Roberto A. Bogomolni

Bacteriorhodopsin: a light-driven proton pump in Halobacterium Halobium.

When grown under low oxygen tension in the light, Halobacterium halobium produces distinct patches in its plasma membrane which can be isolated by differential and sucrose density gradient centrifugation after lysis of the cells by dialysis against distilled water (Oesterhelt and Stoeckenius, 1974). These membrane fragments, which contain 75% protein and 25% lipid by weight, have been named purple membrane because of their characteristic color. This color is due to the protein bacteriorhodopsin, the only protein found in these membranes; it was so named by analogy with the visual pigment rhodopsin, because it contains retinal bound by a Schiff base linkage to an amino group of a lysine residue and shows a broad absorption maximum at 570 nm (Oesterhelt and Stoeckenius, 1971). Isolated purple membrane, when incorporated into lipid vesicles, has been shown to act as a light-driven proton pump, and when mitochondrial ATPase is also incorporated, photophosphorylation can be demonstrated (Racker and Stoeckenius, 1974). In intact cells, photophosphorylation (Danon and Stoeckenius, 1974), lightinduced pH changes (Oesterhelt and Stoeckenius, 1973) and light-induced inhibition of respiration (Oesterhelt and Stoeckenius, 1973) have been observed and all of these effects have action spectra closely corresponding to the absorption spectrum of bacteriorhodopsin (R. A. Bogomolni, R. A. Baker, R. H. Lozier, and W. Stoeckenius, in preparation). These facts suggest that the purple membrane functions in vivo to supplant oxidative phosphorylation as an energy source. They also strongly support a chemiosmotic mechanism for energy transduction. The proton pumping of the purple membrane suggests that bacteriorhodopsin undergoes a light-induced cyclic reaction involving a proton release on one side of the membrane and proton uptake on the opposite side. A light-induced transient shift of the absorption maximum to 412 nm and a concomitant release and uptake of protons have been observed in the purple membrane (Oesterhelt and Stoeckenius, 1973; Oesterhelt and Hess, 1973). A simple two-component system cannot explain the release and

Blue-light-activated histidine kinases: two-component sensors in bacteria.

Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane.

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein

Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

P588, A SECOND RETINAL-CONTAINING PIGMENT IN HALOBACTERIUM HALOBIUM

Abstract— Halobacterium mutant strains with defects in the biosynthesis of various pigments have been isolated. One of these strains, mutant ET‐15, is incapable of producing the light‐driven proton pump bacteriorhodopsin and the carotenoid bacterioruberin. However, ET‐15 synthesizes another photochemically active, retinal‐containing pigment, P588, which mediates light‐induced proton uptake enhanced by uncouplers. P588 and bacteriorhodopsin are simultaneously present in wild‐type cells grown under normal conditions; however, they can be distinguished by the following criteria.

Interactions between a blue-green reversible photoreceptor and a separate UV-B receptor in stomatal guard cells.

Stomatal opening exhibits two main peaks of activity in the visible range-a red peak, mediated by photosynthesis, and a blue peak, mediated by one or more blue light (BL) photoreceptors. In addition, a pronounced peak in the UV-B region has been characterized, as has a smaller UV-A peak. The BL-induced stomatal opening can be reversed by green light (GL). Here we report that UV-B-induced opening is also antagonized by GL. To determine whether UV-B is being absorbed by the BL photoreceptor or by a separate UV-B receptor, the UV-B responses of two different Arabidopsis mutants, npq1 and phot1/phot2, were tested. Both putative BL-photoreceptor mutants exhibited normal stomatal opening in response to UV-B, consistent with the existence of a separate UV-B photoreceptor. Moreover, GL failed to antagonize UV-B-induced stomatal opening in the phot1/phot2 double mutant and only partially antagonized UV-B opening in npq1. Thus, both phot1 and phot 2, as well as zeaxanthin, are required for the normal GL inhibition of UV-B. A model for a photoreceptor network that regulates stomatal opening is presented. Unlike the situation in guard cells, the UV-B bending response of Arabidopsis hypocotyls during phototropism appears to be mediated by phototropins.

Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor

Rhizobium leguminosarum is a soil bacterium that infects root hairs and induces the formation of nitrogen-fixing nodules on leguminous plants. Light, oxygen, and voltage (LOV)-domain proteins are blue-light receptors found in higher plants and many algae, fungi, and bacteria. The genome of R. leguminosarum bv. viciae 3841, a pea-nodulating endosymbiont, encodes a sensor histidine kinase containing a LOV domain at the N-terminal end (R-LOV-HK). R-LOV-HK has a typical LOV domain absorption spectrum with broad bands in the blue and UV-A regions and shows a truncated photocycle. Here we show that the R-LOV-HK protein regulates attachment to an abiotic surface and production of flagellar proteins and exopolysaccharide in response to light. Also, illumination of bacterial cultures before inoculation of pea roots increases the number of nodules per plant and the number of intranodular bacteroids. The effects of light on nodulation are dependent on a functional lov gene. The results presented in this work suggest that light, sensed by R-LOV-HK, is an important environmental factor that controls adaptive responses and the symbiotic efficiency of R. leguminosarum.

The photochemical reactions of bacterial sensory rhodopsin-I. Flash photolysis study in the one microsecond to eight second time window.

Halobacterium halobium Flx mutants are deficient in bacteriorhodopsin (bR) and halorhodopsin (hR). Such strains are phototactic and the light signal detectors are two additional retinal pigments, sensory rhodopsins I and II (sR-I and sR-II), which absorb maximally at 587 and 480 nm, respectively. A retinal-deficient Flx mutant, Flx5R, overproduces sR-I-opsin and does not show any photochemical activity other than that of sR-I after the pigment is regenerated by addition of all-trans retinal. Using native membrane vesicles from this strain, we have resolved a new photointermediate in the sR-I photocycle between the early bathointermediate S610 and the later intermediate S373. The new form, S560, resembles the L intermediate of bR in its position in the photoreaction cycle, its relatively low extinction, and its moderate blue shift. It forms with a half-time of approximately 90 microseconds at 21 degrees C, concomitant with the decay of S610. Its decay with a half-time of 270 microseconds parallels the appearance of S373. From a data set consisting of laser flash-induced absorbance changes (300 ns, 580-nm excitation) measured at 24 wavelengths from 340 to 720 nm in a time window spanning 1 microsecond to 8 s we have calculated the spectra of the photocycle intermediates assuming a unidirectional, unbranched reaction scheme.

FEMTOSECOND TIME-RESOLVED INFRARED LASER STUDY OF THE J-K TRANSITION OF BACTERIORHODOPSIN

Abstract The J − K transition of the bacteriorhodopsin photocycle was monitored by sub-picosecond time-resolved infrared spectroscopy. IR difference spectra in the region between 1670 and 1600 cm −1 were taken at 1.5 and 9 ps, respectively, after photoexcitation of BR 570 at 540 nm. Spectral shifts of the bands at 1607 and 1661 cm −1 reflect the chromophoric conformational changes during the J to K transition. Kinetics, taken at 1640 and 1607 cm −1 show rise times determined by the dephasing times of the vibrational modes. The partial decrease of the bleach signal at 1640 cm −1 is interpreted as a recovery of the vibrationally cooled BR 570 electronic ground state and provides a new method to measure the photocycle quantum yield. The development of the bleach at 1661 cm −1 occurred faster than 500 fs, suggesting an almost instantaneous protein response to the electronic excitation.

Luminescence of Zinc Oxide Crystals Controlled by Electrode Potential and Electrochemical Reactions

The voltage dependence of the green luminescence of ZnO crystals used as electrodes in an electrochemical cell was investigated and compared with the voltage dependence of the photocurrent. When the exciting light is of shorter wavelength than the band edge of ZnO, this luminescence can be varied from its maximal value to complete extinction by applying small positive voltages. The luminescence resulting from excitation within the long wavelength tail of absorption, however, cannot be influenced. The potential dependence of the luminescence has been investigated with differently doped crystals under various conditions. The addition of formic acid to the electrolyte causes an increase of luminescence; this is found to be a consequence of photoelectrochemically induced radical reaction by which electrons are injected into the conduction band of the electrode. The potential dependence of the green luminescence is explained by a mechanism in which the trapping of the holes in recombination centers competes wi...