7.6 (Table 1 and Fig. S3C–F). The Arc3 and Arc4 unfolding midpoint temperatures (Tm,∼110 °C for both proteins) show that the two proteins are more stable than is the NDK from the hyperthermophilic archaeon Archaeoglobus fulgidus at pH 6.0. The bacterial ancestors are less stable than the archaeal ancestors but are as thermostable as, or are more stable than, NDK from the thermophile Thermus thermophilus under both pH conditions.

So, the resurrected proteins are even more thermostable than those found in extant hyperthermofiles.

A bit about making sure the inference that because the proteins are thermostable, the host organism is probably a thermophile, is valid:

Implications for the Environment Temperatures of Ancient Organisms.
The measuredTms of the resurrected NDKs provide experi-mental evidence for the existence of extremely thermally stable ancestral archaea and bacteria, if one assumes that, in general, the denaturation temperature of a protein reflects the environmental temperature of its host organism. Because this assumption is the key premise of our study, we determined the thermal stabilities of extant NDKs from organisms that thrive at various temperatures. A plot of theTms of the extant NDKs as a function of the optimal environmental temperatures of their host organ-isms is shown in Fig. 2A. The Tms correlate strongly with the optimal environmental temperatures [correlation coefficient = 0.96, which is nearly the same as a value (0.91) reported for 56 globular proteins from 16 different families (34)]. Therefore, the stability of each NDK appears to be related directly to its host’s natural environmental temperature.
Gaucher et al. (15, 19) reproduced the ancestor of bacterial elongation factor Tu, and, by virtue of its properties, suggested that the common bacterial ancestor was thermophilic. The an-cestral archaeon also is thought to be a thermophile or a hyper-thermophile. In the most commonly referenced phylogenetic tree composed of small-subunit rRNA sequences, those of hyper-thermophilic archaea are located near the node of their ancestor (2, 3). Theoretical studies also support the thermophilic origin of archaea (10, 35). According to theTms of the reconstructed NDKs (Table 1) and the calibration curve shown in Fig. 2A, the optimal environmental temperatures of the common ancestors of Bacteria and Archaea are∼80–93 °C and∼81–97 °C, respectively.

Having determined that the resurrected proteins don't break apart at high temperature, they turned to finding out the optimum temperature of the enzymatic activity of the NDK's:

Fig3.jpg (97.38 KiB) Viewed 6433 times

Catalytic Properties of Ancestral NDKs.
We next measured the en-zymatic activities of the ancestral NDKs by assessing the extent of γ-phosphate transfer from GTP to ADP to produce GDP and ATP. Fig. 3 depicts the specific activities of the ancestral NDKs as a function of temperature. The specific activities of the hyperthermophilic A. fulgidus and mesophilic Bacillus subtilis NDKs also were measured for comparison. The temperature dependence of the specific activities of the ancestral NDKs more closely resembles that of A. fulgidus NDK than that of the B. subtilis protein. Although B. subtilis NDK functions optimally at 60 °C, the specific activities of the ancestral NDKs and the A. fulgidus NDK all increase as the temperature increases up to 80 °C. A high optimal temperature also has been found for other naturally occurring thermophilic enzymes. The kinetic parameters of the ancestral, A. fulgidus, and T. thermophilus NDKs were obtained from initial velocity experiments at 70 °C that used various concentrations of ADP and 2.5 mM GTP (Table 2). Arc4 has the most unfavorable Km for ADP but the best kcat . Conversely, Bac1 has the best Km for ADP but the worst kcat. Overall, however, the resurrected ancestral and contemporary enzymes have similar kinetic parameters.

Table2.jpg (79.86 KiB) Viewed 6442 times

Unsurprisingly the authors conclude:

Limitations of Ancestral Sequence Reconstruction in Estimating Ancestral Environment Temperatures.
In this study, we found that the ancestral NDKs are extremely thermostable and functioned optimally at high temperatures, and therefore we concluded that the ancestral organisms lived in high-temperature environments.

They then go on to discuss common pitfalls and issues with using Ancestral Sequence Reconstruction both in general an with their exposition in particular, but remain pretty confident their inferences are valid and that the Last Universal Common Ancestor was a thermophile. For anyone with access to the full paper it has a lot of interesting discussion about the many challenges and way to strengthen the assumptions when doing ACR and inferring past environments from the properties of resurrected ancient proteins.

There are other similar papers covering the same material. For example:

Inferring The Palaeoenvironment Of Ancient Bacteria On The Basis Of Resurrected Proteins by Eric A. Gaucher, J. Michael Thomson, Michelle F. Burgan& Steven A. Benner, Nature, 425: 285-288 (18th September 2003) [Full paper downloadable from her]

Gaucher et al, 2003 wrote:Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences1–9 of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55–65 °C. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria10, the distribution of thermophily in derived bacterial lineages11, the inferredG+C content of ancient ribosomal RNA12, and the geological record combined with assumptions concerning molecular clocks13. The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Then there's this paper:

Ancestral Residues Stabilizing 3-Isopropylmalate Dehydrogenase Of An Extreme Thermophile: Experimental Evidence Supporting the Thermophilic Common Ancestor Hypothesis by Junichi Miyazaki, Shuichi Nakaya, Toshiharu Suzuki, Masatada Tamakoshi, Tairo Oshima, and Akihiko Yamagishi, The Journal of Biochemistry, [b]129(5):, 777-782 (2001) [full paper downloadable from here]

Miyazaki et al, 2001 wrote:Ancestral amino acid residues were inferred for 3-isopropylmalate dehydrogenase (IPMDH), and were introduced into the enzyme of an extreme thermophile, Sulfolobus sp. strain 7. The thermostability of the mutant enzymes was compared with that of the wild type enzyme. At least five of the seven mutants tested showed higher thermal stability than the wild type IPMDH. The results are compatible with the hyperthermophilic universal ancestor hypothesis. The results also provide a new method for designing thermostable enzymes. The method only relies on the first dimensional structures of homologous enzymes that can be obtained from genetic databases.

Then we have this:

Thermostability Of Ancestral Mutants Of Caldococcus noboribetus Isocitrate Dehydrogenase by Hisako Iwabata, Keiko Watanabe, Takatoshi Ohkuri†, Shin-ichi Yokobori and Akihiko Yamagishi, FEMS Microbiology Letters, 243(2: 393-398 (February 2005) [Full paper downloadable from here]

Iwabata et al, 2005 wrote:Abstract

We constructed mutant genes of Caldococcus noboribetu sisocitrate dehydrogenase containing ancestral amino acid residues that were inferred using the maximal likelihood method and a composite phylogenetic tree of isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase. The mutant genes were expressed in Escherichia coli and the protein products purified. Thermostabilities, reported as the half-inactivation temperatures, for the purified enzymes were determined and compared with that of the wild-type enzyme. Four of the five mutant enzymes have greater thermal stabilities than wild-type isocitrate dehydrogenase. The results are compatible with the hyperthermophilic universal ancestor (commonote) hypothesis. Incorporation of ancestral residues into a modern-day protein sequence can be used to improve protein thermostability.

Also, we have:

Hyperthermophily And The Origin And Earliest Evolution Of Life by Sara Islas, Ana M. Velasco, Arturo Becerra, Luis Delaye & Antonio Lazcano, International Microbiology, 6(2): 87-94 (June 2003) [Full paper downloadable form here]

Islas et al, 2003 wrote:Abstract The possibility of a high-temperature origin of life has gained support based on indirect evidence of a hot, early Earth and on the basal position of hyperthermophilic organisms in rRNA-based phylogenies. However, although the availability of more than 80 completely sequenced cellular genomes has led to the identification of hyperthermophilic-specific traits, such as a trend towards smaller genomes, reduced protein-encoding gene sizes, and glutamic-acid-rich simple sequences, none of these characteristics are in themselves an indication of primitiveness. There is no geological evidence for the physical setting in which life arose, but current models suggest that the Earth’s surface cooled down rapidly. Moreover, at 100°C the half-lives of several organic compounds, including ribose, nucleobases, and amino acids, which are generally thought to have been essential for the emergence of the first living systems, are too short to allow for their accumulation in the prebiotic environment. Accordingly, if hyperthermophily is not truly primordial, then heat-loving lifestyles may be relics of a secondary adaptation that evolved after the origin of life, and before or soon after separation of the major lineages.

There's also this book chapter:

Experimental Molecular Archeology: Reconstruction Of Ancestral Mutants And Evolutionary History Of Proteins As A New Approach In Protein Engineering by Tomohisa Ogawa and Tsuyoshi Shirai, DOI Identifier.

Ogawa & Shirai, 2013 wrote:1. Introduction

The diversity of life on Earth is the result of perpetual evolutionary processes beginning at life’s origins; evolution is the fundamental development strategy of life. Today, studies of gene and protein sequences, including various genome-sequencing projects, provide insight into these evolutionary processes and events. However, the sequence data obtained is restricted to extant genes and proteins, with the exception of the rare fossil genome samples [1, 2], for example Neanderthal [3], archaic hominin in Siberia [4, 5], and ancient elephants such as mastodon and mammoth [6]. The fossil record, and genome sequences derived from it, has the potential to elucidate ancient, extinct forms of life, acting as missing links to fill evolutionary gaps; however, the sequenced fossil genome is very limited, mainly due to the condition of samples and the challenges of preparing them. Discovering the forms of ancient organisms is one of the major purposes of paleontology, and is valuable in understanding of current life forms as these will be a reflection of their evolutionary history. However, the reconstruction of a living organism from fossils, which would be the ultimate paleontological methodology, is far beyond the currently available technologies, although there has recently been a report of the production of an artificial bacterial cell, using a chemically synthesized genome [7].

Meanwhile, for genes or the proteins they encode, it is already feasible to reconstruct their ancestral forms using phylogenetic trees constructed from sequence data; these techniques may well provide clues to the evolutionary history of certain extant genes and proteins with respect to their ancestors. Although phylogenetic analyses alone, or in combination with protein structure simulations, are useful to analyze structure-function relationships and evolutionary history [8], resurrected ancient recombinant proteins have the potential to provide more direct observations. Production of ancestral or ancient proteins can be achieved comparably easily due to developments in molecular biology and protein engineering techniques, which allow nucleotide or amino acid sequences to be synthesized. Ancestral proteins can be tested in the laboratory using biochemical or biophysical methods, for their activity, stability, specificity, and even three-dimensional structure. Thus, ancestral sequence reconstruction (ASR) has proved a useful experimental tool for studying the diverse structure and function of proteins [9]. To date, such ‘experimental molecular archeology’ using ASR has been applied to several enzymes [10-24], including photo-reactive proteins [25-37], nuclear receptor and transmembrane proteins [38-48], lectins [49-52], viral proteins [53, 54], elongation factor [55-57], paralbmin [58], in addition to a number of peptides [59,60] (Table 1).

Once that paper of yours becomes a free download,I'll add it to the collection.

Thank you cali, it seems the thermophilicty of the last universal ancestor has considerable experimental support.