Acquisition of 1,000 eubacterial genes physiologically transformed a methanogen at the origin of Haloarchaea

Archaebacterial halophiles (Haloarchaea) are oxygen-respiring heterotrophs that derive from methanogens—strictly anaerobic, hydrogen-dependent autotrophs. Haloarchaeal genomes are known to have acquired, via lateral gene transfer (LGT), several genes from eubacteria, but it is yet unknown how many genes the Haloarchaea acquired in total and, more importantly, whether independent haloarchaeal lineages acquired their genes in parallel, or as a single acquisition at the origin of the group. Here we have studied 10 haloarchaeal and 1,143 reference genomes and have identified 1,089 haloarchaeal gene families that were acquired by a methanogenic recipient from eubacteria. The data suggest that these genes were acquired in the haloarchaeal common ancestor, not in parallel in independent haloarchaeal lineages, nor in the common ancestor of haloarchaeans and methanosarcinales. The 1,089 acquisitions include genes for catabolic carbon metabolism, membrane transporters, menaquinone biosynthesis, and complexes I–IV of the eubacterial respiratory chain that functions in the haloarchaeal membrane consisting of diphytanyl isoprene ether lipids. LGT on a massive scale transformed a strictly anaerobic, chemolithoautotrophic methanogen into the heterotrophic, oxygen-respiring, and bacteriorhodopsin-photosynthetic haloarchaeal common ancestor.Halophilic archaebacteria (Haloarchaea) require concentrated salt solutions for survival and can inhabit saturated brine environments such as salt lakes, the Dead Sea, and salterns (1). In rRNA and phylogenomic analyses of informational genes, Haloarchaea always branch well within the methanogens (2–4). Haloarchaea can thus be seen as deriving from methanogen ancestors, but the physiology of methanogens and halophiles could hardly be more different. Methanogens are strict anaerobes, most species are lithoautotrophs that use electrons from H₂ to reduce CO₂ to methane (obligate hydrogenotrophic methanogens), thereby generating a chemiosmotic ion gradient for ATP synthesis in their energy metabolism, although some species can generate methane from reduced C₁ compounds, or acetate in the case of aceticlastic forms (5–7). Their carbon metabolism involves the Wood–Ljungdahl (acetyl-CoA) pathway of CO₂ fixation (5–7). In contrast, Haloarchaea are obligate heterotrophs that typically use O₂ as the terminal acceptor of their electron transport chain, although many can also use alternative electron acceptors such as nitrate in addition to light harnessing via a bacteriorhodopsin-based proton pumping system (8). The evolutionary nature of that radical physiological transformation from anaerobic chemolithoautotroph to aerobic heterotroph is of interest.Many individual reports document that lateral gene transfer (LGT) from eubacteria was involved in the origin of at least some components of haloarchaeal metabolism. These include the operon for gas vesicle formation, which allows Haloarchaea to remain in surface waters (9), the newly identified methylaspartate cycle of acetyl-CoA oxidation (10), various components of the haloarchaeal aerobic respiratory chain (11–18), and proteins involved in the assembly of FeS clusters (19). The sequencing of the first haloarchaeal genome over a decade ago identified some eubacterial genes that possibly could have been acquired by lateral gene transfer (11, 20), and whereas substantial data that would illuminate the origin of haloarchaeal physiology have accumulated since then, those data have not been subjected to comparative evolutionary analysis. Investigating the role of the environment in haloarchaeal genome evolution, Rhodes et al. (21) recently showed that Haloarchaea are indeed far more likely to acquire genes from other halophiles, but they did not address the issues at the focus of our present investigation, namely: How many eubacterial acquisitions are present in haloarchaeal genomes? How was the physiological transformation of methanogens to Haloarchaea affected by LGT? Do those acquisitions trace to the haloarchaeal common ancestor as a single acquisition or not?To discern whether the eubacterial genes in haloarchaeal genomes are the result of multiple independent transfers in individual lineages or the result of a single ancient mass acquisition, here we have analyzed 10 sequenced haloarchaeal genomes—Haloarcula marismortui (22), Halobacterium salinarum (23), Halobacterium sp. (20), Halomicrobium mukohataei (24), Haloquadratum walsbyi (25), Halorhabdus utahensis (26), Halorubrum lacusprofundi (27), Natrialba magadii (28), Natronomonas pharaonis (29), and Haloterrigena turkmmenica (30)—in the context of 65 other archaebacterial and >1,000 eubacterial reference genomes.