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HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.

We investigated the wavelength dependence and photon-fluence rate response relationship for phototaxis of wild-type and a cyanobacterial phytochrome 2 (cph2) mutant in cyanobacterium Synechocystis sp. PCC 6803. Compared to wild-type, the cph2 mutant exhibited maximal activity for positive phototaxis at the near-UV spectral range. Two cysteine to serine substitutions in two chromophore-binding domains showed a similar cph2 mutant phenotype under UV-A. Epistasis of a pixJ mutation over a cph2 mutation implied that pixJ gene acts downstream of the cph2 gene with respect to UV-A-induced positive phototaxis. Therefore, we suggest that Cph2 is essential for the inhibition of positive phototaxis toward UV-A.

Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer complex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the receptor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mechanism might also be relevant for eubacterial chemoreceptor signalling.

The Fourier Transform Infrared (FTIR) spectra of photocycle intermediates of sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were measured. The results of the FTIR experiments indicate considerable conformational movements of pSRII already at the stage of the early K-like intermediate which persist at least during the lifetime of the long lived intermediate. These changes in the amide bond region are more intense than those described for sensory rhodopsin I (SRI) and are quite similar to those observed for rhodopsin. Concomitantly with the deprotonation of the Schiff base a carboxyl group located in a hydrophobic environment is protonated. In analogy to bacteriorhodopsin, this carboxyl group might arise from Asp-75 which probably serves as counter ion to the Schiff base. The protonation reaction differs from the situation observed in SRI where the protonation is pH independent over the range of pH 5–8.

We have found that the sign of phototaxis, i.e. the direction of phototactic migration either towards or away from the light source, of wild-type Chlamydomonas reinhardtii depends on its photosynthetic activity. This accounts for the frequently reported transient positive phototaxis that precedes a negative phototaxis of wild-type cells, as well as the earlier observations that preillumination affects the sign of phototaxis. The bases for our conclusion are as follows. (1) The transient nature of phototaxis was preferentially observable with blue-green actinic light rather than with green actinic light. (2) Red background light induces negative phototaxis under the actinic-light conditions in which, without background light, Chlamydomonas cells show exclusively positive phototaxis. (3) Both the effect of red background light and the transient change in the sign of phototaxis were inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, a specific inhibitor of photosynthesis. The conclusion modifies the accepted view that photosynthesis does not link with the phototaxis of this microorganism, thus constituting a necessary part in elucidating mechanisms of algal phototaxis.

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtil is negatively phototactic in the presence of the native chromophore all- trans retinal. In contrast, analog chromophores such as a ring-acyclic retinal and those in which trans/cis isomerization about the C11C12 double bond was blocked induced predominantly positive pho totaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted Hypothesis [1980, Microbiol. Rev. 44, 572–630] on phototaxis mechanisms.

Isolation of intact photoreceptive organelles (eyespot apparatuses) involved in blue-light mediated photoresponses in a flagellate green alga (Spermatozopsis similis) allowed for the first time the identification of both 11-cis- and all-trans-retinal in a plant cell. Both isomers were identified by HPLC analysis in conjunction with UV spectra. Additionally, reconstitution of a distinct absorption band, centered around 540 nm, was achieved by addition of exogenous 9-cis-retinal to bleached, isolated eyespot apparatuses.

Previous studies using retinal analogs (Wada, A., Sakai, M., Kinumi, T., Tsujimoto, K., Yamauchi, M. and Ito, M. (1994) J. Org. Chem. 59, 6992–6997; Wada, A., Sakai, M., Imamoto, Y., Shichida., Y., Yoshizawa, T. and Ito, M. (1993) Chem. Pharm. Bull. 41, 793–795) revealed that both retinochrome and visual pigments share a common chromophoric conformation in which the ring region of retinal is twisted ca. 50° with respect to the plane of the polyene chain, suggesting a highly conserved 6-s-cis conformation throughout rhodopsin-like pigments in animals. By contrast, 6-s-trans conformation was commonly observed or suggested in archaebacterial rhodopsins examined thus far. Here we have reconstituted in vivo both the photoreceptor for photobehavioral responses of the unicellular alga Chlamydomonas reinhardtii and the second archaeal sensory photoreceptor phoborhodopsin from a series of retinal analogs. Results exclusively point to the conclusion that in both photoreceptors retinal has the coplanar 6-s-trans conformation, though recent molecular cloning studies revealed no homology between Chlamydomonas photoreceptor (chlamyrhodopsin) and archaeal rhodopsins.

The photophobic receptor (psRII) and its transducer pHtrII from Natronobacterium pharaonis were heterologously coexpressed in the phototaxis-deficient Halobacterium salinarium strain Pho81/w which lacks all four bacterial rhodopsins, i.e. the two ion pumps bacteriorhodopsin and halorhodopsin as well as the two sensory pigments SRI and SRII. This genetically transformed Pho81/w strain showed a photophobic response upon illumination with blue light. The action spectrum of the psRII/pHtrII mediated phototactic behavior was determined in the range of 420–600 nm. The shape of the action spectrum was similar to the absorption spectrum of psRII, clearly indicating that the psRII-specific photophobic response in Pho81/w was restored. These results suggest that the pharaonis photoreceptor-transducer complex (psRII/pHtrII) is functionally competent to substitute the corresponding salinarium receptor system. Although the two archaea are phylogenetically quite distant from each other the two signal transduction chains are homologous systems which can replace each other.

In Halobacterium halobium tactic responses towards light and chemoeffectors are accompanied by changes in the methylation level of methyl-accepting chemotaxis proteins (MCP). Taxis towards green light absorbed by the bacteriorhodopsin proton pump appears to be governed by Δ μ H+-sensing. The addition of CCCP, an uncoupler, prevented the increase of MCP methylation in response to green light illumination, but had no effect on CH3-incorporation followed by the addition of the attractants glucose, leucine and histidine. Similarly, CCCP did not change MCP demethylation in response to blue light illumination, a repelling stimuli. The sensitivity to an uncoupler of methylation linked to Δ μ H+-mediated green light taxis is to be expected, while the independence of demethylation caused by the blue light of CCCP is an indication that in the latter case a specific photoreceptor governs phototaxis. Informed processing from the blue light receptor to MCP does not involve a change in the membrane potential.

DCCD and cyanide, the H+-ATPase and respiratory chain inhibitors, increase the sensitivity of Halobacteriwn halobium to the attractant effect of light. This phenomenon requires the presence of bacteriorhodopsin and occurs at high light intensities (about 100 W/m2). Strains lacking both bacteriorhodopsin and halorhodopsin exhibit an increased sensitivity of aerotaxis. These observations are in good agreement with the hypothesis that Δg̃mH reception is one of the mechanisms responsible for phototaxis and aerotaxis in H. halobium.

Phototaxis in the archaeon Halobacterium salinarium is mediated by a stable complex of the photoreceptor sensory rhodopsin I and its transducer HtrI, which relays the light stimulus to the signalling pathway. Removal of the cytoplasmic signalling domain of HtrI eliminated the SRI-specific motor response to light stimulation and led to the loss of the spectroscopically detectable physical interaction of SRI and HtrI. A similar phenotype was obtained by deleting part of a cytoplasmic loop located between the second transmembrane helix of HtrI and the signalling domain. These results indicate that the photochemical behavior of sensory rhodopsin I is not determined by interaction with the transmembrane helices of HtrI per se but functionally coupled to the signalling domain. It is proposed that light excitation of SRI results in a conformational change of the transducer which is conducted by the cytoplasmic loop, an extra module not found in the eubacterial transducer homologues, and activates the signalling domain.

The rhodopsin nature of the photoreceptor for the behavioural light responses in Chlamydomonas has originally been revealed by action spectroscopy. Meanwhile most physiological experiments and the identification of all-trans-retinal in cell extracts favour that this chlamyrhodopsin contains an all-trans-type retinal chromophore with strong similarity to the light sensors SR I and SRII from Halobacteria. Reconstitution of retinal-deficient cells with [3H]retinal identified a single retinal protein with a MW of 30,000. Chlamyrhodopsin triggers a photoreceptor current in the eyespot region resulting in direction changes or phototaxis. Furthermore, when the light stimulus oversteps a critical level, two flagellar currents appear, which are the basis for photophobic responses. The physiological, electrophysiological and biochemical experiments suggest that all behavioural responses are triggered by a single rhodopsin-type receptor.

Archaeal phototaxis is mediated by sensory rhodopsins which form complexes with their cognate transducers. Whereas the receptors sensory rhodopsin I and sensory rhodopsin II (SRII) have been expressed in Escherichia coli (E. coli) only shortened fragments of HtrII from Natronomonas pharaonis (NpHtrII) are available. Here we describe the heterologous expression of full length NpHtrII which was achieved in yields of up to 0.9 mg per litre cell culture. Gel filtration analysis reveals the tendency of the transducer to form dimers and higher-order oligomers which was also observed when complexed to NpSRII. A circular dichroism (CD) spectrum of NpHtrII is comparable to those obtained for the E. coli chemoreceptors indicating a similar folding with predominantly α-helical structure. NpHtrII dissociates from the NpSRII/HtrII complex with an apparent KD of about 0.6 μM. Photocycle kinetics of the complex is comparable to that obtained for NpSRII in complex with a truncated transducer with slight differences in the M-decay. The data indicate that the heterologously expressed NpHtrII adopt a native like structure, providing the means for elucidating transmembrane signal transduction and activation of microbial signalling cascades.

PixD is a blue light using flavin (BLUF)-type blue-light photoreceptor controlling phototaxis in the cyanobacterium Synechocystis sp. PCC6803. The crystal structure of PixD shows a decamer, although in solution an equilibrium is maintained between the dimer and decamer. Because the ratio of these two conformers is altered by illumination, the equilibrium state determines photosensitivity. However, no structural information is available for the PixD dimer. Here, we report a predicted structure for the dimer based on docking simulation, mutagenesis, and mass spectrometry-based cross-linking analyses. The results indicate the importance of the PixD C-terminus for dimer preference and photosensitivity.

Natronobacterium pharaonis can react tactically to photo- and chemostimuli. It moves by rotation of a flagellar bundle which is monopolarly inserted. Under sufficient oxygen supply the photophobic response of N. pharaonis has been measured. The resulting action spectrum matches the absorption spectrum of the purified retinylidene protein psR-II. Retical synthesis could be inhibited by nicotine. Cells grown in the presence of nicotine show a strongly reduced photoresponse, which could be restored by addition of retinal. These data identify psR-II as the receptor for negative phototaxis.

Halobacterium halobium utilizes light both as a stimulus for phototaxis [l] and as an energy source for creating a proton gradient across the cell membrane which in turn produces ATP [2] . The photoreceptive pigment of this bacterium was first investigated by Oesterhelt and Stoeckenius [3] who showed that it is the only protein lying in so-called ‘purple membrane’ of the cell. Since this pigment, like visual pigments, has retinal as a prosthetic group [3] it is called ‘bacteriorhodopsin’. There are two forms of this pigment, dark-adapted bacteriorhodopsin (bRD, : 558 nm) and light-adapted bacteriorhodopsin $ll, x max: 568 nm). While Lozier et al. [4] and Kung et al. [S] have reported on the photochemical reactions of the light-adapted form, the photochemical reactions of the dark-adapted pigment have not been studied. We investigated the photochemical reaction of these forms of bacteriorhodopsin at liquid nitrogen temperatures.