HALORHODOPSIN and PHOTOSENSORY BEHAVIOR IN Halobacterium halobium MUTANT STRAIN L-33

Halobacterium halobium , strain L-33, which is deficient in bacteriorhodopsin (BR) but synthesizes increased amounts of halorhodopsin (HR), shows behavioral responses upon changes in fluence rate with visible light or with UV light. The observations support the earlier report (Schimz et al. , 1982). that BR is not essential for photosensing in H. halobium. In the UV-range, changes in light intensity elicit the maximal response at λ= 370 nm. In the visible range, changes in light intensity show the maximal response at Δ= 565 nm and a secondary peak at Δ= 590 nm. The latter corresponds to the absorption maximum of HR (Δ_max= 588 nm). This light-energy converting retinal pigment of H. halobium thus appears to contribute to photosensory behavior.     

A Microcalorimetric Method for Studying Halobacterium halobium

The application of a microcalorimetric method to the study of extremophiles is described briefly. Using the LKB 2277 Bioactivity Monitor, the growth thermogenic curves of three strains of Halobacterium halobium were determined at 37°C, and compared with the spectrophotometric curves. Then the suitable growth thermokinetic equation was established based on the characteristics of growth thermogenic curves. By using cycle-flow method, all of the growth thermogenic curves of H. halobium strains displayed a brief lag phase before the onset of exponential growth when they were cultured in Halo-2 medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

ULTRAVIOLET QUANTUM YIELDS OF PHOTOPHOSPHORYLATION IN Halobacterium halobium*

Quantum yields of photophosphorylation in Halobacterium halobium were determined for ultraviolet spectral bands between 276 and 365 nm, and at 565 nm wavelength, based on integral spectral cell absorptance, bacteriorhodopsin-specific cell absorptance and the corresponding quantum dose rates. In the ultraviolet, there is an almost linear decline of the quantum yields of photophosphorylation from 365 to 276 nm wavelength, despite the peak absorption of bacteriorhodopsin at 280 nm. The cycling quantum yield for 276 nm excitation of bacteriorhodopsin was determined as 4.5 ± 1.8%, which is about one fourth of the value of 19% for solubilized bacteriorhodopsin. Threshold energy fluence rates of 20 W m^?2 for UV-B radiation typify the photophosphorylation as three orders less sensitive than the sensory UV-B avoidance response that needs 0.02 W m^?2 as the threshold. Thus, UV-B avoidance appears as the dominating strategy for survival of the archaic bacterium H. halobium, rather than possible photoenergetic use of UV-B radiation and photorepair of UV-damage.     

CONSECUTIVE FORMATION OF SENSORY PHOTOSYSTEMS IN GROWING Halobacterium halobium

Abstract— The sensory photosystems PS 370 and PS 565 of Halobacierium halobium are actively degraded in the early growth phase and later resynthesized. Neither degradation nor resynthesis is correlated to the rate of cell division. The reappearance of photosensory activity requires de novo synthesis of proteins which are most likely directly involved in the sensory pathway. PS 370 appears earlier than PS 565 and thus may be studied independently of PS 565, before the latter is synthesized, or by blocking the synthesis of PS 565 with puromycin after PS 370 has appeared. The action spectrum of PS 370 alone shows the same maximum as the spectrum obtained when PS 565 is present. Carotenoids, which act as accessory pigments of PS 370, do not shift its activity peak. Also the maximum of PS 565 is not influenced by PS 370. We conclude that the maxima of the action spectra of PS 370 and PS 56.5 truely reflect the absorption maxima of the sensory retinal pigmentsP–370 andP–565.     

PHOTOREACTIVATION OF Halobacterium halobium: ACTION SPECTRUM AND ROLE OF PIGMENTATION

Abstract— An action spectrum for photoreactivation was measured with Halobacterium halobium R₁m₁ to prove a role of carotenoid pigments in photoreactivation of the bacteria. The action spectrum obtained showed a main peak at 435 nm and a minor peak at about 325 nm. The action spectrum was similar to that of Streptomyces pigment (Eker et al. , 1981) suggesting that the chromophore of the photoreactivating enzyme in Halobacterium halobium is 8-OH-5-deazaflavin. The minor peak may be due to photochemical cleavage of a pyrimidine6–4 hetero adduct. The result indicates that carotenoid pigments do not play a positive role in enhancing photoreactivation. This was confirmed also by comparing the efficiency of photoreactivation at 465 nm among three strains of Halobacterium halobium having different carotenoid pigments; R₁m₁. R₁ and W5002–1.     

PHOTOCHROMIC SYNERGISM OF BACTERIORHODOPSIN- and HALORHODOPSIN-MEDIATED PHOTOPHOSPHORYLATION IN Halobacterium halobium*

Quantitative action spectroscopy was performed in Halobacterium halobium. using four suited pigment mutants, namely the bacteriorhodopsin and halorhodopsin positive mutant strain M-l (BR⁺, HR⁺), the bacteriorhodopsin positive but halorhodopsin negative strain M-18 (BR⁺, HR^-), the bacteriorhodopsin negative but halorhodopsin positive strain L-33 (BR^-, HR⁺), and the bacteriorhodopsin and halorhodopsin negative strain L-07 (BR^-, HR⁺). The approached questions were: First, photoenergetic synergism of halorhodopsin and bacteriorhodopsin in intact cells; second, photochromism and cellular function of the blue light-absorbing intermediates, i.e. M-412 and HR-410 in bacteriorhodopsin and in halorhodopsin, respectively. Dark-adapted cells of mutant strain M-l show wavelength-dependency of quantum yield of photo-phosphorylation, φ_ATP. An 1.4-fold enhancement was found at 575 nm wavelength where the long wavelength absorbance bands of bacteriorhodopsin and halorhodopsin intersect. The enhancement vanished after a 30 min pulse of orange light (600 Wm^-2 bandpass from 495 to 750 nm), but was restored after a 30 min pulse of blue light (100 Wm^-2 bandpass from 325 to 480 nm). Photoreversibility of this enhancement probably reflects phototransformation of halorhodopsin from its ground state into its inactive intermediate, HR-410, and vice versa. The halorhodopsin-mediated enhancement with maximum quantum yield of photophosphorylation, φ_ATP= 0.06, i.e. a quantum requirement of = 17 photons/ATP, is partly substituted by a rise in phosphate potential and explained in terms of a voltage-regulated gating effect on the H⁺-driven ATP-synthase, superimposed on the chemiosmotic mechanism of energy coupling. The blue-absorbing photochromic intermediate, M-412 of bacteriorhodopsin, dissipates light energy upon photoexcitation that is reflected by a spectral decline in quantum yield of photophosphorylation to a minimum value of = 0.01 at 415 nm, i.e. a quantum requirement of = 100 photons/ATP.     

REGENERATION OF BLUE AND PURPLE MEMBRANES FROM DEIONIZED BLEACHED MEMBRANES OF Halobacterium halobium

Abstract— Bleached purple membrane normally binds Ca²⁺ and Mg²⁺, which can be removed by the divalent cation chelator ethylenediaminetetraacetic acid (EDTA). Regeneration of pigments from EDTA-treated bleached membrane (apomembrane) and retinal leads to the formation of blue membrane at pH 4.8, and purple membrane at neutral pH. The pigments take much longer to regenerate than with un-deionized apoprotein. Adding back cations to the deionized apomembrane only partially speeds up the regeneration process. Like native purple membrane, the regenerated purple membrane also undergoes a photocycle and shows a light-induced proton release and uptake, although with much slower kinetics than the native species. Thus, cations control the kinetics of pigment regeneration, and also some aspects of the pigment's conformation which controls the photocycle kinetics. The removal and replacement of the cations is not completely reversible, suggesting the cations are not merely bound in the double layer.     

ULTRAVIOLET RESONANCE RAMAN STUDY ON PURPLE AND BLUE MEMBRANES OF Halobacterium halobium

Abstract— Two hundred and forty and 213 nm excited resonance Raman spectra of purple membrane (PM) and blue membrane (BM) of Halobacterium halobiurn were studied. Generally intense Trp scattering and a strong relative intensity of the W3 band at 1553 cm^-1 in the 240 nm spectrum of PM indicate red-shifted B_b absorptions of some Trp sidechains. A high intensity ratio of Trp doublet at 1360 and 1340 cm^-1 suggests interactions with highly hydrophobic Trp environments. These Trp are not strongly H-bonded and their N₁ sites are located in positions easily reached by solvent water molecules. Tyrosines are also in very hydrophobic environments and H-bonded. The mainchain consists of normal and distorted α-helices whose amide NH are hardly deuterated in D₂O suspension, and some NH exchangeable irregular segments on the membrane surface. Upon acidification, the ratio of Trp doublet with 240 nm excitation decreases concomitant with increase in retinal absorbance at 600 nm, and the W3 relative intensity and overall Trp scattering also decrease. These observations strongly indicate that the counterpart of Trp interactions in PM is the retinal and that the interactions partly diminish upon acidification. The Tyr environment also changes with the color. Although the 240 nm amide I intensity is greater in acid BM than in PM, the change is not related to the color change because the amide I intensity of deionized BM is practically the same as that of PM. The amide I intensity increase in acid BM is ascribable to a structural change of the surface peptides due to acid induced aggregation.     

TRANSFORMATION OF A BOP-HOP-SOP-I-SOP-II-Halobacterium halobium MUTANT TO BOP+: EFFECTS OF BACTERIORHODOPSIN PHOTOACTIVATION ON CELLULAR PROTON FLUXES AND SWIMMING BEHAVIOR

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE SENSORY PHOTORECEPTORS OF Halobacterium halobium REVISITED: ACTION SPECTRA and INFLUENCE OF BACKGROUND LIGHT*

Abstract– Action spectra of the light-dependent behavior of Halobacterium and the effect of background light have been measured with regard to the current hypothesis of Spudich and Bogomolni [Nature 312 ,509–513 (1984)], which proposes sensory rhodopsin I (sRI₅₈₇) to be the receptor for long-wavelength light, and its photoproduct S₃₇₃ to be the receptor for UV light. The action spectrum shows three maxima for attractant responses (prolonged swimming intervals) at 565, 590, and 610 nm, and two maxima for repellent responses (shortened intervals) at 370 and 480 nm. The latter is assigned to sensory rhodopsin II (P-480). All peaks are red-shifted after substitution of the endogeneous retinal by 3, 4-dehydroretinal. The peaks at 590 and 610 nm are suppressed by long-wavelength background light. Ultraviolet background light converts all attractant peaks into repellent peaks. The response at 370 nm is strongly activated by visible background light, the maximal effect occurring with 510 nm. The activated state declines with a half-life of about 1.2 s. In a growing culture, full sensitivity to UV and blue light is restored about 10 h earlier than sensitivity to long-wavelength light. Some of the results cannot easily be explained by the sRI₅₈₇/S₃₇₃ hypothesis. Explanations for the three maxima in the long-wavelength range and for the maximal activation of the UV response by 510 nm light are discussed.     

RESONANCE RAMAN SPECTRA OF THE "BLUE" and THE REGENERATED "PURPLE" MEMBRANES OF Halobacterium halobium

Abstract— We have obtained the resonance Raman spectra of the deionized form of the purple membrane, the so called blue membrane, as well as the purple membrane regenerated by titrating the blue membrane with either Na+, Ca²+ or La³+. All types of regenerated purple membrane have identical Raman spectra which are virtually indistinguishable from the native light-adapted bacteriorhodopsin spectrum. On the other hand, Raman data for the blue membrane suggest that it consists of essentially two pigment forms with absorption maxima around 605 and 570 nm and containing 13-cis and all-trans isomeric configurations of the chromophore. This is consistent with our chromophore extraction results which reveal that the blue membrane consists of 30% 13-cir and 70% all-trans chromophore.     

INTERACTIONS OF BOTH MELITTIN AND ITS SITE-SPECIFIC MUTANTS WITH BACTERIORHODOPSIN OF Halobacterium halobium: SITES OF ELECI'ROSTATIC INTERACI'ION ON MELITI'IN

Abstract Melittin and its site-specific mutants differentially delay the slow-decaying component of the photocycle intermediate M₄₁₂ of bacteriorhodopsin in the purple membrane and the acetylated purple membrane whose several lysine residues are modified. This effect is attributed to the interaction of the total positive charges of melittin or its mutants with the total negative charges of bacteriorhodopsin. The effects of melittin and its mutants on the Triton X-100–solubilized bacteriorhodopsin monomers are somewhat complicated but are associated with their charges. These results show that there is electrostatic interaction between bacteriorhodopsin and melittin and that both N-and C-termini of melittin function as sites of the interaction, with Arg 22 and Arg 24 making a prominent contribution to the effective surface charge of melittin. Melittin, at certain concentrations, partially restores the decreased photoactivity of the bacteriorhodopsin monomers trapped in the Triton-lipid-protein mixed micelles, which suggests that melittin may compete with Triton X-100 for the binding sites on the bacteriorhodopsin monomers. Other kinds of interactions between bacteriorhodopsin and melittin are also indicated. The possible states of melittin in membranes are discussed.     

STEADY HYPSOCHROMIC SHIFT OF PEAK RESPONSIVITY TO ATTRACTANT LIGHT FROM 590 nm TO 560 nm IN Halobacterium halobium*

Abstract— Peak responsivity of photoattraction in Halobacterium halobium cells shows steady hypsochromic shift from 590 nm wavelength under low irradiance conditions to 560 nm under high irradiance conditions. Inversion of the photoattractant response, as dependent on blue vs red background light, is compatible with the known properties of photochromic sensory rhodopsin-I (SR-I) with ground state maximum absorption at 587 nm. Relaxation of the photoattractant response in H. halobium, as a function of wavelength and irradiance, gives a hint at an antagonistic pigment or intermediate state, different from ground state SR-I, with peak sensitivity at 620 nm or even above. The less sensitive photoattractant response at 560 nm persists without photorelaxation and represents the peak responsivity under high irradiance conditions.     

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH-PIGMENT MUTANT OF TOMATO

Abstract— A high-pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light-labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence-response relationships for anthocyanin synthesis after a blue-light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far-red light (FR)-absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6-fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild-type and hp-mutant seedlings, despite the latter accumulating 9-fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence-rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24-h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.     

PHOTOSYNTHETIC EFFICIENCY OF A PHYCOCYANIN-LESS MUTANT OF CYANIDIUM*

Abstract— 1. R-II is a u.v. induced mutant of cyanidium caldarium which lacks the major accessory pigment phycocyanin. 2. Loss of phycocyanin does not impair photosynthesis or the Hill reaction. 3. The action spectrum for the quantum yield indicates an efficiently operating photosynthetic mechanism which is shifted in peak efficiency toward longer wavelengths when compared to the wild-type spectrum. The red drop is also shifted toward the far red in R-II. 4. Although 30 per cent enhancement was obtained in wild-type Cyanidium, no enhancement was observed when phycocyanin was absent. 5. A pigment absorbing at 720 nm has been observed and may possibly represent a chlorophyll-phycocyanin complex.     

LASER PHOTOLYSIS OF THE PURPLE MEMBRANE OF Halobacterium halobium IN THE PHOTOSTATIONARY STATE: THE PHOTOBRANCHING PROCESS FROM THE O640 INTERMEDIATE

Abstract The photobranching process from the O₆₄₀ intermediate (O) in the photocycle of bacteriorhodopsin was studied. The O form accumulated with continuous wave visible light (390–800 nm) irradiation of the acidic (pH 3.9–6.0) purple membrane of Halobacterium halobium at 22°C. The photocycle of O via an L-like (or N-like) intermediate was driven by 630 nm pulsed light. The newly found intermediate has an absorption band in the 450–560 nm region. The "green-light"-absorbing pigment, tentatively called G₅₂₀, was converted to O with a time constant of (1.2 ± 0.2) ms. No M-like species was found in the cycle. The quantum yield of the cycle was estimated to be 0.30 ± 0.15.     

REPAIR OF ULTRAVIOLET-IRRADIATED TRANSFORMING DNA IN A recA MUTANT OF Haemophilus influenzae

—Ultraviolet-irradiated transforming DNA was assayed on a wild-type strain of Haemophilus influenzae strain Rd, on an excision repair-deficient ( uvr-2 ) mutant, on a recombination repair-deficient ( recA4 ) mutant, and on a strain carrying both mutations. The donor DNA had a point mutation genetic marker ( strAl ) and a long nonhomologous plasmid-derived DNA segment inserted in the HP1 prophage. The shape of the inactivation curves suggested that only recombination was responsible for the inverse square root kinetics observed with excision repair-proficient recipients.     

ANALYSIS OF MULTIPLE PHOTORECEPTOR PIGMENTS FOR PHOTOTROPISM IN A MUTANT OF Arabidopsis thaliana

Abstract
The shape of the fluence-response relationship for the phototropic response of the JK224 strain of Arabidopsis thaliana depends on the fluence rate and wavelength of the actinic light. At low fluence rate (0.1 μmol m^-2s^-1), the response to 450-nm light is characterized by a single maximum at about 9 μmol m^-2. At higher fluence rate (0.4 μmol m^-2s^-1), the response shows two maxima, at 4.5 and 9 μmol m^-2. The response to 510-nm light shows a single maximum at 4.5 μmol m^-2. Unilateral preirradiation with high fluence rate (25 μmol m^-2s^-1) 510-nm light eliminates the maximum at 4.5 μmol m^-2 in the fluence response curve to a subsequent unilateral 450-nm irradiation, while the second maximum at 9 μmol m^-2 is unaffected. Based on these results, it is concluded that a single photoreceptor pigment has been altered in the JK224 strain of Arabidopsis thaliana.     

ISOLATION OF PHOTOCHEMICAL REACTION CENTERS FROM A CAROTENOIDLESS MUTANT OF RHODOSPIRILLUM RUBRUM*

Lauryl dimethyl amine oxide was used to isolate reaction centers from a carotenoid-less mutant, strain G-9, of Rhodospirillum rubrum. These reaction centers have absorption spectra and light or chemically induced difference spectra very similar to those obtained from Rhodopseudomonas spheroides, strain R-26. But, unlike those from Rps. spheroides, they are more labile to higher detergent concentrations and to ammonium sulfate. The cytochrome content was estimated to be less than one per 10 P870.