Novel putative photoreceptor and regulatory genes Required for the positive phototactic movement of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803

Synechocystis: sp. PCC 6803 is a unicellular motile cyanobacterium, which shows positive or negative phototaxis on agar plates under lateral illumination. By gene disruption in a substrain showing of positive phototaxis, it was demonstrated that mutants defective in sll0038, sll0039, sll0041, sll0042 or sll0043 lost positive phototaxis but showed negative phototaxis away from the light source. Mutants of sll0040, which is located within the cluster of these genes, retained the capacity of positive phototaxis but to a lesser extent than the parent cells. These genes are homologous to che genes, which are involved in flagellar switching for bacterial chemotaxis. Interestingly, sll0041 (designated pisJ1) is predicted to have a chromophore-binding motif of phytochrome-like proteins and a signaling motif of chemoreceptors for bacterial chemotaxis. It is strongly suggested that the positive phototactic response was mediated by a phytochrome-like photoreceptor and CheA/CheY-type signal transduction system.     

Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

Selection and properties of phototaxis-deficient mutants of Halobacterium halobium

A method for isolating phototaxis-deficient (Pho-) mutants of Halobacterium halobium was developed. The procedure makes use of a flashing repellent light to induce frequent reversals of swimming direction by responsive cells, thereby impeding their migration along a small capillary and resulting in a spatial separation of the parent population and a population enriched for Pho- cells. Two classes of Pho- mutants were obtained by this selection scheme: those which have lost the chemotactic response (Che-) as well as phototaxis sensitivity (general taxis mutants), and those which are defective in steps specific to phototaxis (photosignaling mutants). In the latter class, several retinal synthesis mutants were isolated, as well as a strain which fit the expected properties of a mutant lacking a functional photoreceptor protein. On the basis of spectroscopic and swimming behavior studies, the retinal-containing protein, slow-cycling or sensory rhodopsin (SR), was previously proposed to be a dual-function sensory receptor mediating both attractant and repellent photosensing. The receptor mutant Pho81 fulfills two predictions which provide direct genetic evidence for this proposal. The mutant has lost SR photoactivity as determined by spectroscopic measurements, and it has simultaneously lost both attractant and repellent phototaxis sensitivity. Comparison of [3H]retinal-labeled membrane proteins from the mutant and its SR-containing parent implicated a 25,000 Mr polypeptide as the chromophoric polypeptide of SR.     

Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.     

Comparative study of phototactic and photophobic receptor chromophore properties in Chlamydomonas reinhardtii.

The motile, unicellular, eukaryotic alga Chlamydomonas reinhardtii exhibits two distinct behavioral reactions to light stimuli, phototaxis and the photophobic response. Both are mediated by retinal-containing receptors. This paper focuses on a direct comparison of the two photoresponses and the chromophore requirements for their photoreceptor(s). Using computerized motion analysis assays for phototaxis and photophobic responses by the same populations of cells, we measured the ability of various isomers and analogues of retinal to reconstitute photobehavior in the pigment-deficient mutant FN68. The results indicate that photophobic and phototaxis responses each require chromophores with an all-trans polyene chain configuration, planar ionone ring/polyene chain conformation, and the ability to isomerize around the retinal C13-C14 double bond. One difference between the two behaviors is that the photophobic response becomes highly desensitized after light stimuli to which the phototaxis response does not become desensitized, indicating the existence of at least one distinct step in the photophobic response pathway. A second difference is that the retinal regeneration of the photophobic response but not of phototaxis is inhibited by a 5-membered ring 13-trans-locked analogue. While showing close similarity in the chromophore structural requirements of the two behaviors, the results indicate that differences exist between the two responses at the level of their photoreceptor proteins and/or in their transduction processes.     

Phototactic Orientation in Plasmodia of the Acellular Slime Mold, Physarum polycephalum

Phototactic orientation to lateral light was studied in plasmodiaof the yellow-pigmented strain (CL) and a white mutant of theslime mold Physarum polycephalum. The orientation changes wereperformed by either a turn or a reversal of the internal polarityof the plasmodium. Fluence rate-response curves for white andmonochromatic light showed positive phototaxis with low fluencerates and negative phototaxis with higher ones. The wavelengthdependences for both strains were similar and indicated thepredominant role of a UV/blue light-absorbing photoreceptor,which was not the same as one of the yellow pigments found inthe wild-type strains. Wavelengths >500 nm induced only aninsignificant effect. The control of the motor apparatus andthe possible linkage of the photoresponse with the other sensoryprocesses known in Physarum are discussed. (Received June 3, 1983; Accepted October 11, 1983)     

Light-induced alteration of c-di-GMP level controls motility of Synechocystis sp. PCC 6803

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.     

Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii

Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.     

The role of two plant-derived volatiles in the foraging movement of 1st instar Helicoverpa armigera (Hübner): time to stop and smell the flowers

Positive phototaxis and negative geotaxis are behaviours that 1st instar Helicoverpa armigera use to direct their foraging movement upward towards nutritious new plant growth and reproductive structures. Odours emitted by fruits or seeds can attract larvae directly via chemotaxis. In this study we clarify the effect of leaf and flower odours on foraging 1st instar H. armigera. Using a Y-tube olfactometer we tested for chemotaxis towards two plant volatiles and found larvae were not attracted. Bioassays for phototaxis towards UV, blue, green and white light showed that a green leaf volatile ((Z)-3-hexenyl acetate) and a flower volatile (phenylacetaldehyde) reduced larval phototaxis towards blue light. Feeding on a host plant reduced phototaxis towards blue and green light. We concluded that the upward movement of 1st instars on plants is largely due to phototaxis towards the blue wavelengths of skylight. Plant attributes such as volatile chemicals affect the expression of phototaxis and therefore, indirectly influence larval movement to locate food resources. Handling Editor: Anna-Karin Borg-Karlson     

The pH dependence of photosensory responses in Stentor coeruleus and model system

1. Live Stentor coeruleus exhibits a substantially red-shifted fluorescence maximum, corresponding to the anionic species of the photoreceptor chromophore. No change was observed in either the absorption or fluorescence excitation spectrum, indicating an efficient deprotonation of the photoreceptor pigment upon excitation by light. 2 Changes in external pH exhibit a dramatic effect on the pulmonary response of Stentor. Phototaxis is specifically inhibited at pH less than 6, with loss of photosensory perception which is restored when the pH is returned to pH greater than 6. 3. Fluorescence changes of 9-aminoacridine in suspensions of live Stentor indicate the generation of a pH gradient upon irradiation with light. Both pH gradient and phototaxis were inhibited by the addition of nigericin and p-tri-fluoromethoxy carbonyl cyanide phenylhydrazone (FCCP). 4. Incorporation of the Stentor photoreceptor protein in to artificial liposomes demonstrates the ability of the system to generate pH gradients across model membranes as monitored by the quenching of 9-aminoacridine fluorescence. The effect of external pH on net proton movement in the model system is strikingly similar to the pH dependent of the liver Stentor, thus lending support for transient proton flux being an important mode of light signal processing for photosensory transduction.     

Maintenance of Motility Bias during Cyanobacterial Phototaxis

Signal transduction in bacteria is complex, ranging across scales from molecular signal detectors and effectors to cellular and community responses to stimuli. The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 transduces a light stimulus into directional movement known as phototaxis. This response occurs via a biased random walk toward or away from a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how quickly cells can respond to changes in the presence or directionality of light, or how photoreceptors affect single-cell motility behavior. In this study, we use time-lapse microscopy coupled with quantitative single-cell tracking to investigate the timescale of the cellular response to various light conditions and to characterize the contribution of the photoreceptor TaxD1 (PixJ1) to phototaxis. We first demonstrate that a community of cells exhibits both spatial and population heterogeneity in its phototactic response. We then show that individual cells respond within minutes to changes in light conditions, and that movement directionality is conferred only by the current light directionality, rather than by a long-term memory of previous conditions. Our measurements indicate that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions. Experiments with a photoreceptor (taxD1) mutant suggest a supplementary role of TaxD1 in enhancing movement directionality, in addition to its previously identified role in promoting positive phototaxis. Motivated by the behavior of the taxD1 mutant, we demonstrate using a reaction-diffusion model that diffusion anisotropy is sufficient to produce the observed changes in the pattern of collective motility. Taken together, our results establish that single-cell tracking can be used to determine the factors that affect motility bias, which can then be coupled with biophysical simulations to connect changes in motility behaviors at the cellular scale with group dynamics.     

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild- type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.     

Polarotaxis,gravitaxis and vertical phototaxis in the green flagellate,Euglena gracilis

A fully automatic computer-controlled video analysis system has been used to study the movement of the green unicellular flagellate, Euglena gracilis in a horizontal or vertical cuvette. In darkness, in the absence of gaseous gradients, most cells swim straight upwards. While in a horizontal cuvette the transition between positive and negative phototaxis is found at about 1.5 W m^-2, an excess of 30 W m^-2 is required to reverse the upward swimming (due to the combined stimulus of negative gravitaxis and positive phototaxis) in a vertical cuvette. By studying the swimming direction in horizontal and vertical cuvettes in polarized light irradiated from above or from the side, respectively, the dichroic orientation of the photoreceptor molecules can be determined in three dimensions with respect to the axes of the cell: In a horizontal cuvette, in a linearly polarized beam from above, the cells orient predominantly at an angle of about 30° clockwise off the electric dipole transition moment as seen from above. The behavior in a vertical cuvette with polarized light entering from above indicates that the photoreceptor pigments are dichroically oriented 60° counterclockwise from the flagellar plane (seen from the front end of the cell). Experiments with horizontal polarized light indicate that the photoreceptor transition moment deviates 25° clockwise off the long axis of the cell.Abbreviation PFB paraflagellar body Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Rhodopsin Receptors of Phototaxis in Green Flagellate Algae

Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the signal transduction chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suitable approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique rhodopsin-type protein. It shares common chromophore properties with retinal proteins from archaea. However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual rhodopsins. The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinal-binding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential function of this protein as the phototaxis receptor has been questioned in recent time.     

Isolation and characterisation of chemotactic mutants of Chlamydomonas reinhardtii obtained by insertional mutagenesis

The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independenttransformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.     

Euglena: The Photoreceptor System for Phototaxis*

SYNOPSIS. The photoreceptor structures (eyespot-paraflagellar body-flagellum) for Euglena phototaxis were investigated by electron microscopy. The paraflagellar body—the photoreceptor—is a highly ordered crystalline lamellar structure. Optical diffraction of the electron micrographs and resulting filtered images of the paraflagellar body suggest that it is formed of rods in a helical arrangement. The action spectra for phototaxis, the in situ spectrum by microspectrophotometry of the paraflagellar body, and flavin analysis of the organism indicate that the photoreceptor molecule is a flavoprotein. The phototaxis action spectrum is similar to the spectrum for O₂ evolution and implies that similar molecules participate in the photo-processes. As a result, a photochemical scheme is suggested in which a photo-excited flavin and a cytochrome participate in the photoprocess. The photochemistry and photoreceptor structures for Euglena phototaxis are likened to a photoneuro sensory cell.