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Anatoli Brouchkov, Marsel Kabilov, Svetlana Filippova, Olga Baturina, Victor Rogov, Valery Galchenko, Andrey Mulyukin, Oksana Fursova, Gennady Pogorelko
Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa...
September 12, 2017: Gene
Piotr M Skowron, Brian P Anton, Edyta Czajkowska, Joanna Zebrowska, Ewa Sulecka, Daria Krefft, Joanna Jezewska-Frackowiak, Olga Zolnierkiewicz, Malgorzata Witkowska, Richard D Morgan, Geoffrey G Wilson, Alexey Fomenkov, Richard J Roberts, Agnieszka Zylicz-Stachula
Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5'-GACCGA-3' and 5'-CACCCA-3'. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5'-GACCGA-3' sequence only...
September 6, 2017: Nucleic Acids Research
Gang Sheng, Tasos Gogakos, Jiuyu Wang, Hongtu Zhao, Artem Serganov, Stefan Juranek, Thomas Tuschl, Dinshaw J Patel, Yanli Wang
We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6'U7' and 6'A7' on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step...
September 6, 2017: Nucleic Acids Research
Minwoo Lee, Hyejin Um, Michael W Van Dyke
One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combinatorial selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) and then use this information to bioinformatically identify potential regulated genes. REPSA determined a consensus FadR-binding sequence 5´-TTRNACYNRGTNYAA-3´, which was further characterized using quantitative electrophoretic mobility shift assays...
2017: PloS One
Atsushi Fujita, Yoshio Misumi
We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal(-)) on growth plates containing 3,4-cyclohexenoesculetin β-D-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites (Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T...
September 11, 2017: Extremophiles: Life Under Extreme Conditions
Xiangyun Zang, Meiting Liu, Han Wang, Yihong Fan, Haichang Zhang, Jiawen Liu, Enlu Xing, Xiuhong Xu, Hongtao Li
Cellulose degradation is an important part of global carbon cycle in composting process, and β-Glucosidases complete the final step of cellulose hydrolysis by converting cellobiose to glucose. This work analyzes the succession of β-glucosidase-producing microbial communities that persists throughout composting, evaluates their metabolic activities and community advantage during the various phases of composting. Fungal and bacterial β-glucosidase genes belonging to glycoside hydrolase families GH1 and GH3 amplified from DNA and gene abundance levels were analyzed...
September 11, 2017: Canadian Journal of Microbiology
Abhijit Poddar, Subrata K Das
The earliest microbiological studies on hot springs in India date from 2003, a much later date compared to global attention in this striking field of study. As of today, 28 out of 400 geothermal springs have been explored following both culturable and non-culturable approaches. The temperatures and pH of the springs are 37-99 °C and 6.8-10, respectively. Several studies have been performed on the description of novel genera and species, characterization of different bio-resources, metagenomics of hot spring microbiome and whole genome analysis of few isolates...
September 8, 2017: Archives of Microbiology
Kaiying Cheng, Guangzhi Xu, Hong Xu, Ye Zhao, Yuejin Hua
RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF-dr1088 operon, which is highly conserved in the Deinococcus-Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two-hybrid and pull-down assays. DR1088 exhibited some RecO-like biochemical properties including single/double-stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity...
September 1, 2017: Molecular Microbiology
Inam Ullah Khan, Neeli Habib, Firasat Hussain, Wen-Dong Xian, Arshia Amin, En-Min Zhou, Iftikhar Ahmed, Xiao-Yang Zhi, Wen-Jun Li
A thermophilic bacterial strain, designated YIM 73026T was isolated from a sediment sample collected from a hot spring in Tibet, PR China. The taxonomic position of the novel isolate was investigated by a polyphasic approach. The novel isolate was Gram-stain-negative, aerobic and rod-shaped. Colonies were circular, convex, opaque and yellow. The strain grew at 50-70 °C (optimum, 60 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of up to 1.0 % NaCl (w/v). Comparison of the 16S rRNA gene sequences of YIM 73026T and those of other members of the genus Thermus showed sequence similarities ranging from 91...
August 2017: International Journal of Systematic and Evolutionary Microbiology
Takeo Tomita
Amino acids are essential components in all organisms because they are building blocks of proteins. They are also produced industrially and used for various purposes. For example, L-glutamate is used as the component of "umami" taste and lysine has been used as livestock feed. Recently, many kinds of amino acids have attracted attention as biological regulators and are used for a healthy life. Thus, to clarify the mechanism of how amino acids are biosynthesized and how they work as biological regulators will lead to further effective utilization of them...
August 25, 2017: Bioscience, Biotechnology, and Biochemistry
Yoichiro Togawa, Tatsuo Nunoshiba, Keiichiro Hiratsu
Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination...
August 24, 2017: Molecular Genetics and Genomics: MGG
Sergey A Siletsky, Ilya Belevich, Nikolai P Belevich, Tewfik Soulimane, Mårten Wikström
Two electrogenic phases with characteristic times of ~14μs and ~290μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba3 oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between CuA and heme b. The slow phase includes electron redistribution from both CuA and heme b to heme a3, and electrogenic proton transfer coupled to reduction of heme a3. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a3 is reduced, but there is no proton pumping and no reduction of CuB...
August 12, 2017: Biochimica et Biophysica Acta
Charu Tripathi, Harshita Mishra, Himani Khurana, Vatsala Dwivedi, Komal Kamra, Ram K Negi, Rup Lal
Thermophilic environments represent an interesting niche. Among thermophiles, the genus Thermus is among the most studied genera. In this study, we have sequenced the genome of Thermus parvatiensis strain RL, a thermophile isolated from Himalayan hot water springs (temperature >96°C) using PacBio RSII SMRT technique. The small genome (2.01 Mbp) comprises a chromosome (1.87 Mbp) and a plasmid (143 Kbp), designated in this study as pTP143. Annotation revealed a high number of repair genes, a squeezed genome but containing highly plastic plasmid with transposases, integrases, mobile elements and hypothetical proteins (44%)...
2017: Frontiers in Microbiology
Outi Haapanen, Vivek Sharma
Membrane bound respiratory complex I is the key enzyme in the respiratory chains of bacteria and mitochondria, and couples the reduction of quinone to the pumping of protons across the membrane. Recently solved crystal or electron microscopy structures of bacterial and mitochondrial complexes have provided significant insights into the electron and proton transfer pathways. However, due to large spatial separation between the electron and proton transfer routes, the molecular mechanism of coupling remains unclear...
August 10, 2017: Scientific Reports
J Del Arco, J Cejudo-Sanches, I Esteban, V J Clemente-Suárez, D Hormigo, A Perona, J Fernández-Lucas
Traditionally, enzymatic synthesis of nucleoside-5'-monophosphates (5'-NMPs) using low water-soluble purine bases has been described as less efficient due to their low solubility in aqueous media. The use of enzymes from extremophiles, such as thermophiles or alkaliphiles, offers the potential to increase solubilisation of these bases by employing high temperatures or alkaline pH. This study describes the cloning, expression and purification of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Thermus thermophilus (TtHGXPRT)...
December 15, 2017: Food Chemistry
A V Rayevsky, M Sharifi, M A Tukalo
Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS (PhLeuRS) with misacylated tRNA(Leu) was modeled wherever tRNA's acceptor stem was oriented directly into the editing site...
July 18, 2017: Journal of Molecular Graphics & Modelling
Satomi Fujita, Su-Hee Cho, Ayako Yoshida, Fumihito Hasebe, Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama
LysK is an M20 peptidase family enzyme that hydrolyzes the isopeptide bond between the carrier protein LysW and lysine in order to release lysine, which is the last step of lysine biosynthesis in Thermus thermophilus. In the present study, we determined the crystal structure of LysK in complex with lysine at a resolution of 2.4 Å. The α-amino group of the bound lysine was oriented toward the catalytic center, which was composed of the residues coordinating divalent metal ions for the hydrolysis of the isopeptide bond...
September 16, 2017: Biochemical and Biophysical Research Communications
Andrea Di Luca, Ana P Gamiz-Hernandez, Ville R I Kaila
Complex I functions as the initial electron acceptor in aerobic respiratory chains of most organisms. This gigantic redox-driven enzyme employs the energy from quinone reduction to pump protons across its complete approximately 200-Å membrane domain, thermodynamically driving synthesis of ATP. Despite recently resolved structures from several species, the molecular mechanism by which complex I catalyzes this long-range proton-coupled electron transfer process, however, still remains unclear. We perform here large-scale classical and quantum molecular simulations to study the function of the proton pump in complex I from Thermus thermophilus The simulations suggest that proton channels are established at symmetry-related locations in four subunits of the membrane domain...
August 1, 2017: Proceedings of the National Academy of Sciences of the United States of America
Ana Luisa Ribeiro, Mercedes Sánchez, Aurelio Hidalgo, José Berenguer
Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes...
2017: Methods in Molecular Biology
Pious Thomas, Aparna C Sekhar, Sadiq Pasha Shaik
Molecular and microscopic analyses reveal enormous non-cultivable endophytic bacteria in grapevine field shoots with functional significance. Diverse bacteria enter tissue cultures through surface-sterilized tissues and survive surreptitiously with varying taxonomic realignments. The study was envisaged to assess the extent of endophytic bacterial association with field shoot tissues of grapevine and the likelihood of introduction of such internally colonizing bacteria in vitro adopting molecular techniques targeting the non-cultivable bacterial community...
July 11, 2017: Planta
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