Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties.

Samuel Blanquart,Eric Girard,Mathieu Groussin,Gergely J Szöllosi,Manolo Gouy,Dominique Madern,Aline Le Roy,Bruno Franzetti,Miriam Barlow

doi:10.1093/molbev/msab146

Abstract

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.

Highlights

The adaptation of organisms to fluctuating environments occurred through two evolutionary processes: amino acid replacements that affect protein structure, function and dynamics (Tomatis et al 2008; Liu and Bahar, 2012), and, less frequently, horizontal gene transfer (HGT, the acquisition of a foreign functional coding gene), which can quickly modify the properties of an organism (Fournier et al 2015)
These are the main findings of our study: The last common ancestor malate dehydrogenases (MalDH) of Halobacteria (LCAHa) does not necessitate multi-molar KCl in order to be stable and active, suggesting it was mildly halophilic; the paleo environment at the origin of Halobacteria in Archaea could not be inferred, because the KCl-concentration-dependent stabilities of MalDHs are not correlated with the respective environmental conditions of their hosts; the description of a tradeoff mechanism by which the haloalkaliphilic lineage of MalDH has emerged; and the unexpected experimental outcome that the modern MalDH from Haloarcula marismortui was the result of an ancestral HGT from an early haloalkaliphilic ancestor
We examined several sources of reconstruction artifacts resulting from long branch attraction (LBA) artifacts and compositional biases to obtain a robust and strongly supported halobacteria species phylogeny (Fig.1 and Fig.S1)

Summary

Introduction

The adaptation of organisms to fluctuating environments occurred through two evolutionary processes: amino acid replacements (replacement process, RP) that affect protein structure, function and dynamics (Tomatis et al 2008; Liu and Bahar, 2012), and, less frequently, horizontal gene transfer (HGT, the acquisition of a foreign functional coding gene), which can quickly modify the properties of an organism (Fournier et al 2015). Analytical ultra-centrifugation (AUC) experiments demonstrated how changes of the oligomeric state equilibrium between active conformers could rescue the deleterious effect of low salt concentration in some lineages These are the main findings of our study: The last common ancestor MalDH of Halobacteria (LCAHa) does not necessitate multi-molar KCl in order to be stable and active, suggesting it was mildly halophilic; the paleo environment at the origin of Halobacteria in Archaea could not be inferred, because the KCl-concentration-dependent stabilities of MalDHs are not correlated with the respective environmental conditions of their hosts; the description of a tradeoff mechanism by which the haloalkaliphilic lineage of MalDH has emerged; and the unexpected experimental outcome that the modern MalDH from Haloarcula marismortui (the most studied halophilic enzyme to date) was the result of an ancestral HGT from an early haloalkaliphilic ancestor. Stability and activity of MalDHs facing extreme salt conditions did not evolve concomitantly, and discuss, here, how these findings establish a new understanding of halophilic adaptation in MalDHs

Results

Discussion

Materials and Methods