Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay.

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.

Critical roles of astrocytic-CCL2-dependent monocyte infiltration in a DJ-1 knockout mouse model of delayed brain repair.

Monocyte-derived macrophages play a role in the repair of the injured brain. We previously reported that a deficiency of the Parkinson's disease (PD)-associated gene DJ-1 delays repair of brain injury produced by stereotaxic injection of ATP, a component of damage-associated molecular patterns. Here, we show that a DJ-1 deficiency attenuates monocyte infiltration into the damaged brain owing to a decrease in C-C motif chemokine ligand 2 (CCL2) expression in astrocytes. Like DJ-1-knockout (KO) mice, CCL2 receptor (CCR2)-KO mice showed defects in monocyte infiltration and delayed recovery of brain injury, as determined by 9.4 T magnetic resonance imaging analysis and immunostaining for tyrosine hydroxylase and glial fibrillary acid protein. Notably, transcriptome analyses showed that genes related to regeneration and synapse formation were similarly downregulated in injured brains of DJ-1-KO and CCR2-KO mice compared with the injured wild-type brain. These results indicate that defective astrogliosis in DJ-1-KO mice is associated with decreased CCL2 expression and attenuated monocyte infiltration, resulting in delayed repair of brain injury. Thus, delayed repair of brain injury could contribute to the development of PD. MAIN POINTS: A DJ-1 deficiency attenuates infiltration of monocytes owing to a decrease in CCL2 expression in astrocytes, which in turn led to delay in repair of brain injury.

Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins.

The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR. We present the novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Previously, the biosynthesis of a retinal precursor, ß-carotene, has been introduced into many different organisms. We extended this system to the prototrophic E. coli expression strain BL21 in conjunction with the inducible expression of a ß-dioxygenase and proteo-opsin. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern.

Enhancement of radionuclide bio-decontamination by screening highly efficient microalgae for Sr biomineralization.

In this study, a new strategy for improving the radionuclide bio-decontamination (RBD) activity of microalgae by screening a better strain with high potential for biomineral production has been proposed. A noninvasive dielectrophoresis (DEP)-based microalgae screening microplatform has been used to select the highly capable microalgae in RBD. Microalgae (Chlorella vulgaris KMMCC9) with a high degree of competence in strontium (Sr) removal were successfully segregated against Chlorella vulgaris KCTC AG10002 that has relatively weak Sr removal activity under an AC electric field. C. vulgaris KMMCC9 with higher Sr biomineral competence (HSC) was also successfully segregated against others with lower Sr biomineral competence (LSC). Furthermore, after the screening and large-scale cultivation of C. vulgaris KMMCC9 with HSC, the microalgae showed highly effective Sr bio-decontamination in both non-radioactive and radioactive Sr contaminated water compared to wild-type (WT).

Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system.

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymerization, with the latter altering outer vesicle morphology. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.

Mistic-fused expression of algal rhodopsins in Escherichia coli and its photochemical properties.

BACKGROUND: Since algal rhodopsins, the eukaryotic seven-transmembrane proteins, are generally difficult to express in Escherichia coli, eukaryotic cells have been used for heterologous expression. Mistic, a membrane-associated protein that was originally discovered in Bacillus subtilis, has been shown to improve the expression levels of many foreign integral membrane proteins in E. coli when used as a fusion partner linked to the N-terminus of cargo proteins. METHODS: Here, we expressed two algal rhodopsins with N- and C-terminal Mistic domains in E. coli-Acetabularia rhodopsin I (ARI) and Chlamydomonas sensory rhodopsin B (CSRB, channel rhodopsin 2). UV/VIS spectroscopy, pH titration of proton acceptor residue, laser-induced photolysis and electrophysiological measurement were used for investigating important residues in proton transport and spectroscopic characters of the proteins. RESULTS: Protein yield of two algal rhodopsins was enhanced, obtaining 0.12mg of Mistic-ARI and 0.04mg of Mistic-CSRB per liter of culture. Spheroplast expression Mistic-ARI had outward proton-pumping activity, indicating protein functionality. Asp89 of ARI changed its protonation state by light absorption, and Asp100 was important for O(600) formation. Electrophysiology revealed that both residues took part in proton transport. The spectroscopic analyses of Mistic-CSRB revealed its characteristics. CONCLUSIONS: Fusion to the membrane-integrating protein Mistic can enhance overexpression of eukaryotic type I rhodopsins in E. coli. GENERAL SIGNIFICANCE: These findings indicate that Mistic fusion and E. coli expression method could be an effective, low cost technique for studying eukaryotic membrane proteins. This may have useful implications, for example, in studying structural characteristics and optogenetics for rhodopsins.

Photosynthetic biomineralization of radioactive Sr via microalgal CO2 absorption.

Water-soluble radiostrontium ((90)Sr) was efficiently removed as a carbonate form through microalgal photosynthetic process. The immobilization of soluble (90)Sr radionuclide and production of highly-precipitable radio-strontianite ((90)SrCO3) biomineral are achieved by using Chlorella vulgaris, and the biologically induced mineralization drastically decreased the (90)Sr radioactivity in water to make the highest (90)Sr removal ever reported. The high-resolution microscopy revealed that the short-term removal of soluble (90)Sr by C. vulgaris was attributable to the rapid and selective carbonation of (90)Sr together with the consumption of dissolved CO2 during photosynthesis. A small amount of carbonate in water could act as Sr(2+) sinks through the particular ability of the microalga to make the carbonate mineral of Sr stabilized firmly at the surface site.

ATP regeneration system using E. coli ATP synthase and Gloeobacter rhodopsin and its stability.

Great efforts in using non-photosynthetic bacteria as light-utilizing bacteria for producing biomaterials have been developed recently as increasing interest in renewable resources such as light energy. With respect to producing bio-materials industrially such as food ingredients and amino acids, huge amount of adenosine-5'-triphosphate (ATP) is required. In this work, we developed a bio-ATP-synthesis system using ATP synthase of Escherichia coil as a biocatalyst and a microbial rhodopsin which is from primitive cyanobacteria, Gloeobacter violaceus. Gloeobacter rhodopsin (GR) is a light-driven proton pump. Besides electro-chemical gradient produced by cellular respiration system, GR produces a proton gradient using light illumination which is used in additional driving force of synthesizing ATP by ATP synthase. Inverted membrane vesicle was prepared so that it could be incorporated with both of GR and ATP synthase and produced ATP in the exterior side of the vesicle in the presence of light. Since inverted membrane vesicle does not contain precursors for ATP, we added ADP and inorganic phosphate (P(i)). Then, we measured the amounts of ATP produced by ATP synthase in the presence of light. As the average value of 6 samples, 4.79 x 10(-2) micromole of ATP produced for 1 microg of GR per minute. Also, we measured again after 7 days and 65 days, respectively, in order to check the stability of the bio-ATP-synthesis system. Amount of ATP produced decayed double-exponentially and an expected value of half-life of the system was 1.5 days and 39.7 days. Our results demonstrate that ATP was regenerated successfully by using GR and ATP synthase. However, the stability of ATP synthase should be increased to use this system industrially in the near future.