Descent of Bacteria and Eukarya From an Archaeal Root of Life

The 3 biological domains delineated based on small subunit ribosomal RNAs (SSU rRNAs) are confronted by uncertainties regarding the relationship between Archaea and Bacteria, and the origin of Eukarya. The similarities between the paralogous valyl-tRNA and isoleucyl-tRNA synthetases in 5398 species estimated by BLASTP, which decreased from Archaea to Bacteria and further to Eukarya, were consistent with vertical gene transmission from an archaeal root of life close to Methanopyrus kandleri through a Primitive Archaea Cluster to an Ancestral Bacteria Cluster, and to Eukarya. The predominant similarities of the ribosomal proteins (rProts) of eukaryotes toward archaeal rProts relative to bacterial rProts established that an archaeal parent rather than a bacterial parent underwent genome merger with bacteria to generate eukaryotes with mitochondria. Eukaryogenesis benefited from the predominantly archaeal accelerated gene adoption (AGA) phenotype pertaining to horizontally transferred genes from other prokaryotes and expedited genome evolution via both gene-content mutations and nucleotidyl mutations. Archaeons endowed with substantial AGA activity were accordingly favored as candidate archaeal parents. Based on the top similarity bitscores displayed by their proteomes toward the eukaryotic proteomes of Giardia and Trichomonas, and high AGA activity, the Aciduliprofundum archaea were identified as leading candidates of the archaeal parent. The Asgard archaeons and a number of bacterial species were among the foremost potential contributors of eukaryotic-like proteins to Eukarya.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri. Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 fromMethanopyrus kandleri

Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such asMethanopyrus kandlericontain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 fromM. kandleriin apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified.

COMPARATIVE ANALYSIS OF METABOLIC NETWORKS IN MESOPHILIC AND THERMOPHILIC ARCHAEA METHANOGENS BASED ON MODULARITY

Metabolic networks are useful representations of the metabolic capabilities of cells. A comparison of metabolic networks across species is essential to better understand how evolutionary pressures shape these networks. By comparing the set of reactions that are expected to occur in an organism with the set of reactions in reference metabolic pathways, it is possible to infer the main metabolic functions of an organism. In this paper, the metabolic networks of the mesophilic archaeon Methanosarcina acetivorans and the thermophilic archaeon Methanopyrus kandleri have been reconstructed based on the KEGG LIGAND database, followed by four topological statistical analyses of the nodes in the two networks to compare their metabolic networks. The values of average degree and characteristic path length are very small but clustering coefficient is relatively large. The results show that the complete metabolic networks of M. acetivorans and M. kandleri possessed "small-world" network properties. Then we used Girvan–Newman modular algorithm to identify hub modules and compared hub modules with non-hub modules, respectively. The results show that M. kandleri metabolic network has a better modular organization than the M. acetivorans network. M. acetivorans includes 39 modules, 25 modules of them are independent, and 15 modules are functionally pure. On the other hand, M. kandleri includes 30 modules. Among them, there are 20 independent modules, and 14 of them are functionally pure. These results further indicated that the present approach for identifying modules yields modules that have biologically significant functions. We also identified hub modules of the metabolic networks and found that these hub modules are carbohydrate metabolism and amino acid metabolism. The conclusions obtained from such studies provide a broad overview of the similarities and differences between organism's metabolic networks. These will be very helpful for further research on thermostability of methanogens.

Positive, Neutral and Negative Interactions in Cocultures between Pyrococcus furiosus and Different Methanogenic Archaea

The model organism Pyrococcus furiosus has recently been reported to interact with Methanopyrus kandleri in coculture, suggesting a H2 symbiosis. In the current study we further investigated this hypothesis by growing P. furiosus with four other hyperthermophilic methanogens providing evidence that the organisms did not only exert positive effects ( P. furiosus/ Methanocaldococcus villosus and P. furiosus/ Methanocaldococcus infernus) on each other, but also neutral ( P. furiosus/ Methanocaldococcus jannaschii) and even inhibitory interactions ( P. furiosus/ Methanotorris igneus) were detected suggesting interspecies relationships not only based on H2 symbiosis. Using various microscopic techniques we further analyzed the coculture with the highest positive interactions ( P. furiosus/ M. villosus) concerning its growth behavior on various surfaces, which turned out to be in stark contrast to the previous reported coculture of P. furiosus/ M. kandleri. This communication provides new insights into possible interactions of extremophilic Archaea in cocultures and again raises the question if and how hyperthermophilic Archaea communicate besides metabolic intermediates like H2.

The Mode of Cell Wall Growth in Selected Archaea Is Similar to the General Mode of Cell Wall Growth in Bacteria as Revealed by Fluorescent Dye Analysis

ABSTRACTThe surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilicArchaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth inArchaeaby subculturing stained cells. The data obtained show that incorporation of new cell wall material inArchaeafollows the pattern observed forBacteria: in the coccoid speciesPyrococcus furiosusincorporation was in the region of septum formation while for the rod-shaped speciesMethanopyrus kandleriandMethanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used forin situanalyses at temperatures up to 100°C.