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Mieke M E Huijbers, Ilona van Alen, Jenny W Wu, Arjan Barendregt, Albert J R Heck, Willem J H van Berkel
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ¹-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket...
January 16, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Saheb Dutta, Nilashis Nandi
Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (ttSerRS) bound with tttRNASer and dimeric ThrRS from Escherichia coli (ecThrRS) bound with ectRNAThr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively...
January 10, 2018: Journal of Biomolecular Structure & Dynamics
Nina Ihling, Natalie Bittner, Sylvia Diederichs, Maximilian Schelden, Anna Korona, Georg Theo Höfler, Alexander Fulton, Karl-Erich Jaeger, Kohsuke Honda, Hisao Ohtake, Jochen Büchs
Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and non-inducing conditions...
January 5, 2018: Biotechnology Progress
Atsuko Nakanishi, Jun-Ichi Kishikawa, Masatada Tamakoshi, Kaoru Mitsuoka, Ken Yokoyama
Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit...
January 8, 2018: Nature Communications
Edoardo D'Imprima, Ralf Salzer, Ramachandra M Bhaskara, Ricardo Sánchez, Ilona Rose, Lennart Kirchner, Gerhard Hummer, Werner Kühlbrandt, Janet Vonck, Beate Averhoff
Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane...
December 27, 2017: ELife
C Denise Okafor, Manish C Pathak, Crystal E Fagan, Nicholas C Bauer, Megan F Cole, Eric A Gaucher, Eric A Ortlund
Rationally engineering thermostability in proteins would create enzymes and receptors that function under harsh industrial applications. Several sequence-based approaches can generate thermostable variants of mesophilic proteins. To gain insight into the mechanisms by which proteins become more stable, we use structural and dynamic analyses to compare two popular approaches, ancestral sequence reconstruction (ASR) and the consensus method, used to generate thermostable variants of Elongation Factor Thermo-unstable (EF-Tu)...
December 16, 2017: Structure
Mounia Akassou, Denis Groleau
The objective of this study was to determine the potential of Thermus thermophilus HB8 for accumulating a high level of extracellular, thermostable amylolytic enzyme. Initial production tests indicated clearly that only very low levels of amylolytic activity could be detected, solely from cells after extraction using the mild, non-ionic detergent Triton X-100. A sequential optimization strategy, based on statistical designs, was used to enhance greatly the production of extracellular amylolytic activity to achieve industrially attractive enzyme titers...
December 19, 2017: Extremophiles: Life Under Extreme Conditions
M El-Fadel, F Sleem, J Hashisho, P E Saikaly, I Alameddine, S Ghanimeh
This study examines the performance and fouling potential of flat sheet (FS) and hollow fiber (HF) membrane bioreactors (MBRs) during the treatment of high strength landfill leachate under varying solid retention times (SRT = 5-20 days). Mixed-liquor bacterial communities were examined over time using 16S rRNA gene sequence analysis in an attempt to define linkages between the system performance and the microbial community composition. Similarly, biofilm samples were collected at the end of each SRT to characterize the microbial communities that evolved on the surface of the FS and HF membranes...
December 14, 2017: Waste Management
Ramganesh Selvarajan, Timothy Sibanda, Memory Tekere
Microbial mats are occasionally reported in thermal springs and information on such mats is very scarce. In this study, microbial mats were collected from two hot springs (Brandvlei (BV) and Calitzdorp (CA)), South Africa and subjected to scanning electron microscopy (SEM) and targeted 16S rRNA gene amplicon analysis using Next Generation Sequencing (NGS). Spring water temperature was 55°C for Brandvlei and 58°C for Calitzdorp while the pH of both springs was slightly acidic, with an almost identical pH range (6...
December 15, 2017: MicrobiologyOpen
Lisa M Steinberg, Rachel E Kronyak, Christopher H House
Future long-term manned space missions will require effective recycling of water and nutrients as part of a life support system. Biological waste treatment is less energy intensive than physicochemical treatment methods, yet anaerobic methanogenic waste treatment has been largely avoided due to slow treatment rates and safety issues concerning methane production. However, methane is generated during atmosphere regeneration on the ISS. Here we propose waste treatment via anaerobic digestion followed by methanotrophic growth of Methylococcus capsulatus to produce a protein- and lipid-rich biomass that can be directly consumed, or used to produce other high-protein food sources such as fish...
November 2017: Life Sciences in Space Research
Laura Alvarez, Nieves G Quintáns, Alba Blesa, Ignacio Baquedano, Mario Mencía, Carlos Bricio, José Berenguer
Denitrification in Thermus thermophilus is encoded by the nitrate respiration conjugative element (NCE) and nitrite and nitric oxide respiration (nic) gene clusters. A tight coordination of each cluster's expression is required to maximize anaerobic growth, and to avoid toxicity by intermediates, especially nitric oxides (NO). Here, we study the control of the nitrite reductases (Nir) and NO reductases (Nor) upon horizontal acquisition of the NCE and nic clusters by a formerly aerobic host. Expression of the nic promoters PnirS, PnirJ, and PnorC, depends on the oxygen sensor DnrS and on the DnrT protein, both NCE-encoded...
December 1, 2017: Genes
Kenji Konishi, Shigeru Ueda, Miki Kawano, Susumu Osawa, Tomohiro Tamura, Eisaku Hokazono, Yuzo Kayamori, Shin-Ichi Sakasegawa
Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC homologue was identified in Thermus thermophilus HB8. The gene from T. thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45 min at 70°C. The Km values for NMN and ATP were calculated to be 0...
November 23, 2017: Journal of Bioscience and Bioengineering
Wei-Yang Ooi, Yuko Murayama, Vladimir Mekler, Leonid Minakhin, Konstantin Severinov, Shigeyuki Yokoyama, Shun-Ichi Sekine
RNA polymerase (RNAP) is a major target of gene regulation. Thermus thermophilus bacteriophage P23-45 encodes two RNAP binding proteins, gp39 and gp76, which shut off host gene transcription while allowing orderly transcription of phage genes. We previously reported the structure of the T. thermophilus RNAP•σA holoenzyme complexed with gp39. Here, we solved the structure of the RNAP•σA holoenzyme bound with both gp39 and gp76, which revealed an unprecedented inhibition mechanism by gp76. The acidic protein gp76 binds within the RNAP cleft and occupies the path of the template DNA strand at positions -11 to -4, relative to the transcription start site at +1...
November 20, 2017: Nucleic Acids Research
Shion A Lim, Susan Marqusee
The amino acid sequence encodes the energy landscape of a protein. Therefore, we expect evolutionary mutations to change features of the protein energy landscape, including the conformations adopted by a polypeptide as it folds to its native state. Ribonucleases H (RNase H) from Escherichia coli and Thermus thermophilus both fold via a partially folded intermediate in which the core region of the protein (helices A-D and strands 4-5) is structured. Strand 1, however, uniquely contributes to the T. thermophilus RNase H folding intermediate (Icore+1 ), but not the E...
November 20, 2017: Biopolymers
J Norambuena, Y Wang, T Hanson, J M Boyd, T Barkay
Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low molecular weight and protein thiols may be important cell components used in Hg resistance. To date, the role of low molecular weight thiols in Hg-detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low molecular weight thiol metabolism. This mer operon encodes for an enzyme involved in methionine biosynthesis, Oah...
November 17, 2017: Applied and Environmental Microbiology
Hai-Ying Jiang, Jing-E Ma, Juan Li, Xiu-Juan Zhang, Lin-Miao Li, Nan He, Hai-Yang Liu, Shu-Yi Luo, Zheng-Jun Wu, Ri-Chou Han, Jin-Ping Chen
The crocodile lizard is a critically endangered reptile, and serious diseases have been found in this species in recent years, especially in captive lizards. Whether these diseases are caused by changes in the gut microbiota and the effect of captivity on disease remains to be determined. Here, we examined the relationship between the gut microbiota and diet and disease by comparing the fecal microbiota of wild lizards with those of sick and healthy lizards in captivity. The gut microbiota in wild crocodile lizards was consistently dominated by Proteobacteria (∼56...
2017: Frontiers in Microbiology
Gota Kawai, Yuri Aoki, Maina Otsu, Naomi Koike, Gen-Ichi Sampei
By using the data obtained by the DNA microarray analysis for the intergenic regions applied to RNA samples extracted from Thermus thermophilus HB8, seven small non-coding RNAs, TtR-1 to TtR-7, were found to be expressed in the cells growing in rich and/or minimal media. By analysing the time course of the expression for the cell growth in combination with the sequence comparison to the known RNAs, two RNAs, TtR-1 and TtR-2, are suggested to be riboswitches. The existence of the seven RNAs and the exact sequence and length, ranging 77-284 nt, were confirmed by the next-generation sequencing...
July 4, 2017: Journal of Biochemistry
Nataliya Miropolskaya, Andrey Feklistov, Vadim Nikiforov, Andrey Kulbachinskiy
Bacterial RNA polymerase (RNAP) is an RNA-synthesizing molecular machine and a target for antibiotics. In transcription, RNAP can interact with DNA sequence-specifically, during promoter recognition by the σ-containing holoenzyme, or nonspecifically, during productive RNA elongation by the core RNAP. We describe high-affinity single-stranded DNA aptamers that are specifically recognized by the core RNAP from Thermus aquaticus. The aptamers interact with distinct epitopes inside the RNAP main channel, including the rifamycin pocket, and sense the binding of other RNAP ligands such as rifamycin and the σ(A) subunit...
October 30, 2017: Biochemical and Biophysical Research Communications
Juan Wang, Hui Chen, Ming Tang
Survival rate at low temperature becomes a crucial strategy since temperature change often leads to fluctuations in the insect population. Microbes play important roles in the process of resisting low temperature. In this study, we analyzed gut bacterial communities from Chinese white pine beetle Dendroctonus armandi which remained overwintering process under natural conditions from October 2015 to January 2016, monthly, in the Qinling Mountains, Shaanxi, China using Illumina MiSeq sequencing. A total of 835,227 high-quality sequences and 48 singleton operational taxonomic units were obtained...
October 27, 2017: Scientific Reports
Margarida M Santana, Juan M Gonzalez, Cristina Cruz
Many publications highlight the importance of nitric oxide (NO) in plant-bacteria interactions, either in the promotion of health and plant growth or in pathogenesis. However, the role of NO in the signaling between bacteria and plants and in the fate of their interaction, as well as the reconstruction of their interactive evolution, remains largely unknown. Despite the complexity of the evolution of life on Earth, we explore the hypothesis that denitrification and aerobic respiration were responsible for local NO accumulation, which triggered primordial antagonistic biotic interactions, namely the first phytopathogenic interactions...
2017: Frontiers in Microbiology
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