Role of the response oscillator in inverse responses of Halobacterium halobium to weak light stimuli.

Under certain conditions Halobacterium halobium organisms respond to a weak attractant light stimulus with a repellent response and to a weak repellent stimulus with an attractant response. The appearance of inverse responses depends on the stimulus strength, on the interval length between spontaneous reversals, and on the moment of stimulation during the interval. Although the cells are absolutely refractory to repellent stimuli for 500 ms after a reversal, repellent responses can be evoked even during that period if they are inverse responses to weak attractant stimuli. Simultaneous attractant and repellent stimuli cancel each other even when one of them leads to an inverse response, indicating that normal cellular signals occur at the site of signal integration. We postulate that the inverse responses are caused by certain properties of a cellular oscillator for which we previously postulated a role in response regulation and sensory control in halobacteria (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985).     

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.     

Integration of photosensory signals in Halobacterium halobium.

Stimulation of Halobacterium halobium through its sensory photosystems, PS 370 and PS 565, leads either to a prolonged or to a shortened interval between two reversals of the swimming direction of the cell, the attractant or repellent response. Stimuli are integrated to yield the same response regardless through which photosystem they are given. Simultaneously elicited attractant and repellent signals cancel each other at any time during a reversal interval, even in the period of refractoriness shortly after a reversal, when the cell is insensitive to repellent stimuli. Successively applied stimuli are less completely integrated. The net response depends on the moment of stimulation during the interval, on the sequence of stimuli, and on the delay between them. Integration of successively applied effective stimuli (after refractoriness) is to a great extent explained in terms of a cellular oscillator (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985) which is changed in opposite directions by attractant and repellent signals. Some conclusions on the shape of the oscillator after its disturbance by a stimulus can be made. Integration of signals during refractoriness leads us to postulate an additional step before the oscillator in the sensory pathway. Cancelling of simultaneous opposite signals is thought to proceed at this integrator. It also takes part in the integration of successively evoked signals. At this step signals rapidly decline within 10 ms, and the total life time (at least of repellent signals) does not exceed 1.2 s.     

The lifetime of photosensory signals in Halobacterium halobium and its dependence on protein methylation

Halobacteria spontaneously reverse their swimming direction about every 10 s. This behavioral pattern is transiently disturbed upon stimulation through sensory photosystems of different spectral sensitivity. As a result of stimulation, a single swimming interval is either prolonged (attractant response) or shortened (repellent response). Thereafter the cell returns to its autonomous reversal rhythm, i.e., it quickly adapts. Method are presented to determine the lifetime of repellent as well as of attractant cellular signals at the site of signal integration, using particular stimulation programs. Independent of the photosystem through which the signals were generated, the total lifetime of a repellent signal was 1.3 s. The decay of the signal was rapid during the first 100 ms and slow thereafter. The lifetime of an attractant signal was about 4 s and likewise did not depend on the photosystems. The degree of methylation of membrane proteins was increased by attractant stimuli and decreased by repellent stimuli. Inhibition of protein methylation by homocysteine was accompanied by a slowdown of the decay of both the repellent and attractant signal. A mutant strain with an increased demethylation also gave increased signal lifetimes. A lowered Ca2+ concentration, which activates methylation in vivo, led to shortened signal lifetimes. Methylation is proposed to be the mechanism which limits the signal lifetime and thereby allows the cells to adapt.     

Oscillating signals in the sensory pathway of halobacteria induced by periodic light stimuli

The swimming behaviour of Halobacterium salinarium can be modulated by light. Changes of the light intensity that induce reversals of the swimming direction are called repellent stimuli, those that suppress reversals, which otherwise would occur spontaneously from time to time, are called attractant stimuli. Bacteria were stimulated by periodic pulse-like stimuli, and the frequency of induced reversals was recorded. Stimulation with a period length between 16 and 6.5 s let the cells reverse periodically with the frequency of the external force. After the stimulation had been stopped, the cells continued to reverse periodically for 3 to 9 periods which, however, switched to a value of about 6 to 8 s, independent of the frequency of preceding stimulation. This endogeneous oscillation was most distinct when the stimulation period either equalled the endogeneous period or was twice or half of its length. During the endogeneous oscillation, the responsiveness to an attractant stimulus showed a pronounced phase-dependence. These results point to the oscillation of a signal in the sensory pathway which, different from our former assumption, seems to be not self-sustained but has to be set going by external stimulation. Received: 14 January 1998 / Revised version: 9 April 1998 / Accepted: 17 May 1998     

Evidence that the long-lifetime photointermediate of s-rhodopsin is a receptor for negative phototaxis in Halobacterium halobium

The effect of blue background light on behavioral response of Halobacterium halobium to step-like stimulation with green-orange attractant light was examined. The results strongly support the previously proposed hypothesis that a long-lifetime photointermediate of s-rhodopsin is the photoreceptor for repellent light: the step-like increase in green-orange light was convertible from attractant stimulus to repellent one, when the cells were constantly illuminated with blue light. No difference of the threshold intensity of the blue background light was observed between the mutant strain that lacks both bacteriorhodopsin and halorhodopsin and the wild type strain, suggesting that the two light-driven ion pumps are not participant in sensing attractant light.     

Effects of cGMP,calcium and reversible methylation on sensory signal processing in halobacteria

Cyclic GMP and Ca²⁺ change the following parameters of sensory behavior of Halobacterium halobium in opposite directions: the interval between two spontaneous reversals of the swimming direction, the phases of increasing and decreasing responsiveness to light stimuli during an interval and the length of the refractory period. Inhibition of the methylation of membrane proteins by blocking the methyltransferase increases the attractant response and prolongs the time during which successive stimuli are integrated. A decrease of the external Ca²⁺ concentration, which activates methylation, has the opposite effect. Inhibition of methylation also prevents the entrainment of the reversal frequency by rhythmic repellent stimuli. The result suggest that cGMP and Ca²⁺ may be antagonistic components of an oscillator, which generates the autonomous rhythm of flagellar reversals, while methylation determines the life-time of sensory signals at an integration link prior to the oscillator, and thereby allows adaptation. Ca²⁺ is thought to act on the methylation system in a feedback loop.     

Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I.

Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II).     

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.     

Entrainment and temperature dependence of the response oscillator in Halobacterium halobium.

Halobacterium halobium spontaneously reverses its swimming direction with a frequency of about 0.1 s-1. The self-sustained periodicity can be entrained by rhythmic light stimuli within a limited range between 0.13 and 0.3 s-1. Increase of temperature increases the reversal frequency and shortens the time to reach maximal responsiveness to attractant stimuli during a cycle. Also, the period of absolute refractoriness to repellent stimuli is shortened. The results illustrate characteristic properties of biological oscillators.     

Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.     

Photosensing and Processing of Sensory Signals in Halobacterim halobium

Halobacteria detect changes in light intensity by retinal proteins, the number and identity of which are not yet unequivocally established. The sensory receptors are different from those for light energy conversion. The cells having no preferred swimming direction spontaneously reverse about every 10 s. An oscillator model has been proposed to explain this periodicity. Depending on wavelength and sign, a stimulus leads either to one prolonged interval between two reversals, the attractant response, or to a shortened interval, the repellent response. Sensory signals generated by stimulation of P-565 and of P-370 are integrated at a common link. Signals from other receptors may be processed by separate links. The nature of the sensory signals is not known, but the membrane potential can be excluded as a candidate. On the basis of the oscillator hypothesis the output signals of the integration links act on the oscillator and thus shift the time at which it triggers a reversal of the flagellar motor. Experiments indicate that cGMP and calcium play antagonistic roles in the oscillatory activity. Reversible methylation of specific membrane proteins influences the time during which successive signals are integrated. This reaction is assumed to terminate the lifetime of the excitatory signals and thus to allow the system to adapt.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (K_eq) for the conformational transition in different mutants and mutant-suppressor pairs.     

Model of bacterial band formation in aerotaxis

Aerotaxis is a particular form of "energy taxis". It is based on a largely elusive signal transduction machinery. In aerotaxis, oxygen dissolved in water plays the role of both attractant (at moderate concentrations) and repellent (at high and low concentrations). Cells swimming from favorable oxygen concentrations into regions with unfavorable concentrations increase the frequency of reversals, turn back into the favorable domain, and become effectively trapped there. At the same time, bacteria consume oxygen, creating an oxygen gradient. This behavior leads to a pattern formation phenomenon: bacteria self-organize into a dense band at a certain distance from the air-water interface. We incorporate experimental observations of the aerotactic bacterium, Azospirillum brasilense, into a mathematical model. The model consists of a system of differential equations describing swimming bacterial cells and diffusing oxygen. The cells' frequency of reversals depends on the concentration of oxygen and its time derivative while oxygen is depleted by the bacteria. We suggest a hypothetical model of energy sensing mediated by aerotactic receptors Aer and Tsr. Computer simulations and analysis of the model equations allow comparisons of theoretical and experimental results and provide insight into the mechanisms of bacterial pattern formation and underlying signal transduction machinery. We make testable predictions about position and density of the bacterial band.     

Excitatory signaling in bacterial probed by caged chemoeffectors.

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.     

Effect of methionine on chemotaxis by Bacillus subtilis.

Bacillus subtilis, like Escherichia coli and Salmonella typhimurium, carries out chemotaxis by modulating the relative frequency of smooth swimming and tumbling. Like these enteric bacteria, methionine auxotrophs starved for methionine show an abnormally long-period of smooth swimming after addition of attractant. This "hypersensitive" state requires an hour of starvation for its genesis, which can be hastened by including alanine, a strong attractant, in starvation medium. Susceptibility to repellent, which causes transient tumbling when added, if anything, increases slightly by starvation for methionine. The results are interpreted by postulating the existence of a methionine-derived structure that hastens recovery of attractant-stimulated bacteria back to normal.     

Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli.

The chemoreceptors responsible for the repellent response of Escherichia coli to phenol were investigated. In the absence of all four known methyl-accepting chemoreceptors (Tar, Tsr, Trg, and Tap), cells showed no response to phenol. However, when Trg, which mediates the attractant response to ribose and galactose, was introduced via a plasmid, the cells acquired a repellent response to phenol. About 1 mM phenol induced a clear repellent response; this response was suppressed by 1 mM ribose. Thus, Trg mediates the repellent response to phenol. Mutant Trg proteins with altered sensing for ribose and galactose showed a normal response to phenol, indicating that the interaction site for phenol differs from that for the ribose- and galactose-binding proteins. Tap, which mediates the attractant response to dipeptides, mediated a weaker repellent response to phenol. Tsr, which mediates the attractant response to serine, mediated an even weaker response to phenol. Trg and Tap were also found to function as intracellular pH sensors. Upon a pH decrease, Trg mediated an attractant response, whereas Tap mediated a repellent response. These results indicate that all the receptors in E. coli have dual functions, mediating both attractant and repellent responses.     

Evidence for an intermediate methyl-acceptor for chemotaxis in Bacillus subtilis

Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol. We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves. First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation. Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent. Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment. We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming). Its demethylation during the period of smooth swimming affords adaptation.     

A one-instant mechanism of phototaxis in the cyanobacterium Phormidium uncinatum

Abstract The front portion ('head') of a Phormidium uncinatum trichome responds to a step-down in light intensity [10], whereas the rear end ('tail') responds to step-up stimuli [11]. We studied this phenomenon further and found that: (i) illumination of the head caused a reversal within 1 min in only 6% of the trichomes, whereas illumination of the tail produced a reversal in 56% of trichomes; (ii) if a light spot trained on the head of a trichome was moved together with the trichome, there were no reversals for > 20 min, while the normal rate of spontaneous reversals was once per 3–5 min. Shifting the light spot from the head to the tail caused a reversal within 1–2 min; (iii) both the step-up response of the tail and phototaxis were suppressed by an inhibitor of methylation, ethionine, but not by inhibitors of photosynthesis (DCMU, DBMIB); phototaxis and the step-up response of the tail were absent in red light ( λ > 670 nm). It was concluded that trichomes of P. uncinatum possess a one-instant mechanism of phototaxis, which involves a simultaneous comparison of light intensities between two parts of the organism.