scholarly journals Functional metagenomics of the Thioredoxin superfamily

2020 ◽  
pp. jbc.RA120.016350
Author(s):  
Sebastian Nilewski ◽  
Marharyta Varatnitskaya ◽  
Thorsten Masuch ◽  
Anna Kusnezowa ◽  
Manuela Gellert ◽  
...  
Keyword(s):  
Redox Potential ◽  
Sequence Data ◽  
Global Ocean ◽  
Phylogenetic Relation ◽  
Functional Metagenomics ◽  
Expression Library ◽  
Intramolecular Disulfide Bond ◽  
Ocean Sampling ◽  
Metagenomic Approach

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging, because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22’000 thioredoxins found in the Global Ocean Sampling dataset. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (∆trxA), isomerase (∆dsbC), or oxidase (∆dsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins, but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

scholarly journals Reconsideration of the ‘well-known’ hypotrichous ciliate Pleurotricha curdsi (Shi et al., 2002) Gupta et al., 2003 (Ciliophora, Sporadotrichida), with notes on its morphology, morphogenesis and molecular phylogeny

2015 ◽  
Vol 65 (Pt_9) ◽  
pp. 3216-3225 ◽  
Author(s):  
Xiaoteng Lu ◽  
Chen Shao ◽  
Yuhe Yu ◽  
Alan Warren ◽  
Jie Huang
Keyword(s):  
Sequence Data ◽  
Southern China ◽  
Phylogenetic Analyses ◽  
Large Body ◽  
Small Subunit ◽  
Variable Number ◽  
Ssu Rdna ◽  
Intermediate Form ◽  
Rdna Sequence Data

The oxytrichid species Pleurotricha curdsi (Shi et al., 2002) Gupta et al., 2003, isolated from a tributary of the Yangtze River in the Mudong district of Chongqing, southern China, was reinvestigated with emphasis on its morphology, morphogenesis and small-subunit (SSU) rDNA-based phylogeny. Compared with three previously described populations, the Mudong population of P. curdsi is characterized by its large body size, 170–295 × 65–110 μm in vivo, and by having a variable number of right marginal rows, either two or three. Likewise, the number of right marginal rows anlagen (RMA) is also variable, i.e. usually two, but sometimes several small extra anlagen that give rise to the formation of the third row, are present to the left of the RMAs. We posit that the Mudong population is an intermediate form between the three previously described populations. Phylogenetic analyses based on the SSU rDNA sequence data show that all populations of P. curdsi cluster with the type species of the genus, Pleurotricha lanceolata, in a clade nested within the Oxytrichidae.

scholarly journals Fluorescent probes for in vivo redox potential measurement and thiol rich protein function

2006 ◽  
Vol 20 (4) ◽  
Author(s):  
Phani Kumar Pullela ◽  
Taurai Chiku ◽  
Michael J Carvan ◽  
Daniel S Sem
Keyword(s):  
Redox Potential ◽  
Fluorescent Probes ◽  
Protein Function ◽  
Potential Measurement

scholarly journals Different processes shape prokaryotic and picoeukaryotic assemblages in the sunlit ocean microbiome

2018 ◽  
Author(s):  
Ramiro Logares ◽  
Ina M. Deutschmann ◽  
Caterina. R. Giner ◽  
Anders K. Krabberød ◽  
Thomas S. B. Schmidt ◽  
...  
Keyword(s):  
Ecosystem Function ◽  
Sequence Data ◽  
Scale Effects ◽  
Dispersal Limitation ◽  
Global Scale ◽  
Global Ocean ◽  
Ecosystem Functions ◽  
Ecological Processes ◽  
Surface Ocean

ABSTRACTThe smallest members of the sunlit-ocean microbiome (prokaryotes and picoeukaryotes) participate in a plethora of ecosystem functions with planetary-scale effects. Understanding the processes determining the spatial turnover of this assemblage can help us better comprehend the links between microbiome species composition and ecosystem function. Ecological theory predicts thatselection,dispersalanddriftare main drivers of species distributions, yet, the relative quantitative importance of these ecological processes in structuring the surface-ocean microbiome is barely known. Here we quantified the role of selection, dispersal and drift in structuring surface-ocean prokaryotic and picoeukaryotic assemblages by using community DNA-sequence data collected during the global Malaspina expedition. We found that dispersal limitation was the dominant process structuring picoeukaryotic communities, while a balanced combination of dispersal limitation, selection and drift shaped prokaryotic counterparts. Subsequently, we determined the agents exerting abiotic selection as well as the spatial patterns emerging from the action of different ecological processes. We found that selection exerted via temperature had a strong influence on the structure of prokaryotic communities, particularly on species co-occurrences, a pattern not observed among communities of picoeukaryotes. Other measured abiotic variables had limited selective effects on microbiome structure. Picoeukaryotes presented a higher differentiation between neighbouring communities and a higher distance-decay when compared to prokaryotes, agreeing with their higher dispersal limitation. Finally, drift seemed to have a limited role in structuring the sunlit-ocean microbiome. The different predominance of ecological processes acting on particular subsets of the ocean microbiome suggests uneven responses to environmental change.SIGNIFICANCE STATEMENTThe global ocean contains one of the largest microbiomes on Earth and changes on its structure can impact the functioning of the biosphere. Yet, we are far from understanding the mechanisms that structure the global ocean microbiome, that is, the relative importance of environmentalselection,dispersaland random events (drift). We evaluated the role of these processes at the global scale, based on data derived from a circumglobal expedition and found that these ecological processes act differently on prokaryotes and picoeukaryotes, two of the main components of the ocean microbiome. Our work represents a significant contribution to understand the assembly of marine microbial communities, providing also insights on the links between ecological mechanisms, microbiome structure and ecosystem function.

scholarly journals Metagenomic Approach: Transforming In-Silico Research for Improved Biogas Production

2019 ◽  
Vol 7 (1) ◽  
pp. 6-11 ◽  
Author(s):  
Roushney Fatima Mukti ◽  
Sanjida Sakhawat Sinthee
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Sequence Data ◽  
Biogas Production ◽  
Biogas Plant ◽  
Rational Approach ◽  
Production Scale ◽  
Specific Element ◽  
Metagenomic Approach ◽  
Metagenome Sequence

The complexity of the microbial communities and metabolic pathways involved in the microbiological process of biogas production is poorly understood and numerous microorganisms in the fermentation sample of the biogas plant are still unclassified or unknown. The structure and function of microbial communities and the effects of the addition of trace elements are needed to be known, to control and channel the energy sources microbes produce and to capture and store the useful by-products or for targeted screening of novel enzymes. In this review, we discussed an emerging idea that Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production. The composition and gene content of a biogas-producing consortium can be determined through metagenomic approach which allows the design of the optimal microbial community structure for any biogas plant for the significant progress in the efficacy and economic improvement of biogas production and biofertilizer of either balanced nutrition or rich in specific element for plant growth produced from the sludge of biogas plant. Biogas-producing microbial community from different production-scale biogas plants supplied with different raw materials as substrates can be analyzed by polyphasic approach to find out the best raw material composition for biogas production. The phylogenetic structure of the microbial community residing in a fermentation sample from a biogas plant can be analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Int. J. Appl. Sci. Biotechnol. Vol 7(1): 6-11

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
Author(s):  
Peter L. Wang
Keyword(s):  
Directed Evolution ◽  
High Efficiency ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Strand Breaks ◽  
E Coli ◽  
Hybrid Genes ◽  
Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

Analysis of 23S rRNA genes in metagenomes – A case study from the Global Ocean Sampling Expedition

2011 ◽  
Vol 34 (6) ◽  
pp. 462-469 ◽  
Author(s):  
Pelin Yilmaz ◽  
Renzo Kottmann ◽  
Elmar Pruesse ◽  
Christian Quast ◽  
Frank Oliver Glöckner
Keyword(s):  
Global Ocean ◽  
Rrna Genes ◽  
23S Rrna ◽  
Global Ocean Sampling ◽  
Ocean Sampling ◽  
23S Rrna Genes

A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp)

2000 ◽  
Vol 351 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Wei KONG ◽  
Susumu SHIOTA ◽  
Yixin SHI ◽  
Hiroaki NAKAYAMA ◽  
Koji NAKAYAMA
Keyword(s):  
Escherichia Coli ◽  
Peroxidase Activity ◽  
Discrete Group ◽  
Sequence Data ◽  
Accession Number ◽  
Gene Encoding ◽  
Reaction Cycle ◽  
Mutant Proteins ◽  
E Coli

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo. The sequence data presented have been deposited in the GenBank/EMBL/DDBJ nucleotide sequence databases under the accession number AB037598.

scholarly journals Identification of Group A Streptococcus Antigenic Determinants Upregulated In Vivo

2005 ◽  
Vol 73 (9) ◽  
pp. 6026-6038 ◽  
Author(s):  
Kowthar Y. Salim ◽  
Dennis G. Cvitkovitch ◽  
Peter Chang ◽  
Darrin J. Bast ◽  
Martin Handfield ◽  
...  
Keyword(s):  
Murine Model ◽  
Vibrio Vulnificus ◽  
Group A Streptococcus ◽  
Hypothetical Protein ◽  
Invasive Disease ◽  
Antigenic Determinants ◽  
Expression Library ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) causes a range of diseases in humans, from mild noninvasive infections to severe invasive infections. The molecular basis for the varying severity of disease remains unclear. We identified genes expressed during invasive disease using in vivo-induced antigen technology (IVIAT), applied for the first time in a gram-positive organism. Convalescent-phase sera from patients with invasive disease were pooled, adsorbed against antigens derived from in vitro-grown GAS, and used to screen a GAS genomic expression library. A murine model of invasive GAS disease was included as an additional source of sera for screening. Sequencing DNA inserts from clones reactive with both human and mouse sera indicated 16 open reading frames with homology to genes involved in metabolic activity to genes of unknown function. Of these, seven genes were assessed for their differential expression by quantitative real-time PCR both in vivo, utilizing a murine model of invasive GAS disease, and in vitro at different time points of growth. Three gene products—a putative penicillin-binding protein 1A, a putative lipoprotein, and a conserved hypothetical protein homologous to a putative translation initiation inhibitor in Vibrio vulnificus—were upregulated in vivo, suggesting that these genes play a role during invasive disease.

scholarly journals The principal site of glycation of human complement factor B

1991 ◽  
Vol 274 (2) ◽  
pp. 473-480 ◽  
Author(s):  
M A Niemann ◽  
A S Bhown ◽  
E J Miller
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Data ◽  
Complement Factor ◽  
Human Complement ◽  
Factor B ◽  
Complement Factor B ◽  
Functional Regions ◽  
High Concentrations

Accumulating amino acid sequence data have made it increasingly evident that many essential complement proteins have potentially modifiable lysine residues in putative critical functional regions. Evidence is now presented that glucose is covalently attached to lysine-266 of purified human complement Factor B as a result of glycation. Purified B was treated with NaB3H4, which reduces such bound glucose to a mixture of radiolabelled hexitols. Amino acid analysis revealed the expected radiolabelled hexitol-lysine epimers. In addition, fluorography of dried gels resolving the major high-molecular-mass h.p.l.c.-fractionated CNBr-cleavage peptides of NaB3H4-reduced B indicated that this radioactivity was specifically associated with the 15 kDa fragment derived from the N-terminal region of fragment Bb. Amino acid sequence analysis suggested that the C-terminal lysine (residue 266 of B) of the N-terminal Lys-Lys doublet of this peptide is preferentially modified. If such glycation can subsequently be shown to occur in vivo, then perhaps this modification might also be found to affect the functional activity of B and offer a potential explanation for some of the immunopathological complications of diseases exposing key plasma proteins, such as this active-site-containing proteinase of the multimeric alternative-complement-pathway C3/C5 convertases, to long-term high concentrations of glucose, such as the decreased resistance to infection and impaired chemotaxis and phagocytosis characteristic of diabetes.

Excitation pressure regulates the activation energy for recombination events in the photosystem II reaction centres of Chlamydomonas reinhardtii

2007 ◽  
Vol 85 (6) ◽  
pp. 721-729 ◽  
Author(s):  
Tessa Pocock ◽  
P. V. Sane ◽  
S. Falk ◽  
N. P.A. Hüner
Keyword(s):  
Activation Energy ◽  
Photosystem Ii ◽  
Redox Potential ◽  
Chlamydomonas Reinhardtii ◽  
Reaction Center ◽  
Growth Conditions ◽  
High Irradiance ◽  
Reaction Centres ◽  
Excitation Pressure

Using in vivo thermoluminescence, we examined the effects of growth irradiance and growth temperature on charge recombination events in photosystem II reaction centres of the model green alga Chlamydomonas reinhardtii. We report that growth at increasing irradiance at either 29 or 15 °C resulted in comparable downward shifts in the temperature peak maxima (TM) for S2QB– charge pair recombination events, with minimal changes in S2QA– recombination events. This indicates that such growth conditions decrease the activation energy required for S2QB– charge pair recombination events with no concomitant change in the activation energy for S2QA– recombination events. This resulted in a decrease in the ΔTM between S2QA– and S2QB– recombination events, which was reversible when shifting cells from low to high irradiance and back to low irradiance at 29 °C. We interpret these results to indicate that the redox potential of QB was modulated independently of QA, which consequently narrowed the redox potential gap between QA and QB in photosystem II reaction centres. Since a decrease in the ΔTM between S2QA– and S2QB– recombination events correlated with growth at increasing excitation pressure, we conclude that acclimation to growth under high excitation pressure narrows the redox potential gap between QA and QB in photosystem II reaction centres, enhancing the probability for reaction center quenching in C. reinhardtii. We discuss the molecular basis for the modulation of the redox state of QB, and suggest that the potential for reaction center quenching complements antenna quenching via the xanthophyll cycle in the photoprotection of C. reinhardtii from excess light.

Sign in / Sign up

Export Citation Format

Share Document