Hiroaki Tomioka

Action spectrum of the photoattractant response of Halobacterium halobium in early logarithmic growth phase and the role of sensory rhodopsin

Abstract Photoattractant response was measured in a relatively carotenoid-poor strain derived from the mutant of Halobacterium halobium that lacks both bacteriorhodopsin and halorhodopsin (strain Flx 3 ). No photoattractant response was observed in the cells at logarithmic growth stage, coinciding with the fact that there was no sensory rhodopsin in membrane fraction prepared from the cells in logarithmic growth stage as measured by flash photholysis experiment. When all- trans -retinal was added to the cell suspension or the membrane suspension, the phototactic activity or the photocycling due to sensory rhodopsin appeared rapidly. This indicates that apoprotein of sensory rhodopsin had been formed in the cells at the growth stage, and suggests that the photoattractant response was mediated by sensory rhodopsin. The action spectrum of the photoattractant response resembled sensory rhodopsin absorption at wavelengths than 600 nm, but was distorted at shorter wavelengths by the photorepellent system that was found recently.

Properties and the primary structure of a new halorhodopsin from halobacterial strain mex

A new halorhodopsin-like pigment from the new halobacterial strain mex (Otomo, J., Tomoika, H. and Sasabe, H. (1992) J. Gen. Microbiol. 138, 1027-1037) was partially purified, and its amino acid sequence from helices A to G was determined using PCR technique. Two arginine residues in the A-B interhelix loop segment, a series of six amino acid residues (EMPAGH) in the B-C interhelix segment and most of the residues near the Schiff base of the retinal were found to be conserved in three halorhodopsins (halobium, pharaonis and mex). This result strongly suggests that these residues are essential for anion pumping function in halorhodopsin. The light-induced ion-pump measurements have shown that the selectivity of anion transport between chloride and nitrate in mex halorhodopsin is lower than that of halobium halorhodopsin, but higher than that of pharaonis halorhodopsin. The number of amino acid residues in the B-C interhelix loop segments is different in each halorhodopsin, and it correlates with their anion (chloride and nitrate) selectivity. These results suggest that the length of the B-C segment affects the selectivity of anion transport in halorhodopsin.

Comparative study of primary photochemical events of two retinal proteins, bacteriorhodopsin and halorhodopsin, by use of subpicosecond time-resolved spectroscopy

Abstract The primary photochemical events of bacteriorhodopsin (bR) were compared with those of halorhodopsin (hR) by subpicosecond time-resolved spectroscopy. The absorption and stimulated emission spectra display similar features. It is proposed that the isomerization reaction and relaxation to the fluorescent state occur simultaneously from the Franck—Condon excited state. The quantum yield of cis—trans isomerization in bR is about twice that in hR.

Isolation of photochemically active archaebacterial photoreceptor, pharaonis phoborhodopsin from Natronobacterium pharaonis

A photoreceptor, pharaonis phoborhodopsin for the negative phototaxis of extremely halophilic and alkalophilic archaebacterium, Natronobacterium pharaonis was isolated in a photochemically active state. A detailed examination of the chromatographic separation made it possible to separate contaminating proteins, such as cytochromes. The procedure resulted in a 2938-fold enrichment with a yield of 15.5%. The isolated pharaonis phoborhodopsin had an absorption maximum at 498 nm, an A280/A498 ratio of 1.27 and a single band near 24 kDa on the SDS-polyacrylamide gels. The isolated pharaonis phoborhodopsin underwent a photochemical reaction after flash excitation. The photocyclic reaction closely resembled that of the membrane-bound pharaonis phoborhodopsin.

Photochemical intermediate of third rhodopsin-like pigment in Halobacterium halobium by simultaneous illumination with red and blue light.

We found new photochemical intermediate of third rhodopsin-like pigment (tR) or slow cycling rhodopsin-like pigment (sR) in Halobacterium halobium, which was produced by simultaneous illumination with red and blue light. This illumination is employed for measurements of negative phototaxis. The formation of this intermediate is fast. (With the instrument used, it could not be measured.) The half-time of its decay is ca 150 msec in 4 M NaCl, pH 7.0 at 20 degrees C. The maximum of absorbance is located at 510-530 nm.

A new anion-sensitive biosensor using an ion-sensitive field effect transistor and a light-driven chloride pump, halorhodopsin

A new biosensor sensitive to chloride anion using a light-driven chloride pump protein, halorhodopsin (hR), and an ion-sensitive field effect transistor (ISFET) has been developed. Membrane vesicles of halophilic bacteria containing hR were immobilized in the matrix of polyvinylbutyral resin on the surface of the ISFET. The gate voltage of this device changed in the min time scale under yellow light illumination. The response for chloride anion increased according to the increase of chloride anion concentration in the bulk aqueous phase. In the dark, the gate potential did not change even in the presence of chloride anion. These chloride-dependent gate potential changes of the hR-ISFET indicate that the chloride pumping by hR is active on the ISFET and that ISFET detects the light-dependent chloride transport by hR.

Phototaxis and the Second Sensory Pigment in Halobacterium halobium

Halobacterium halobium is an archaebacterium well known for its unique light energy-transducing apparatus, bacteriorhodopsin. Usually the bacterium is energized by respiration. When the cells grow so dense that the supply of oxygen becomes insufficient, the cells produce a large amount of bacteriorhodopsin and utilize the energy of visible light. Therefore, bacteriorhodopsin appears to be an emergency apparatus [1]. The cell growing in such a dense culture is attracted to visible light. The cells accumulate at a spot of green-yellow light. On the other hand, UV and violet light are avoided by the cells [2]. The behavioral response of the cells to light is similar to that of enteric bacteria to chemical substances [3], namely, the frequency of the change in swimming direction is increased or decreased when a repellent stimulus or an attractant one is introduced to the cell, respectively. The difference lies in the way the change of swimming direction is brought about. Halobacterium halobium has flagella in both poles of the cell and swims indifferently in either direction. Therefore, the bacterium simply reverses the swimming direction by reversing the direction of flagellar rotation when repellent light is applied (step-up Photophobic response) or attractant light is taken away (step-down Photophobic response). The photoattractant and the photorepellent response are controlled by different photosystems. The photorepellent system that shows maximal response of the cells to 370nm light was named photosystem 370 (PS370) [2].

Halide Anion Sensor

We studied biosensors that consisted of an electrochemical device and functional biomolecules. We recognized that membrane proteins, which play a role in energy conversion, environmental sensing, and information transmission, could be candidates for bio-recognition elements in the biosensor. In this study, halorhodopsin (hR), a light-driven chloride anion pump in the cytoplasmic membrane of Halobacterium halobium [1], was chosen as a halide anion recognition element, and it was coupled with an ion-sensitive field effect transistor (ISFET) so as to construct an anion-sensing system. In this system, hR-containing vesicles were immobilized on the surface of the ISFET. Under yellow light illumination, this hR-ISFET sensor showed a halide anion-dependent response.

Photoreceptors of halophilic archaebacterium phototaxis. Phoborhodopsin and sensory rhodopsin.

Extremely halophilic archaebacterium Halobacterium halobium cells show phototaxis to visible hight. The phototaxis requires vitamine A aldehyde, a retinal. Protein pigments containing retinal are the photoreceptors of the phototaxis. Phoborhodopsin (pR) is a photoreceptor of the negative phototaxis. Sensory rhodopsin (sR) is a photoreceptor of the positive phototaxis and S373, a transient photoproduct of sR, is another photoreceptor of the negative phototaxis. These photoreceptors are too unstable to isolate. Haloalkalophilic archaebacterium Natronobacterium pharaonis cells have pharaonis phoborhodopsin (ppR), which is also a photoreceptor of a negative phototaxis. PPR is solubilized and isolated in an active state, A methylation/demethylation system is involved in the signalling chain from photoreceptor to flagellar motor switch.

Flash Photolysis Study on Sensory Rhodopsin and Phoborhodopsin

At least four retinylidene-proteins have been discovered so far in the membrane of Halobacterium halobium. They are bacteriorhodopsin (BR), halorhodopsin (HR), sensory rhodopsin (SR) and phoborhodopsin (PR). The first to be discovered was bacteriorhodopsin (BR), which functions as a light-driven H -pump (for a review see [1]). On illumination, BR extrudes H from the inside to the outside and creates the so-called proton motive force, which is a driving force for ATP synthesis, transport of amino acids and other energy-requiring processes.