Menaquinone Biosynthesis Research Articles

Suppression subtraction hybridization analysis revealed regulation of some cell cycle and toxin genes in Alexandrium catenella by phosphate limitation

Molecular mechanisms regulating responses to phosphate (P) limitation in dinoflagellates are poorly understood. Here we investigated gene differential expression Alexandrium catenella strain ATHK using suppression subtractive hybridization (SSH) and reverse-transcription quantitative PCR (qRT-PCR) techniques. SSH revealed a score of genes responded to P-limitation, and comparison with EST sequencing results indicated superior efficiency of SSH in identifying differentially expressed genes. Among the P-limitation responsive genes were the up-regulated stress-related genes, the down-regulated phosphorus-transferring genes, and a number of other genes previously undocumented for dinoflagellates. Most notably, the previously undocumented cell cycle inhibitory regulator fizzy/cell division cycle 20-related protein gene was up-regulated while putatively cell cycle promoting genes (e.g. calcium-dependent protein kinase) down-regulated, consistent with the decrease in growth rate and cell cycle arrest in G1 phase under P-limitation observed microscopically and flow cytometrically respectively. Besides, both SSH and qRT-PCR showed that saxitoxin related genes, menaquinone biosynthesis methyltransferase, an unknown gene and some other genes were also significantly induced by P-limitation. Our results demonstrate the utility of SSH in gene regulation research and provide a set of promising genes for further studies to understand the regulation of cell division (and hence population growth) and toxin production by P-limitation in A. catenella and likely other dinoflagellates.

Genome-Based Discovery of a Novel Membrane-Bound 1,6-Dihydroxyphenazine Prenyltransferase from a Marine Actinomycete

Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated Streptomyces sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in E. coli and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms.

Multitarget Drug Discovery for Tuberculosis and Other Infectious Diseases

We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.

The Futalosine Pathway Played an Important Role in Menaquinone Biosynthesis during Early Prokaryote Evolution

Menaquinone (MK) is an important component of the electron-transfer system in prokaryotes. One of its precursors, 1,4-dihydroxy-2-naphthoate, can be synthesized from chorismate by the classical MK pathway. Interestingly, in some bacteria, chorismate can also be converted to 1,4-dihydroxy-6-naphthoate by four enzymes encoded by mqnABCD in an alternative futalosine pathway. In this study, six crucial enzymes belonging to these two independent nonhomologous pathways were identified in the predicted proteomes of prokaryotes representing a broad phylogenetic distribution. Although the classical MK pathway was found in 32.1% of the proteomes, more than twice the proportion containing the futalosine pathway, the latter was found in a broader taxonomic range of organisms (18 of 31 phyla). The prokaryotes equipped with the classical MK pathway were almost all aerobic or facultatively anaerobic, but those with the futalosine pathway were not only aerobic or facultatively anaerobic but also anaerobic. Phylogenies of enzymes of the classical MK pathway indicated that its genes in archaea were probably acquired by an ancient horizontal gene transfer from bacterial donors. Therefore, the organization of the futalosine pathway likely predated that of the classical MK pathway in the evolutionary history of prokaryotes.

Chlamydia exploit the mammalian tryptophan-depletion defense strategy as a counter-defensive cue to trigger a survival state of persistence

We previously proposed that in Chlamydiaceae rapid vegetative growth and a quiescent state of survival (persistence) depend upon alternative protein translational profiles dictated by host tryptophan (Trp) availability. These alternative profiles correspond, respectively, with a set of chlamydial proteins having higher-than-predicted contents of Trp (“Up-Trp” selection), or with another set exhibiting lower-than-predicted contents of Trp (“Down-Trp” selection). A comparative evaluation of Chlamydiaceae proteomes for Trp content has now been extended to a number of other taxon families within the Chlamydiales Order. At the Order level, elevated Trp content occurs for transporters of nucleotides, S-adenosylmethionine (SAM), dicarboxylate substrates, and Trp itself. For Trp and nucleotide transporters, this is even more pronounced in other chlamydiae families (Parachlamydiaceae, Waddliaceae, and Simkaniaceae) due to extensive paralog expansion. This suggests that intracellular Trp availability served as an ancient survival cue for enhancement or restraint of chlamydial metabolism in the common Chlamydiales ancestor. The Chlamydiaceae Family further strengthened Up-Trp selection for proteins that function in cell division, lipopolysaccharide biosynthesis, and methyltransferase reactions. Some proteins that exhibit Up-Trp selection are uniquely present in the Chlamydiaceae, e.g., cytotoxin and the paralog families of polymorphic membrane proteins (Pmp's). A striking instance of Down-Trp selection in the Chlamydiaceae is the chorismate biosynthesis pathway and the connecting menaquinone pathway. The newly recognized 1,4-dihydroxy-6-napthoate pathway of menaquinone biosynthesis operates in Chlamydiaceae, whereas the classic 2-napthoate pathway is used in the other Chlamydiales families. Because of the extreme Down-Trp selection, it would appear that menaquinone biosynthesis is particularly important to the integrity of the persistent state maintained under conditions of severe Trp limitation, and may thus be critical for perpetuation of chronic disease states.

Energetics of pathogenic bacteria and opportunities for drug development.

The emergence and spread of drug-resistant pathogens and our inability to develop new antimicrobials to overcome resistance has inspired scientists to consider new targets for drug development. Cellular bioenergetics is an area showing promise for the development of new antimicrobials, particularly in the discovery of new anti-tuberculosis drugs where several new compounds have entered clinical trials. In this review, we have examined the bioenergetics of various bacterial pathogens, highlighting the versatility of electron donor and acceptor utilisation and the modularity of electron transport chain components in bacteria. In addition to re-examining classical concepts, we explore new literature that reveals the intricacies of pathogen energetics, for example, how Salmonella enterica and Campylobacter jejuni exploit host and microbiota to derive powerful electron donors and sinks; the strategies Mycobacterium tuberculosis and Pseudomonas aeruginosa use to persist in lung tissues; and the importance of sodium energetics and electron bifurcation in the chemiosmotic anaerobe Fusobacterium nucleatum. A combination of physiological, biochemical, and pharmacological data suggests that, in addition to the clinically-approved target F1Fo-ATP synthase, NADH dehydrogenase type II, succinate dehydrogenase, hydrogenase, cytochrome bd oxidase, and menaquinone biosynthesis pathways are particularly promising next-generation drug targets. The realisation of cellular energetics as a rich target space for the development of new antimicrobials will be dependent upon gaining increased understanding of the energetic processes utilised by pathogens in host environments and the ability to design bacterial-specific inhibitors of these processes.

Structural enzymology ofHelicobacter pylorimethylthioadenosine nucleosidase in the futalosine pathway

The recently discovered futalosine pathway is a promising target for the development of new antibiotics. The enzymes involved in this pathway are crucial for the biosynthesis of the essential prokaryotic respiratory compound menaquinone, and as the pathway is limited to few bacterial species such as the gastric pathogen Helicobacter pylori it is a potential target for specific antibiotics. In this report, the crystal structure of an H. pylori methylthioadenosine nucleosidase (MTAN; an enzyme with broad specificity and activity towards 6-amino-6-deoxyfutalosine), which is involved in the second step of menaquinone biosynthesis, has been elucidated at a resolution of 1.76 Å and refined with R factors of Rwork = 17% and Rfree = 21%. Activity studies on the wild type and active-site mutants show that the hydrolysis of 6-amino-6-deoxyfutalosine follows a mechanism similar to that of Escherichia coli MTAN. Further evidence for this mode of action is supplied by the crystal structures of active-site mutants. Through the use of reaction intermediates, the structures give additional evidence for the previously proposed nucleosidase mechanism. These structures and the confirmed reaction mechanism will provide a structural basis for the design of new inhibitors targeting the futalosine pathway.

Both Terminal Oxidases Contribute to Fitness and Virulence during Organ-Specific Staphylococcus aureus Colonization

ABSTRACTIn their recent article, Hammer et al. (N. D. Hammer, M. L. Reniere, J. E. Cassat, Y. Zhang, A. O. Hirsch, M. Indriati Hood, and E. P. Skaar, mBio 4:e00241-13, 2013) described the dual functions of the two terminal oxidases encoded by cydBA and qoxABCD in Staphylococcus aureus. The aerobic growth of cydB or qoxB single mutant bacteria was barely affected. However, a cydB qoxB double mutant was completely unable to respire and exhibited the small-colony variant phenotype that is typical of menaquinone and heme biosynthesis mutants. The authors found that the two terminal oxidases play a role in pathogenesis. In a systemic mouse infection model, it turned out that in the cydB mutant the bacterial burden was significantly decreased in the heart, kidneys, and liver, while in the qoxB mutant it was decreased only in the liver. These results illustrate that both terminal oxidases contribute to fitness and virulence, representing promising candidates for the development of antimicrobials.

Molecular Modeling and Docking Studies of O-Succinylbenzoate Synthase of M. tuberculosis—a Potential Target for Antituberculosis Drug Design

Menaquinone is a lipid-soluble naphthoquinone that is essential for various pivotal functions of bacteria. Naphthoquinone is synthesized from chorismate of the shikimate pathway in microorganisms. Due to its absence in humans and animals, menaquinone biosynthesis has been an attractive target for development of antibiotics against a number of important microbial pathogens, such as Mycobacterium tuberculosis (Mtb). In shikimate pathway, O-succinylbenzoate synthase (OSBS) plays a major role and is one of the major potential drug targets. For Mtb-OSBS, a systematic study was conducted to get an insight about Mtb-OSBS enzyme and the corresponding inhibitors using in silico methods. The 3-D model of Mtb-OSBS was built using structure coordinates of Thermobifida fusca. O-succinylbenzoate synthase, the model, was further refined. The active site amino acids have been identified by comparing the template sequence with the Mtb-OSBS sequence. We identified that Lys(108), Asn(140), Asp(138), Lys(110), Glu(189), Ser(236), Asp(188), Arg(27), Tyr(52), and Ser(237) are highly conserved, and these may play a vital role as active residues, similar to that in template protein. As per the competitive binding of substrate (2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC)), we screened the SHCHC through AutoDock 4.0. The SHCHC molecule was further modified structurally and optimized through PRODRG server. Docking of the 12 lead molecules for best interactions with Mtb-OSBS has given an insight that all the lead molecules have shown interactions with active site amino acids of Mtb-OSBS. MD simulation analysis report has shown the stable conformation annotations of Mtb-OSBS. These hypothetical studies create another way to develop more potential drugs against the deadly mycobacterium.

Menaquinone and Iron Are Essential for Complex Colony Development in Bacillus subtilis

Cells of undomesticated species of Bacillus subtilis frequently form complex colonies during spreading on agar surfaces. Given that menaquinone is involved in another form of coordinated behavior, namely, sporulation, we looked for a possible role for menaquinone in complex colony development (CCD) in the B. subtilis strain NCIB 3610. Here we show that inhibition of menaquinone biosynthesis in B. subtilis indeed abolished its ability to develop complex colonies. Additionally some mutations of B. subtilis which confer defective CCD could be suppressed by menaquinone derivatives. Several such mutants mapped to the dhb operon encoding the genes responsible for the biosynthesis of the iron siderophore, bacillibactin. Our results demonstrate that both menaquinone and iron are essential for CCD in B. subtilis.

Divergent Evolution of Ligand Binding in the o-Succinylbenzoate Synthase Family

Thermobifida fusca o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily that catalyzes a step in menaquinone biosynthesis, has an amino acid sequence that is 22 and 28% identical with those of two previously characterized OSBS enzymes from Escherichia coli and Amycolatopsis sp. T-1-60, respectively. These values are considerably lower than typical levels of sequence identity among homologous proteins that have the same function. To determine how such divergent enzymes catalyze the same reaction, we determined the structure of T. fusca OSBS and identified amino acids that are important for ligand binding. We discovered significant differences in structure and conformational flexibility between T. fusca OSBS and other members of the enolase superfamily. In particular, the 20s loop, a flexible loop in the active site that permits ligand binding and release in most enolase superfamily proteins, has a four-amino acid deletion and is well-ordered in T. fusca OSBS. Instead, the flexibility of a different region allows the substrate to enter from the other side of the active site. T. fusca OSBS was more tolerant of mutations at residues that were critical for activity in E. coli OSBS. Also, replacing active site amino acids found in one protein with the amino acids that occur at the same place in the other protein reduces the catalytic efficiency. Thus, the extraordinary divergence between these proteins does not appear to reflect a higher tolerance of mutations. Instead, large deletions outside the active site were accompanied by alteration of active site size and electrostatic interactions, resulting in small but significant differences in ligand binding.

Deamination of 6-Aminodeoxyfutalosine in Menaquinone Biosynthesis by Distantly Related Enzymes

Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10(5) M(-1) s(-1), Km values of 0.9-6.0 μM, and kcat values of 1.2-8.6 s(-1). Adenosine, 2'-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after β-strand 1 to be partially responsible for the substrate specificity of Sav2595.

Structure of isochorismate synthase DhbC from Bacillus anthracis.

The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg(2+)-dependent catalytic mechanism.

Dependence of the Product Chain-Length on Detergents for Long-Chain E-Polyprenyl Diphosphate Synthases

Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and nonionic detergents, we discovered considerable variability. Examination of a series of nonionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain-lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast, incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain-lengths.

Global transcriptome analysis of Escherichia coli exposed to immobilized anthraquinone-2-sulfonate and azo dye under anaerobic conditions

The immobilization of quinone compounds is regarded as a promising strategy to accelerate anaerobic decolorization of xenobiotic compounds azo dyes in the presence of quinone-reducing microorganisms. However, little is known about the basic response of these microorganisms to immobilized quinones in the presence of azo dyes. In the present study, whole-genome DNA microarrays were used to investigate a quinone-reducing bacterium Escherichia coli K-12 transcription response to immobilized anthraquinone-2-sulfonate (AQSim) reduction and azo dye acid red 18 (AR 18) decolorization. Transcriptome analysis showed that AQSim was more accessible for the cells of E. coli K-12 than AR 18. Despite there being some differences between AQSim and soluble AQS mediated decolorization of AR 18, AQSim reduction and AR 18 decolorization, more similarity could be observed in the four processes. Among over 60 % shared genes, several groups of genes exhibited high expression levels, including those genes encoding terminal reductases, menaquinone biosynthesis, formate dehydrogenases and outer membrane proteins. Especially, nrfABCD, frdBCD and dsmABC encoding terminal reductases were significantly upregulated. Further gene deletion experiments demonstrated that the above three groups of genes were involved in AQSim-mediated AR 18 decolorization. In addition, significant upregulation of stress response genes was observed, which indicated the adaptation of E. coli K-12 to AQSim and AR 18 exposures.

In Vitro Reconstitution of the Radical S-Adenosylmethionine Enzyme MqnC Involved in the Biosynthesis of Futalosine-Derived Menaquinone

The radical S-adenosylmethionine enzyme MqnC catalyzes conversion of dehypoxanthine futalosine (DHFL) to the unique spiro compound cyclic DHFL in the futalosine pathway for menaquinone biosynthesis. This study describes the in vitro reconstitution of [4Fe-4S] cluster-dependent MqnC activity and identifies the site of abstraction of a hydrogen atom from DHFL by the adenosyl radical.

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.

Identification of a hotdog fold thioesterase involved in the biosynthesis of menaquinone in Escherichia coli.

Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ΔybgC and ΔentH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.

Enzyme‐Substrate Interactions in the Helicobacter pylori 5′‐Methylthioadenosine/S‐adenosylhomocysteine Nucleosidase

5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase (MTAN) is a multi‐functional bacterial enzyme that catalyzes the hydrolysis of the N‐glycosidic bond of many adenosine‐based metabolites. This hydrolytic activity places MTAN at the hub of at least seven different metabolic pathways, which includes the purine salvage pathway, the methionine salvage pathway, various S‐adenosylmethionine (SAM)‐dependent methylation pathways, polyamine biosynthesis, the production of quorum sensing molecules and menaquinone biosynthesis. The bacterial species such as Campylobacter and Helicobacter are dependent on MTAN for menaquinone synthesis, an essential metabolite for bacterial viability. As MTAN is absent in humans but essential for bacteria like Helicobacter pylori, it is an excellent target for the development of new treatments for Helicobacter infections. To structurally characterize the interactions between MTAN and its various substrates, complexes of an inactive mutant of the Helicobacter pylori MTAN with two known substrates, 5′‐methylthioadenosine (MTA) and S‐adenosylhomocysteine (SAH) were formed and crystallized. The crystal structures of mutant MTAN with SAH and MTA bound in its active site were solved to 1.2 and 1.6 Å, respectively. The MTAN‐SAH co‐crystal structure represents the first visualization of interactions between the homocysteine moiety of SAH and the 5′‐alkylthiol‐binding sub‐site of the MTAN active site. The co‐crystal structure of wild‐type MTAN with products, adenine and S‐ribosylhomocysteine, was also determined to 1.54 Å resolution. The similarities and differences in these three structures highlight features that can be exploited to design H. pylori specific drugs.

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.