Acquisition of 1,000 eubacterial genes physiologically transformed a methanogen at the origin of Haloarchaea Shijulal Nelson-Sathia, Tal Daganb, Giddy Landana,b, Arnold Janssenc, Mike Steeld, James O. McInerneye, Uwe Deppenmeierf, and William F. Martina,1 aInstitute of Molecular Evolution, bInstitute of Genomic Microbiology, cMathematisches Institut, Heinrich Heine University, 40225 Düsseldorf, Germany; dBiomathematics Research Centre, University of Canterbury, Private Bag 4800, Christchurch, New Zealand; eDepartment of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland; and fInstitute of Microbiology and Biotechnology, University of Bonn, 53115 Bonn, Germany Edited* by W. Ford Doolittle, Dalhousie University, Halifax, NS, Canada, and approved October 25, 2012 (received for review May 29, 2012) Archaebacterial halophiles (Haloarchaea) are oxygen-respiring involved in the assembly of FeS clusters (19). The sequencing of heterotrophs that derive from methanogens—strictly anaerobic, the first haloarchaeal genome over a decade ago identified some hydrogen-dependent autotrophs. Haloarchaeal genomes are known eubacterial genes that possibly could have been acquired by lat- to have acquired, via lateral gene transfer (LGT), several genes eral gene transfer (11, 20), and whereas substantial data that from eubacteria, but it is yet unknown how many genes the Hal- would illuminate the origin of haloarchaeal physiology have ac- oarchaea acquired in total and, more importantly, whether inde- cumulated since then, those data have not been subjected to pendent haloarchaeal lineages acquired their genes in parallel, or comparative evolutionary analysis. Investigating the role of the as a single acquisition at the origin of the group. Here we have environment in haloarchaeal genome evolution, Rhodes et al. studied 10 haloarchaeal and 1,143 reference genomes and have (21) recently showed that Haloarchaea are indeed far more likely identified 1,089 haloarchaeal gene families that were acquired by to acquire genes from other halophiles, but they did not address a methanogenic recipient from eubacteria. The data suggest that the issues at the focus of our present investigation, namely: How these genes were acquired in the haloarchaeal common ancestor, many eubacterial acquisitions are present in haloarchaeal genomes? EVOLUTION not in parallel in independent haloarchaeal lineages, nor in the How was the physiological transformation of methanogens to common ancestor of haloarchaeans and methanosarcinales. The Haloarchaea affected by LGT? Do those acquisitions trace to the 1,089 acquisitions include genes for catabolic carbon metabolism, haloarchaeal common ancestor as a single acquisition or not? membrane transporters, menaquinone biosynthesis, and com- To discern whether the eubacterial genes in haloarchaeal plexes I–IV of the eubacterial respiratory chain that functions in genomes are the result of multiple independent transfers in the haloarchaeal membrane consisting of diphytanyl isoprene individual lineages or the result of a single ancient mass ac- ether lipids. LGT on a massive scale transformed a strictly anaero- quisition, here we have analyzed 10 sequenced haloarchaeal bic, chemolithoautotrophic methanogen into the heterotrophic, genomes—Haloarcula marismortui (22), Halobacterium salina- oxygen-respiring, and bacteriorhodopsin-photosynthetic haloarch- rum (23), Halobacterium sp. (20), Halomicrobium mukohataei aeal common ancestor. (24), Haloquadratum walsbyi (25), Halorhabdus utahensis (26), Halorubrum lacusprofundi (27), Natrialba magadii (28), Natro- alophilic archaebacteria (Haloarchaea) require concen- nomonas pharaonis (29), and Haloterrigena turkmmenica (30)— Htrated salt solutions for survival and can inhabit saturated in the context of 65 other archaebacterial and >1,000 eubac- brine environments such as salt lakes, the Dead Sea, and salterns terial reference genomes. (1). In rRNA and phylogenomic analyses of informational genes, Haloarchaea always branch well within the methanogens (2–4). Results and Discussion Haloarchaea can thus be seen as deriving from methanogen We first clustered the 172,531 proteins encoded in the chromo- ancestors, but the physiology of methanogens and halophiles somes of 75 archaebacterial genomes into families using the could hardly be more different. Methanogens are strict anae- standard Markov cluster (MCL) procedure (31) yielding 16,061 robes, most species are lithoautotrophs that use electrons from protein families. Comparison with 1,078 completely sequenced H2 to reduce CO2 to methane (obligate hydrogenotrophic metha- eubacterial genomes delivered 1,479 protein families that are nogens), thereby generating a chemiosmotic ion gradient for present in at least two Haloarchaea and contain archaebacterial ATP synthesis in their energy metabolism, although some species and eubacterial homologs (Fig. 1A). Gene trees for the protein can generate methane from reduced C1 compounds, or acetate families were reconstructed using maximum likelihood inference in the case of aceticlastic forms (5–7). Their carbon metabolism (Methods). – involves the Wood Ljungdahl (acetyl-CoA) pathway of CO2 Of 1,479 trees, 1,089 (73%) uncovered Haloarchaea as mono- fixation (5–7). In contrast, Haloarchaea are obligate heterotrophs phyletic and rooting within (or branching next to) eubacterial that typically use O2 as the terminal acceptor of their electron rather than archaebacterial homologs (Fig. 1B). For 414 of these transport chain, although many can also use alternative electron trees, no homologs at all were detected in nonhalophilic acceptors such as nitrate in addition to light harnessing via a bac- teriorhodopsin-based proton pumping system (8). The evolutionary nature of that radical physiological transformation from anaerobic Author contributions: T.D., U.D., and W.F.M. designed research; S.N.-S. and T.D. per- chemolithoautotroph to aerobic heterotroph is of interest. formed research; S.N.-S., G.L., A.J., M.S., and J.O.M. analyzed data; and S.N.-S., G.L., and Many individual reports document that lateral gene transfer W.F.M. wrote the paper. (LGT) from eubacteria was involved in the origin of at least The authors declare no conflict of interest. some components of haloarchaeal metabolism. These include *This Direct Submission article had a prearranged editor. the operon for gas vesicle formation, which allows Haloarchaea Freely available online through the PNAS open access option. to remain in surface waters (9), the newly identified methylaspartate 1To whom correspondence should be addressed. E-mail: [email protected]. cycle of acetyl-CoA oxidation (10), various components of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. haloarchaeal aerobic respiratory chain (11–18), and proteins 1073/pnas.1209119109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1209119109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A Korarchaeota Nanoarchaeota Thaumarchaeota Thermoprotei Thermococci Methanopyri Methanobacteria Methanococci Methanosarcinales Methanocellales Methanomicrobiales Haloarchaea Archaeoglobi Thermoplasmata Eubacterial acquisitions in haloarchaeal genomes [trees] B C 2 (292) 0 500 1000 1500 3 (225) 4 ( 142) 952 (64.4%) 137(9.2%) 390 (26.4%) 5 (122) Single acqusition Single replacement Non-monophyletic 6 (107) 7 (101) Acquisitions Replacements 8 (115) Non-monophyletic 9 (106) 10 (269) Haloarchaea Eubacteria Other archaebacteria Presence in haloarchaeal genomes 0 50 100 150 200 250 300 Eubacterial acquisitions in haloarchaeal genomes [trees] Fig. 1. (A) Number of shared genes between 1,078 bacterial genomes and 75 archaebacterial genomes. (B) Types of phylogenetic trees obtained with respect to the relationship of Haloarchaea, nonhalophilic archaea, and eubacterial genes. (C) Types of phylogenetic trees detailed by the number of haloarchaeal taxa. archaebacteria. An additional 538 families had only very dis- gene trees for imported genes would be very different from one − tant homologs (E values >10 10 or amino acid identity <30%) in another as opposed to the case of single acquisition, where trees some nonhalophilic archaebacteria, together we designate these for imports should be the same due to vertical inheritance from 952 cases as “acquisitions.” An additional 137 genes yielded trees the haloarchaeal common ancestor. Moreover, trees for ances- in which Haloarchaea branch within eubacteria to the exclusion trally acquired eubacterial imports should not only be similar to of readily detectable archaebacterial homologs, we designate each other, they should also be similar to trees for endogenous these genes as “replacements”; acquisitions and replacements haloarchaeal genes that are shared only with other arch- we designate collectively as “imports” (Fig. 1B). The 390 cases of aebacteria, which we call recipient genes. There are 364 hal- Haloarchaea nonmonophyly included 76 trees in which one oarchaeal recipient genes that are present as single copies in all haloarchaeon branched deviantly and 105 trees in which the Hal- 10 Haloarchaea sampled and 109 haloarchaeal imports that are oarchaea were split into two groups of two or more species. Because present as single copies in all 10 Haloarchaea (Fig. 2A), LGT is common in prokaryotes (32, 33), among haloarchaeans in providing comparable tree sets. To avoid oversampling, the particular (21), these 181 gene trees could well depict secondary H. salinarum and the Halobacterium sp. genomes