Metabolic and evolutionary patterns in the extremely acidophilic archaeon Ferroplasma acidiphilum YT

Ferroplasmaceae represent ubiquitous iron-oxidising extreme acidophiles with a number of unique physiological traits. In a genome-based study of Ferroplasma acidiphilum YT, the only species of the genus Ferroplasma with a validly published name, we assessed its central metabolism and genome stability during a long-term cultivation experiment. Consistently with physiology, the genome analysis points to F. acidiphilum YT having an obligate peptidolytic oligotrophic lifestyle alongside with anaplerotic carbon assimilation. This narrow trophic specialisation abridges the sugar uptake, although all genes for glycolysis and gluconeogenesis, including bifunctional unidirectional fructose 1,6-bisphosphate aldolase/phosphatase, have been identified. Pyruvate and 2-oxoglutarate dehydrogenases are substituted by ‘ancient’ CoA-dependent pyruvate and alpha-ketoglutarate ferredoxin oxidoreductases. In the lab culture, after ~550 generations, the strain exhibited the mutation rate of ≥1.3 × 10−8 single nucleotide substitutions per site per generation, which is among the highest values recorded for unicellular organisms. All but one base substitutions were G:C to A:T, their distribution between coding and non-coding regions and synonymous-to-non-synonymous mutation ratios suggest the neutral drift being a prevalent mode in genome evolution in the lab culture. Mutations in nature seem to occur with lower frequencies, as suggested by a remarkable genomic conservation in F. acidiphilum YT variants from geographically distant populations

Search for Primitive Methanopyrus Based on Genetic Distance Between Val- and Ile-tRNA Synthetases

Since evidence indicates that the Last Universal Common Ancestor (LUCA) was phylogenetically closest to Methanopyrus kandleri among living organisms with elucidated genomes, this study has been directed to a search for the most primitive Methanopyrus lineage. For this purpose, the divergence of valyl-tRNA synthetase (ValRS) and isoleucyl-tRNA synthetase (IleRS) was employed as a measure of primitivity. Comparison of Methanopyrus kandleri and the Methanopyrus isolates GC34 and GC37 from the Pacific Ocean and KOL6, TAG1, TAG11, and SNP6 from the Atlantic Ocean established that the Pacific lineages are more primitive than the Atlantic lineages. Both the groups, however, are younger than environmental genomes from the Kairei Field of Central Indian Ridge in the Indian Ocean. These results showed that different Methanopyrus isolates differ significantly with respect to ValRS-IleRS divergence. On this basis, genomes giving rise to the ValRS and IleRS gene fragments from the Central Indian Ridge represent the most primitive Methanopyrus, phylogenetically the oldest living lineage closest to LUCA

Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA

Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially α-helical protein possibly containing a short N-terminal β-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of ∼43.5 ± 3.5 nm (136 ± 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon–helix–helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins

Cooperation between Catalytic and DNA Binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) <i>Proc. Natl. Acad. Sci. U.S.A.</i> <i>99</i>, 13510–13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix–hairpin–helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium <i>Bacillus stearothermophilus</i>. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases

Sugar Metabolism of the First Thermophilic Planctomycete Thermogutta terrifontis: Comparative Genomic and Transcriptomic Approaches

Xanthan gum, a complex polysaccharide comprising glucose, mannose and glucuronic acid residues, is involved in numerous biotechnological applications in cosmetics, agriculture, pharmaceuticals, food and petroleum industries. Additionally, its oligosaccharides were shown to possess antimicrobial, antioxidant, and few other properties. Yet, despite its extensive usage, little is known about xanthan gum degradation pathways and mechanisms. Thermogutta terrifontis, isolated from a sample of microbial mat developed in a terrestrial hot spring of Kunashir island (Far-East of Russia), was described as the first thermophilic representative of the Planctomycetes phylum. It grows well on xanthan gum either at aerobic or anaerobic conditions. Genomic analysis unraveled the pathways of oligo- and polysaccharides utilization, as well as the mechanisms of aerobic and anaerobic respiration. The combination of genomic and transcriptomic approaches suggested a novel xanthan gum degradation pathway which involves novel glycosidase(s) of DUF1080 family, hydrolyzing xanthan gum backbone beta-glucosidic linkages and beta-mannosidases instead of xanthan lyases, catalyzing cleavage of terminal beta-mannosidic linkages. Surprisingly, the genes coding DUF1080 proteins were abundant in T. terrifontis and in many other Planctomycetes genomes, which, together with our observation that xanthan gum being a selective substrate for many planctomycetes, suggest crucial role of DUF1080 in xanthan gum degradation. Our findings shed light on the metabolism of the first thermophilic planctomycete, capable to degrade a number of polysaccharides, either aerobically or anaerobically, including the biotechnologically important bacterial polysaccharide xanthan gum