scholarly journals A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Masumi Hasegawa ◽  
Toshiaki Hosaka ◽  
Keiichi Kojima ◽  
Yosuke Nishimura ◽  
Yu Nakajima ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
Genomic Analysis ◽  
Proton Pumping ◽  
Spectroscopic Characteristics ◽  
Light Utilization ◽  
Photochemical Properties ◽  
Microbial Rhodopsin ◽  
Unique Clade ◽  
Biased Distribution

Abstract Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H+ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from Calothrix sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 420
Author(s):  
Yi Ma ◽  
Liu Cui ◽  
Meng Wang ◽  
Qiuli Sun ◽  
Kaisheng Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
High Yield ◽  
Wild Type ◽  
High Quality ◽  
Efficient Protection ◽  
Bacterial Ghosts ◽  
Cell Envelopes ◽  
Extracellular Structures ◽  
First Time

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

scholarly journals Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria

1996 ◽  
Vol 318 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Susan K BUCHANAN ◽  
John E. WALKER
Keyword(s):  
Complex I ◽  
Large Scale ◽  
Gel Filtration ◽  
Atp Synthesis ◽  
Lipid Vesicles ◽  
Proton Pumping ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Bovine Heart ◽  
F1fo Atpase

A new chromatographic procedure has been developed for the isolation of F1Fo-ATPase and NADH:ubiquinone oxidoreductase (complex I) from a single batch of bovine heart mitochondria. The method employed dodecyl β-Δ-maltoside, a monodisperse, homogeneous detergent in which many respiratory complexes exhibit high activity, for solubilization and subsequent purification by ammonium sulphate fractionation and column chromatography. A combination of anion-exchange, gel-filtration, and dye-ligand affinity chromatography was used to purify both complexes to homogeneity. The F1Fo-ATPase preparation contains only the 16 known subunits of the enzyme. It has oligomycin-sensitive ATP hydrolysis activity and, as demonstrated elsewhere, when reconstituted into lipid vesicles it is capable of ATP-dependent proton pumping and of ATP synthesis driven by a proton gradient [Groth and Walker (1996) Biochem. J. 318, 351–357]. The complex I preparation contains all of the subunits identified in other preparations of the enzyme, and has rotenone-sensitive NADH:ubiquinone oxidoreductase and NADH:ferricyanide oxidoreductase activities. The procedure is rapid and reproducible, yielding 50–80 mg of purified F1Fo-ATPase and 20–40 mg of purified complex I from 1 g of mitochondrial membranes. Both preparations are devoid of phospholipids, and gel filtration and dynamic light scattering experiments indicate that they are monodisperse. Therefore, the preparations fulfil important prerequisites for structural analysis.

scholarly journals BatchGenAna: a batch platform for large-scale genomic analysis of mammalian small RNAs

2009 ◽  
Vol 3 (8) ◽  
pp. 336-348
Author(s):  
Xiaomin Ying ◽  
You Jung Kim ◽  
Yiqing Mao ◽  
Ming Liu ◽  
Yanyan Hou ◽  
...  
Keyword(s):  
Small Rnas ◽  
Large Scale ◽  
Genomic Analysis

The preparation and photochemical properties of some 1,3-Diphenyl-2-pyrazolines containing a heteroaromatic substituent

1977 ◽  
Vol 30 (3) ◽  
pp. 629 ◽  
Author(s):  
J Lin ◽  
DE Rivett ◽  
JFK Wilshire
Keyword(s):  
Photochemical Reaction ◽  
Large Scale ◽  
Polar Solvent ◽  
Methanolic Solution ◽  
Pyrazoline Ring ◽  
Photochemical Properties

Several 1,3-diphenyl- and 1,3,5-triphenyl-2-pyrazolines containing a heteroaromatic substituent (viz. imidazol-1-yl, pyrazol-1-yl and 1,2,4- triazol-1-yl) have been synthesized. When compared with their unsubstituted analogues, these compounds (a) absorbed more intensely at a longer wavelength, (b) exhibited greater fluorescence in a polar solvent (methanol) and (c) possessed similar stability to light. ��� Large-scale irradiations of two 1,3,5-triphenyl-2-pyrazolines (viz. unsubstituted and that containing the 1,2,4-triazol-1-yl substituent) in methanolic solution revealed that, although dehydrogenation of the 2-pyrazoline ring was the major photochemical reaction, significant oxidation to the corresponding chalcone also occurred. In addition, phenol was detected among the irradiation products. ��� Several sulphonated analogues of the abovementioned pyrazolines were prepared. When applied to wool, these compounds exhibited excellent fluorescent whitening properties.

scholarly journals Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1295
Author(s):  
Archana P. Thankamony ◽  
Reshma Murali ◽  
Nitheesh Karthikeyan ◽  
Binitha Anu Varghese ◽  
Wee S. Teo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Triple Negative Breast Cancer ◽  
Large Scale ◽  
Triple Negative ◽  
Genomic Analysis ◽  
Aurora Kinase ◽  
Cell Cycle Regulators ◽  
Tumor Models ◽  
Tnbc Subtype

The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1–Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1–Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

scholarly journals Comparative genomic analysis of Methanimicrococcus blatticola provides insights into host adaptation in archaea and the evolution of methanogenesis

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Courtney M. Thomas ◽  
Najwa Taib ◽  
Simonetta Gribaldo ◽  
Guillaume Borrel
Keyword(s):  
Gut Microbiota ◽  
Large Scale ◽  
Genomic Analysis ◽  
Host Adaptation ◽  
Comparative Genomic Analysis ◽  
Comparative Genomic ◽  
Methyl Branch ◽  
Mechanisms Of Adaptation ◽  
Hydrogenotrophic Methanogens ◽  
Almost All

AbstractOther than the Methanobacteriales and Methanomassiliicoccales, the characteristics of archaea that inhabit the animal microbiome are largely unknown. Methanimicrococcus blatticola, a member of the Methanosarcinales, currently reunites two unique features within this order: it is a colonizer of the animal digestive tract and can only reduce methyl compounds with H2 for methanogenesis, a increasingly recognized metabolism in the archaea and whose origin remains debated. To understand the origin of these characteristics, we have carried out a large-scale comparative genomic analysis. We infer the loss of more than a thousand genes in M. blatticola, by far the largest genome reduction across all Methanosarcinales. These include numerous elements for sensing the environment and adapting to more stable gut conditions, as well as a significant remodeling of the cell surface components likely involved in host and gut microbiota interactions. Several of these modifications parallel those previously observed in phylogenetically distant archaea and bacteria from the animal microbiome, suggesting large-scale convergent mechanisms of adaptation to the gut. Strikingly, M. blatticola has lost almost all genes coding for the H4MPT methyl branch of the Wood–Ljungdahl pathway (to the exception of mer), a phenomenon never reported before in any member of Class I or Class II methanogens. The loss of this pathway illustrates one of the evolutionary processes that may have led to the emergence of methyl-reducing hydrogenotrophic methanogens, possibly linked to the colonization of organic-rich environments (including the animal gut) where both methyl compounds and hydrogen are abundant.

scholarly journals Precision nomenclature for the new genomics

GigaScience ◽  
2019 ◽  
Vol 8 (8) ◽  
Author(s):  
Harris A Lewin ◽  
Jennifer A Marshall Graves ◽  
Oliver A Ryder ◽  
Alexander S Graphodatsky ◽  
Stephen J O'Brien
Keyword(s):  
Large Scale ◽  
Genomic Analysis ◽  
End Users ◽  
Dna Fragments ◽  
Current State ◽  
Scientific Disciplines ◽  
Genome Assemblies

Abstract The confluence of two scientific disciplines may lead to nomenclature conflicts that require new terms while respecting historical definitions. This is the situation with the current state of cytology and genomics, which offer examples of distinct nomenclature and vocabularies that require reconciliation. In this article, we propose the new terms C-scaffold (for chromosome-scale assemblies of sequenced DNA fragments, commonly named scaffolds) and scaffotype (the resulting collection of C-scaffolds that represent an organism's genome). This nomenclature avoids conflict with the historical definitions of the terms chromosome (a microscopic body made of DNA and protein) and karyotype (the collection of images of all chromosomes of an organism or species). As large-scale sequencing projects progress, adoption of this nomenclature will assist end users to properly classify genome assemblies, thus facilitating genomic analysis.

scholarly journals Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5538
Author(s):  
Zhongxuan Li ◽  
Qiang Cheng ◽  
Henan Guo ◽  
Rijun Zhang ◽  
Dayong Si
Keyword(s):  
Pichia Pastoris ◽  
High Performance ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
E Coli ◽  
Cell Membrane Permeabilization ◽  
Bactericidal Activities ◽  
The Individual ◽  
Industrial Level

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

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