Publications

For fertilization to occur in mammals, sperm cells must travel a long way through the female genital tract, overcoming numerous obstacles to reach the egg. In recent years it became clear that sperm arrival at the egg is not coincidental but rather that sperm cells must be guided, challenging long-standing beliefs. Three navigation means have been discovered: two active and highly sensitive meanschemotaxis and thermotaxisand one passive means, rheotaxis. This review critically examines and puts into perspective the data accumulated over the past two decades about these navigation means and their underlying mechanisms. It questions whether these multiple navigation means are redundant or complementary, demonstrates how they work in harmony, and surveys successful attempts to harness them for improving artificial fertilization outcomes.

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extractstri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or switch, limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motors direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.

Recently, various opsin types, known to be involved in vision, were demonstrated to be present in human and mouse sperm cells and to be involved there in thermosensing for thermotaxis. In vision, each opsin type is restricted to specific cells. The situation in this respect in sperm cells is not known. It is also not known whether or not both signaling pathways, found to function in sperm thermotaxis, are each activated by specific opsins, as in vision. Here we addressed these questions. Choosing rhodopsin and melanopsin as test cases and employing immunocytochemical analysis with antibodies against these opsins, we found that the majority of sperm cells were stained by both antibodies, indicating that most of the cells contained both opsins. By employing mutant mouse sperm cells that do not express melanopsin combined with specific signaling inhibitors, we furthermore demonstrated that rhodopsin and melanopsin each activates a different pathway. Thus, in mammalian sperm thermotaxis, as in vision, rhodopsin and melanopsin each triggers a different signaling pathway but, unlike in vision, both opsin types coexist in the same sperm cells.

Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliGone of the motor's switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling. Here we show that the FrdA subunit in the presence of fumarate is sufficient for binding to FliG and for clockwise enhancement. We further demonstrate by in vitro binding assays and super-resolution microscopy in vivo that the mechanism by which fumarate-occupied FrdA enhances clockwise rotation involves its preferential binding to the clockwise state of FliG (FliG _cw). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdAFliG _cw interaction is driven by the positive electrostatic potential generated by FrdA and the negatively charged areas of FliG. They further demonstrated that fumarate changes FrdA's conformation to one that can bind to FliG _cw. These findings also show that the reason for the failure of the succinate dehydrogenase flavoprotein SdhA (an almost-identical analog of FrdA shown to bind to FliG equally well) to enhance clockwise rotation is that it has no binding preference for FliG _cw. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG ⁰ between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.

Quinol:fumarate reductase (QFR) is an integral membrane protein and a member of the respiratory Complex II superfamily. Although the structure of Escherichia coli QFR was first reported almost twenty years ago, many open questions of catalysis remain. Here we report two new crystal forms of QFR, one grown from the lipidic cubic phase and one grown from dodecyl maltoside micelles. QFR crystals grown from the lipid cubic phase processed as P1, merged to 7.5 Å resolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P2 ₁, merged to 3.35 Å resolution, and displayed a unique crystal packing. This latter crystal form provides the first view of the E. coli QFR active site without a dicarboxylate ligand. Instead, an unidentified anion binds at a shifted position. In one of the molecules in the asymmetric unit, this is accompanied by rotation of the capping domain of the catalytic subunit. In the other molecule, this is associated with loss of interpretable electron density for this same capping domain. Analysis of the structure suggests that the ligand adjusts the position of the capping domain.

Recent studies demonstrated the dependence of speed adaptation in Escherichia coli on acetylation of the chemotaxis signaling molecule CheY. Here, we examined whether CheY acetylation is involved in chemotactic adaptation. A mutant lacking the acetylating enzyme acetyl-CoA synthetase (Acs) requires more time to adapt to attractant stimulation, and vice versa to repellent stimulation. This effect is avoided by conditions that favor production of acetyl-CoA, thus enabling Acs-independent CheY autoacetylation, or reversed by expressing Acs from a plasmid. These findings suggest that CheY should be acetylated for ordinary adaptation time, and that the function of this acetylation in adaptation is to enable the motor to shift its rotation to clockwise. We further identify the enzyme phosphotransacetylase as a third deacetylase of CheY in E. coli.

Chemoreceptor methylation and demethylation has been shown to be at the core of the adaptation mechanism in Escherichia coli chemotaxis. Nevertheless, mutants lacking the methylation machinery can adapt to some extent. Here we carried out an extensive quantitative analysis of chemotactic and chemokinetic methylation-independent adaptation. We show that partial or complete adaptation of the direction of flagellar rotation and the swimming speed in the absence of the methylation machinery each occurs in a small fraction of cells. Furthermore, deletion of the main enzyme responsible for acetylation of the signaling molecule CheY prevented speed adaptation but not adaptation of the direction of rotation. These results suggest that methylation- independent adaptation in bacterial chemotaxis involves chemokinetic adaptation, which is dependent on CheY acetylation.

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate: ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol: fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of

In mammals, sperm guidance in the oviduct appears essential for successful sperm arrival at the oocyte. Hitherto, three different potential sperm guidance mechanisms have been recognized: thermotaxis, rheotaxis, and chemotaxis, each of them using specific stimuli - a temperature gradient, fluid flow, and a chemoattractant gradient, respectively. Here, we review sperm behavioral in these mechanisms and indicate commonalities and differences between them.

STUDY QUESTIONWhat is the behavioral mechanism underlying the response of human spermatozoa to a temperature gradient in thermotaxis?SUMMARY ANSWERHuman spermatozoa swim up a temperature gradient by modulating their speed and frequencies of hyperactivation events and turns.WHAT IS KNOWN ALREADYCapacitated human spermatozoa are capable of thermotactically responding to a temperature gradient with an outcome of swimming up the gradient. This response occurs even when the gradient is very shallow.STUDY DESIGN, SIZE, DURATIONHuman sperm samples were exposed to a fast temperature change. A quantitative analysis of sperm motility parameters, flagellar wave propagation, and directional changes before, during, and after the temperature change was carried out.PARTICIPANTS/MATERIALS, SETTING, METHODSThe swimming behavior of 44 human sperm samples from nine healthy donors was recorded under a phase-contrast microscope at 75 and 2000 frames/s. A temperature shift was achieved by using a thermoregulated microscope stage. The tracks made by the cells were analyzed by a homemade computerized motion analysis system and ImageJ software.MAIN RESULTS AND THE ROLE OF CHANCEA temperature shift from 31 to 37°C resulted in enhanced speed and a lower frequency of turning events. These were reflected in a 35 ± 1% (mean ± SEM) increase of the straight-line velocity, 33 ± 1% increase of the average path velocity, 11 ± 1% increase of the curvilinear velocity, 20 ± 1% increase of the wobble, and 4 ± 1% increase of the linearity. Qualitatively, the inverse trend was observed in response to a 37-to-31°C shift. In addition, the amplitude of flagellar waves increased close to the sperm head, resulting in higher side-to-side motion of the head and, often, hyperactivation. This increase in the extent of sperm hyperactivation was reflected in an increase in the average (mean ± SEM) fractal dimension from 1.15 ± 0.01 to 1.29 ± 0.01 and in the percentage of hyperactivated spermatozoa from 3 ± 1% to 19 ± 2%. These changes in hyperactivation were observed less often in sperm populations that had not been incubated for capacitation. All these changes partially adapted within 310 min, meaning that following the initial change and while being kept at the new temperature, the values of the measured motility parameters slowly and partially returned toward the original values. These results led us to conclude that spermatozoa direct their swimming in a temperature gradient by modulating the frequency of turns (both abrupt turns as in hyperactivation events and subtle turns) and speed in a way that favors swimming in the direction of the gradient.LIMITATIONS, REASONS FOR CAUTIONThe conclusions were made on the basis of results obtained in temporal and steep temperature gradients. The conclusions for spatial, shallow gradients were made by extrapolation.WIDER IMPLICATIONS OF THE FINDINGSThis is the first study that reveals the behavior of human spermatozoa in thermotaxis. This behavior is very similar to that observed during human sperm chemotaxis, suggesting commonality of guidance mechanisms in mammalian spermatozoa. This study further substantiates the function of hyperactivation as a means to direct spermatozoa in guidance mechanisms.

Summary: Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K91 repression is manifested in the conformational dynamics of the β4α4 loop, shifted toward an active state upon mutation. Removal of β4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination, we further suggest that K109 acetylation is actively involved in generating clockwise rotation.

In the recent two decades mammalian spermatozoa were demonstrated to perform chemotaxis, thermotaxis and rheotaxis. It is believed that in the Fallopian tube spermatozoa are first guided to the fertilization site by long-range mechanisms, thermotaxis and rheotaxis, and there they are guided to the egg by two processes of chemotaxis, considered a short-range mechanism. The occurrence of chemotaxis and thermotaxis in additional locations along the female genital tract cannot be excluded.

Sperm thermotaxis, the active orientation of sperm swimming according to a temperature gradient has been suggested to act as a long-range guidance mechanism in the oviduct during fertilization, between the cooler sperm storage site and the warmer fertilization site. In this process capacitated spermatozoa can sense even very shallow temperature gradients. They respond to the changing temperature by modulating their flagellar beating. The outcome is a higher frequency of turns and hyperactivation events when the temperature drops, and a rather linear swimming when they sense a temperature increase. In this way they are guided towards the warmer temperature.

Objective To characterize the nature of the human oocyte-derived chemoattractant. Design Laboratory in vitro study. Setting Academic research institute. Patient(s) Ten healthy sperm donors. Oocyte-conditioned media from women undergoing IVF treatment because of male factor infertility. Intervention(s) Sperm samples were processed by the migration-sedimentation technique. Oocyte-conditioned media were collected 2-3 hours after oocyte stripping. Main Outcome Measure(s) Sperm chemotaxis was assayed in a μ-slide chamber according to the direction of swimming relative to that of the chemical gradient. Result(s) Oocyte-conditioned media treated with proteases did not lose their chemotactic activity; on the contrary, they became more active, with the activity shifted to lower concentrations. When oocyte-conditioned media were subjected to hexane extraction, chemotactic activity was found in both the hydrophobic and aqueous phases. Known mammalian sperm chemoattractants were ruled out as oocyte-derived chemoattractants. Conclusion(s) Our results suggest that the oocyte-derived chemoattractant is a hydrophobic nonpeptide molecule that, in an oocyte-conditioned medium, is associated with a carrier protein that enables its presence in a hydrophilic environment.

On the basis of the finding that capacitated (ready to fertilize) rabbit and human spermatozoa swim towards warmer temperatures by directing their movement along a temperature gradient, sperm thermotaxis has been proposed to be one of the processes guiding these spermatozoa to the fertilization site. Although the molecular mechanism underlying sperm thermotaxis is gradually being revealed, basic questions related to this process are still open. Here, employing human spermatozoa, we addressed the questions of how wide the temperature range of thermotaxis is, whether this range includes an optimal temperature or whether spermatozoa generally prefer swimming towards warmer temperatures, whether or not they can sense and respond to descending temperature gradients, and what the minimal temperature gradient is to which they can thermotactically respond. We found that human spermatozoa can respond thermotactically within a wide temperature range (at least 29-41°C), that within this range they preferentially accumulate in warmer temperatures rather than at a single specific, preferred temperature, that they can respond to both ascending and descending temperature gradients, and that they can sense and thermotactically respond to temperature gradients as low as

Biased motion of motile cells in a concentration gradient of a chemoattractant is frequently studied on the population level. This approach has been particularly employed in human sperm chemotactic assays, where the fraction of responsive cells is low and detection of biased motion depends on subtle differences. In these assays, statistical measures such as population odds ratios of swimming directions can be employed to infer chemotactic performance. Here, we report on an improved method to assess statistical significance of experimentally determined odds ratios and discuss the strong impact of data correlations that arise from the directional persistence of sperm swimming.

Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays in vitro, we provided evidence for the interaction of F ₀F₁ ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F₀F₁ ATP synthase with FliG is important for the function of the switch of bacterial flagella.

When mammalian spermatozoa become capacitated they acquire, among other activities, chemotactic responsiveness and the ability to exhibit occasional events of hyperactivated motility-a vigorous motility type with large amplitudes of head displacement. Although a number of roles have been proposed for this type of motility, its function is still obscure. Here we provide evidence suggesting that hyperactivation is part of the chemotactic response. By analyzing tracks of spermatozoa swimming in a spatial chemoattractant gradient we demonstrate that, in such a gradient, the level of hyperactivation events is significantly lower than in proper controls. This suggests that upon sensing an increase in the chemoattractant concentration capacitated cells repress their hyperactivation events and thus maintain their course of swimming toward the chemoattractant. Furthermore, in response to a temporal concentration jump achieved by photorelease of the chemoattractant progesterone from its caged form, the responsive cells exhibited a delayed turn, often accompanied by hyperactivation events or an even more intense response in the form of flagellar arrest. This study suggests that the function of hyperactivation is to cause a rather sharp turn during the chemotactic response of capacitated cells so as to assist them to reorient according to the chemoattractant gradient. On the basis of these results a model for the behavior of spermatozoa responding to a spatial chemoattractant gradient is proposed.

Bacteria can move by a variety of means, the most common one by rotating their flagella. This movement is often directed towards favourable chemicals (chemoattractants) or away from unfavourable chemicals (chemorepellents), a process termed chemotaxis. This modulation of swimming direction is the outcome of controlled changes in the direction of flagellar rotation. Therefore, mechanistically, the essence of bacterial chemotaxis is to control the direction of flagellar rotation. This control is done by a sophisticated signal transduction system, involving a small protein, CheY, which shuttles back and forth between the receptor complexes clustered at the pole of the cell and the flagellar motor complexes around the cell. These interactions are modulated by phosphorylation and acetylation. The excitatory signalling process involves amplification. The adaptation signalling involves methylation of the receptors. Even though bacterial chemotaxis is considered the best understood signalling system at the molecular level, many major questions are still waiting to be resolved. Key Concepts: In bacteria, chemotaxis is the motile response to stimuli and it serves as a means of cellenvironment and cell-to-cell communication. Bacteria sense stimulant gradients (chemoattractants and chemorepellents) temporally rather than spatially. The chemotaxis-specific receptors are clustered at the bacterial poles. This clustering is essential for signalling and amplification. Bacteria swim by rotating their flagella. The direction of rotation determines the swimming mode. Therefore, the essence of bacterial chemotaxis is to control the direction of flagellar rotation. The direction of flagellar rotation is modulated by the signalling molecule, CheY, according to changes in receptor occupancy. CheY function is modulated by phosphorylation at both the excitatory and adaptation signalling phases. The latter also involves receptor carboxy methylation. Phosphorylated CheY binds to the switch at the base of the flagellar motor and, thereby, changes the direction of rotation.

The ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications - phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non-enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphatase CheZ and the switch protein FliM - its target at the flagellar switch complex. Acetylation repressed the binding to all three proteins. These results suggest that both phosphorylation and acetylation determine CheY's ability to bind to its target proteins, thus providing two levels of regulation, fast and slow respectively. The fast level is modulated by environmental signals (e.g. chemotactic and thermotactic stimuli). The slow one is regulated by the metabolic state of the cell and it determines, at each metabolic state, the fraction of CheY molecules that can participate in signalling.

Capacitated human and rabbit spermatozoa can sense temperature differences as small as those within the oviduct of rabbits and pigs at ovulation, and they respond to them by thermotaxis (i.e., by swimming from the cooler to the warmer temperature). The molecular mechanism of sperm thermotaxis is obscure. To reveal molecular events involved in sperm thermotaxis, we took a pharmacological approach in which we examined the effect of different inhibitors and blockers on the thermotactic response of human spermatozoa. We found that reducing the intracellular, but not extracellular, Ca²⁺ concentration caused remarkable inhibition of the thermotactic response. The thermotactic response was also inhibited by each of the following: La³⁺, a general blocker of Ca²⁺ channels; U73122, an inhibitor of phospholipase C (PLC); and 2-aminoethoxy diphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate receptors (IP₃R) and store-operated channels. Inhibitors and blockers of other channels had no effect. Likewise, saturating concentrations of the chemoattractants for the known chemotaxis receptors had no effect on the thermotactic response. The results suggest that the IP₃R Ca ²⁺ channel, located on internal Ca²⁺ stores, operates in sperm thermotaxis, and that the response is mediated by PLC and requires intracellular Ca²⁺. They also suggest that the thermosensors for thermotaxis are not the currently known chemotaxis receptors.

BACKGROUNDA major question in mammalian sperm chemotaxis is whether the cells sense a chemoattractant gradient by comparing the chemoattractant concentration between time points or between spatial points.METHODSTo resolve this question, we exposed human spermatozoa to a temporal chemoattractant gradient under conditions of no spatial gradient by rapidly mixing the cells with progesterone or bourgeonal on a microscope slide and analyzing their swimming with motion analysis software.RESULTSThe cells responded within seconds with an increase in velocity and lateral head displacement, and with a decrease in the linearity of swimming, becoming hyperactivated at the peak of the response. All the responses were transient, lasting for a number of seconds. Essentially similar results were obtained upon intracellular photorelease of cyclic adenosine monophosphate or cyclic guanosine monophosphate, which are thought to be involved in mediating the chemotactic response.CONCLUSIONThese results suggest that human spermatozoa sense and respond to a temporal chemoattractant gradient. On the basis of these observations, we propose a potential model for the chemotactic response of spermatozoa in a spatial chemoattractant gradient.

Chemotaxis is a basic guidance mechanism employed by cells and organisms to move toward beneficial targets or environments and to avoid undesired ones. This mechanism, which is prevalent from bacteria to human beings, consists of two basic processes. One is the formation by diffusion of a concentration gradient of a specific chemical. The other is the sensation of this gradient by a cell/organism and the modification of its direction of movement up or down the chemical gradient (the chemical being defined as chemoattractant or chemorepellent, respectively). Although chemotaxis is a universal mechanism and so are the steps that constitute it (gradient sensing by receptors, signal transduction and amplification, and movement response), the processes that underlie this mechanism are diverse.

BACKGROUND: Human spermatozoa appear to be guided by chemotaxis to the oocyte in the female genital tract. While one of the sources of sperm chemoattractants is the cumulus cells that surround the oocyte, the identity of the chemoattractant secreted from them is unknown. Progesterone, recognized to be secreted from cumulus cells, was demonstrated, at the pM concentration range, to be a chemoattractant for human spermatozoa. Here, we examined whether this steroid is the cumulus-originated chemoattractant for human spermatozoa. METHODS: Human cumulus cells were cultured, and the cultured medium was demonstrated to be chemotactically active. Progesterone was then eliminated from the medium by a specific anti-progesterone antibody, and the residual chemotactic activity was assessed. RESULTS: The rate of progesterone secretion from the cells decreased with time. Removal of progesterone from the cumulus-cultured medium resulted in total loss of the chemotactic activity of the medium. Furthermore, the cumulus-cultured medium could substitute for progesterone in stimulating changes in the intracellular Ca²⁺ concentration in the spermatozoa, and the changes were very similar to those caused by measured progesterone concentrations in the medium. CONCLUSIONS: Taken together, the results suggest that progesterone is the main, if not the sole, chemoattractant secreted by human cumulus cells.

The mechanism of function of the bacterial flagellar switch, which determines the direction of flagellar rotation and is essential for chemotaxis, has remained an enigma for many years. Here we show that the switch complex associates with the membrane-bound respiratory protein fumarate reductase (FRD). We provide evidence that FRD binds to preparations of isolated switch complexes, forms a 1:1 complex with the switch protein FliG, and that this interaction is required for both flagellar assembly and switching the direction of flagellar rotation. We further show that fumarate, known to be a clockwise/switch factor, affects the direction of flagellar rotation through FRD. These results not only uncover a new component important for switching and flagellar assembly, but they also reveal that FRD, an enzyme known to be primarily expressed and functional under anaerobic conditions in Escherichia coli, nonetheless, has important, unexpected functions under aerobic conditions.

CheY, the excitatory response regulator in the chemotaxis system of Escherichia coli, can be modulated by two covalent modifications: phosphorylation and acetylation. Both modifications have been detected in vitro only. The role of CheY acetylation is still obscure, although it is known to be involved in chemotaxis and to occur in vitro by two mechanisms-acetyl-CoA synthetase-catalyzed transfer of acetyl groups from acetate to CheY and autocatalyzed transfer from AcCoA. Here, we succeeded in detecting CheY acetylation in vivo by three means-Western blotting with a specific anti-acetyl-lysine antibody, mass spectrometry, and radiolabeling with [¹⁴C]acetate in the presence of protein-synthesis inhibitor. Unexpectedly, the level and rate of CheY acetylation in vivo were much higher than that in vitro. Thus, before any treatment, 9-13% of the lysine residues were found acetylated, depending on the growth phase, meaning that, on average, essentially every CheY molecule was acetylated in vivo. This high level was mainly the outcome of autoacetylation. Addition of acetate caused an incremental increase in the acetylation level, in which acetyl-CoA synthetase was involved too. These findings may have far-reaching implications for the structure-function relationship of CheY.

The detection of chemotaxis-related changes in the swimming behavior of mammalian spermatozoa in a spatial chemoattractant gradient has hitherto been an intractable problem. The difficulty is that the fraction of responsive cells in the sperm population is very small and that the large majority of the cells, though non-responsive, are motile too. Assessment of the chemotactic effects in a spatial gradient is also very sensitive to the quality of sperm tracking. To overcome these difficulties we propose a new approach, based on the analysis of the distribution of instantaneous directionality angles made by spermatozoa in a spatial gradient versus a no-gradient control. Although the use of this parameter does not allow identification of individual responding cells, it is a reliable measure of directionality, independent of errors in cell tracking caused by cell collisions, track crossings, and track splitting. The analysis identifies bias in the swimming direction of a population relative to the gradient direction, It involves statistical χ² tests of the very large sample of measured angles, where the critical χ² values are adjusted to the sample size by the bootstrapping procedure. The combination of the newly measured parameter and the special analysis provides a highly sensitive method for the detection of a chemotactic response, even a very small one.

Chemotaxis is a basic recognition process, governed by protein network that translates molecular-based information on the surrounding environment into a guided motional response of the recipient cell or organism. This process is prevalent from bacteria to human beings. Some of the chemotaxis systems - like that of the bacterium Escherichia coli - are well established; others - like that of mammalian sperm cells - are at their relatively early stages of research. In contrast to mammalian sperm chemotaxis, where studies have so far been limited to the phenomenological level primarily, the model of bacterial chemotaxis is known down to the angstrom resolution. Despite this difference in depth of understanding, many fundamental questions are open not only in the new but also in the old chemotaxis fields of research, and recent advances in them are raising additional intriguing questions. This review summarizes some of these surprises and previously unasked or overlooked questions, and as such it offers a guided tour through conceptual changes in chemotaxis.

BACKGROUND: Earlier studies demonstrated that macrophages phagocytize spermatozoa in the female genital tract of mammals. In spite of this phagocytosis, fecundity is not affected, raising questions of how the resulting decrease in the number of spermatozoa does not reduce the fertilization rate and of the role of this phagocytosis. We hypothesized that its role is to rid the female genital tract of spermatozoa past their fertilizing stage (post-capacitated spermatozoa). Here we examined whether, indeed, phagocytosis is restricted to post-capacitated spermatozoa. METHODS: Spermatozoa were incubated for 22 h either in a medium that allows them to become capacitated and then post-capacitated, or in a medium that prevents them from acquiring these states. These sperm populations were compared for their susceptibilities to macrophage phagocytosis. RESULTS: Phagocytosis was significantly higher (P ≪ 0.001) in the sperm population containing post-capacitated spermatozoa. Vitality, motility, the acrosomal status and the proportion of capacitated cells did not affect phagocytosis. CONCLUSION: Post-capacitated spermatozoa are, probably, preferentially phagocytized by macrophages.

Thermotaxis - movement directed by a temperature gradient - is a prevalent process, found from bacteria to human cells. In the case of mammalian sperm, thermotaxis appears to be an essential mechanism guiding spermatozoa, released from the cooler reservoir site, towards the warmer fertilization site. Only capacitated spermatozoa are thermotactically responsive. Thermotaxis appears to be a long-range guidance mechanism, additional to chemotaxis, which seems to be short-range and likely occurs at close proximity to the oocyte and within the cumulus mass. Both mechanisms probably have a similar function-to guide capacitated, ready-to-fertilize spermatozoa towards the oocyte. The temperature difference between the site of the sperm reservoir and the fertilization site is generated at ovulation by a temperature drop at the former. The molecular mechanism of sperm thermotaxis waits to be revealed.

One of the processes by which CheY, the excitatory response regulator of chemotaxis in Escherichia coli, can be activated to generate clockwise flagellar rotation is by acetyl-CoA synthetase (Acs)-mediated acetylation. Deletion of Acs results in defective chemotaxis, indicating the involvement of Acs-mediated acetylation in chemotaxis. To investigate whether Acs is the sole acetylating agent of CheY, we purified the latter from a Δacs mutant. Mass spectrometry analysis revealed that this protein is partially acetylated in spite of the absence of Acs, suggesting that CheY can be post-translationally acetylated in vivo by additional means. Using [¹⁴C]AcCoA in the absence of Acs, we demonstrated that one of these means is autoacetylation, with AcCoA serving as an acetyl donor and with a rate similar to that of Acs-mediated acetylation. Biochemical characterization of autoacetylated CheY and mass spectrometry analysis of its tryptic digests revealed that its acetylated lysine residues are those found in CheY acetylated by Acs, but the acetylation-level distribution among the acetylation sites was different. Like CheY acetylated by Acs, autoacetylated CheY could be deacetylated by Acs. Also similarly to the case of Acs-mediated acetylation, the phosphodonors of CheY, CheA and acetyl phosphate, each inhibited the autoacetylation of CheY, whereas the phosphatase of CheY, CheZ, enhanced it. A reduced AcCoA level interfered with chemotaxis to repellents, suggesting that CheY autoacetylation may be involved in chemotaxis of E. coli. Interestingly, this interference was restricted to repellent addition and was not observed with attractant removal, thus endorsing our earlier suggestion that the signaling pathway triggered by repellent addition is not identical to that triggered by attractant removal.

Contrary to the prevalent view, there seems to be no competition in the mammalian female genital tract among large numbers of sperm cells that are racing towards the egg. Instead, small numbers of the ejaculated sperm cells enter the Fallopian tube, and these few must be guided to make the remaining long, obstructed way to the egg. Here, we review the mechanisms by which mammalian sperm cells are guided to the egg.

Background: Earlier studies demonstrated a small temperature difference between the sperm storage and fertilization sites within the oviducts of rabbits and pigs. Our aim was to reveal the time dependence of this temperature difference relative to ovulation, and to determine how this difference is generated - by temperature elevation at one of these sites or by temperature decrease at the other site. Methods: The temperature at the sperm storage site (at the isthmus near the uterotubal junction) and at the fertilization site (the isthmic-ampullary junction) of rabbit oviducts were measured before, during, and after ovulation by two probes, connected to digital thermometers. Rectal temperature was constantly measured and served as a control for body temperature. Results: The temperature difference between the fertilization site and the storage site was 0.8±0.2°C before ovulation. This difference increased at ovulation, reaching 1.6±0.1°C after ovulation (P

Background: Human sperm chemotaxis to pre-ovulatory follicular fluid is well established in vitro. However, it is not known whether the female's oocyte-cumulus complex secretes sperm chemoattractants subsequent to ovulation (for enabling sperm chemotaxis within the Fallopian tube) and, if so, which of these cell types - the oocyte or the cumulus oophorus - is the physiological origin of the secreted chemoattractant. Methods: By employing a directionality-based chemotaxis assay, we examined whether media conditioned with either individual, mature (metaphase II) human oocytes or the surrounding cumulus cells attract human sperm by chemotaxis. Results: We observed sperm chemotaxis to each of these media, suggesting that both the oocyte and the cumulus cells secrete sperm chemoattractants. Conclusions: These observations suggest that sperm chemoattractants are secreted not only prior to ovulation within the follicle, as earlier studies have demonstrated, but also after oocyte maturation outside the follicle, and that there are two chemoattractant origins: the mature oocyte and the surrounding cumulus cells.

CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.

Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E. coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient ≈3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.

The time course of the level of A23187-induced acrosome reaction between human and rabbit spermatozoa was compared. It was extended in the former (a periodic ovulator) and short in the latter (an induced ovulator). This finding suggests that the capacitated state is programmed to maximize the prospects that an ovulated egg will meet spermatozoa in the best functional state.

In bacteria, the chemotactic signal is greatly amplified between the chemotaxis receptors and the flagellar motor. In Escherichia coli, part of this amplification occurs at the flagellar switch. However, it is not known whether the amplification results from cooperativity of CheY binding to the switch or from a post-binding step. To address this question, we purified the intact switch complex (constituting the switch proteins FliG, FliM, and FliN and the scaffolding protein FliF) in quantities sufficient for biochemical work and used it to investigate whether the binding of CheY to the switch complex is cooperative. As a negative control, we used complexes of switchless basal bodies, formed from the proteins FliF and FliG and similarly isolated. Using double-labeling centrifugation assays for binding, we found that CheY binds to the isolated, intact switch complex in a phosphorylation-dependent manner. We observed no significant phosphorylation-dependent binding to the negative control of the switchless basal body. The dissociation constant for the binding between the switch complex and phosphorylated CheY (CheY∼P) was 4.0 ± 1.1 μM, well in line with the published range of CheY∼P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient ≈ 1). This lack of CheY∼P-switch complex binding cooperativity, taken together with earlier in vivo studies suggesting that the dependence of the rotational state of the motor on the fraction of occupied sites at the switch is sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol. Biol. 312, 699-709), indicates that the chemotactic signal is amplified within the switch, subsequent to the CheY∼P binding.

When mammalian sperm cells enter the female genital tract, many of them are attacked and phagocytosed by leukocytes and epithelial cells. Although this intriguing phenomenon is known for almost five decades, there is no satisfactory explanation for it. Here, on the basis of recent information on the nature of the capacitated stage of mammalian sperm cells, that is, the sperm's stage of readiness for fertilizing the egg, I put forward the hypothesis that the phagocytosed sperm cells are post-capacitated cells. These cells, which lost their fertilizing ability and became functionless, apparently recruit leukocytes and then undergo apoptosis and phagocytosis and, thereby, are removed from the female genital tract. This fast removal probably prevents severe inflammation that could have been caused by necrotic products of sperm cells that remain functionless in the tract.

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10-4 dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.

This chapter gives a summary of capacitation, which is an additional \u201cmaturation\u201d process that spermatozoa must undergo to acquire fertilization potential. Sperm capacitation is a prerequisite for the acrosome reaction that is a release of proteolytic enzymes enabling sperm penetration through the egg coat. Capacitation can be initiated by incubating spermatozoa in any medium that retains them in a competent state, provided that it is free of seminal fluid. The removal of seminal fluid is essential, because it contains decapacitating factors that inhibit capacitation. This is possibly one of the reasons for the observation that ejaculated spermatozoa of many species are more resistant to in vitro capacitation than are epididymal spermatozoa. The inhibitory effect of seminal fluid on capacitation became evident when it was shown that on exposure to seminal fluid, capacitated spermatozoa loses both their ability to undergo induced acrosome reaction and their fertilizing potentialthat is, they become \u201cdecapacitated\u201d. This chapter finally concludes with a remark that mammalian spermatozoa should undergo two ripening stages for acquiring fertilization potential: maturation in the male reproductive tract and capacitation in the female genital tract or in vitro. Sperm motility and fertility are acquired during maturation, and the abilities to find the egg and penetrate it are acquired during capacitation. Capacitation is not involved in the actual transfer of the sperm genetic material to the egg. Its role is to prepare the conditions for such a transfer and to ensure that spermatozoa reaching the egg are those endowed with the capacity to penetrate and fertilize it.

One of the major questions in bacterial chemotaxis is how the switch, which controls the direction of flagellar rotation, functions. It is well established that binding of the signaling molecule CheY to the switch protein FliM shifts the rotation from the default direction, counterclockwise, to clockwise. How this shift is done is still a mystery. Our aim in this study was to determine the correlation between the fraction of FliM molecules in the clockwise state (i.e. occupied by CheY) and the probability of clockwise rotation. For this purpose we gradually expressed, from a plasmid, a clockwise FliM mutant protein in cells that express, from the chromosome, wild-type FliM but no chemotaxis proteins. We verified that plasmid-borne FliM exchanges chromosomal FliM in the switch. Surprisingly, a substantial clockwise probability was not obtained before the large majority of the FliM molecules in the switch were clockwise molecules. Thereafter, the rise in clockwise probability was very steep. These results suggest that an increase in the clockwise probability requires a high level of FliM occupancy by CheY ∼ P. They further suggest that the steep increase in clockwise rotation upon increasing CheY levels, reported in several studies, is due, at least in part, to cooperativity of post-binding interactions within the switch. We also carried out the inverse experiment, in which wild-type FliM was gradually expressed in a background of a clockwise fliM mutant. In this case, the level of the clockwise mutant protein, required for establishing a certain clockwise probability, was lower than in the original experiment. If our system (in which the ratio between the rotational states of FliM in the switch is established by slow exchange) and the native system (in which the ratio is established by fast changes in FliM occupancy) are comparable, the results suggest that hysteresis is involved in the switch function. Such a situation might reflect a damping mechanism, which prevents a situation in which fluctuations in the phosphorylation level of CheY throw the switch from one direction of rotation to the other.

Bacterial chemotaxis is the phenomenon in which bacteria actively modulate their direction of movement so as to approach chemoattractants (favourable, usually nutritious chemicals) and avoid chemorepellents (unfavourable, usually noxious chemicals).

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl-CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine-for-lysine substitution at residue 92 of CheY - one of the acetylation sites. The Δacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring-forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs-mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys-92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.

Sperm chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime between 50 min and 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in man) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the duration of time over which capacitated spermatozoa would be available in the female reproductive tract. The sperm chemoattractants have not been identified but they appear to be heat-stable peptides. The in vivo location of sperm chemotaxis is not known; a number of possible locations are discussed.

CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modem nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.

Communication between spermatozoa and egg before contact by chemotaxis appears to be prevalent throughout the animal kingdom. In non-mammalian species, sperm chemotaxis to factors secreted from the egg is well documented. In mammals, sperm chemotaxis to follicular factors in vitro has been established in humans and mice. The attractants of female origin in non-mammalian species are heat-stable peptides or proteins of various sizes, or other small molecules, depending on the species. Species specificity of the attractants in non-mammalian species may vary from high species specificity, through specificity to families with no specificity within a family, to absence of specificity. The mammalian sperm attractants have not been identified but they appear to be heat-stable peptides. The claim that progesterone is the attractant for human spermatozoa has failed to be substantiated, neither have claims for other mammalian sperm attractants been verified. The molecular mechanism of sperm chemotaxis is not known. Models involving modulation of the intracellular Ca²⁺ concentration have been proposed for both mammalian and non-mammalian sperm chemotaxis. The physiological role of sperm chemotaxis in non-mammalian species appears to differ from that in mammals. In non-mammalian species, sperm chemotaxis strives to bring as many spermatozoa as possible to the egg. However, in mammals, the role appears to be recruitment of a selective population of capacitated ('ripe') spermatozoa to fertilize the egg.

Follicular fluid (FF) induces sperm chemotaxis in human spermatozoa. Progesterone also causes sperm accumulation. However, sperm accumulation can be caused by chemotaxis, chemokinesis, and trapping of various kinds. It has been suggested that progesterone also induces chemotaxis in human spermatozoa. In view of the physiological significance of sperm chemotaxis in human fertilization and its potential clinical implications, it is important to determine unequivocally whether chemotaxis is induced by progesterone and, if so, whether progesterone in FF is the chemoattractant. To resolve these questions we looked for characteristic changes in the direction of sperm swimming toward pure progesterone as well as toward FF before and after progesterone removal. Progesterone caused sperm accumulation and hyperactivation-like motility, but it caused very few changes in the direction of sperm swimming that are characteristic of chemotaxis. Removal of progesterone (and other steroids) from FF by charcoal treatment abolished the sperm hyperactivation-like motility but not sperm chemotaxis. These results suggest that while progesterone might be a weak chemoattractant, it is not the major chemoattractant in FF. Progesterone probably causes human sperm accumulation mainly by inducing hyperactivation-like motility and, as a consequence, sperm trapping.

Much progress has been made in recent years in establishing mammalian sperm chemotaxis and understanding sperm capacitation. Thus far, chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime of 50 min to 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in humans) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the time during which capacitated spermatozoa are available in the female reproductive tract. The sperm chemoattractants have not been identified, but they appear to be heat-stable peptides. Although the molecular mechanism and the in vivo location of sperm chemotaxis are not known, a number of possible mechanisms and locations are discussed.

A key process in human fertilization is bringing the two gametes together, so that the complex molecular events involved in sperm and egg interaction can begin. Does nature allow fertilization to occur only as a consequence of a chance collision, or is there a precontact sperm-egg communication? This review summarizes the bioassays used in testing human spermatozoa for chemotaxis, emphasizing the necessity to distinguish between chemotaxis and other accumulation-causing processes, and the results obtained. It demonstrates that human sperm chemotaxis to a follicular factor(s) does occur, at least in vitro, and that only capacitated spermatozoa are chemotactically responsive. Substances that have been proposed as attractants for human spermatozoa are reassessed. The potential role of sperm chemotaxis in vivo is discussed. Faulty precontact sperm-egg communication may be one of the causes of male infertility, female infertility, or both. On the other hand, interfering with human sperm chemotaxis may represent an exciting new approach to contraception.

We describe a chemotactic-like response of Escherichia coli strains lacking most of the known chemotaxis machinery but containing high levels of the response regulator CheY. The bacteria accumulated in aspartate-containing capillaries, they formed rings on tryptone-containing semisolid agar, and the probability of counterclockwise flagellar rotation transiently increased in response to stimulation with aspartate (10^-10-10^-5M; the response was inverted at > 10^-4M). The temporal response was partial and delayed, as was the response of a control wild-type strain having a high CheY level. α-Methyl-DL-aspartate, a non-metabolizable analogue of aspartate as well as other known attractants of E. Coli, glucose and, to a lesser extent, galactose, maltose and serine caused a similar response. So did low concentrations of acetate and benzoate (which, at higher concentrations, act as repellents for wild-type E. coli). Other tested repellents such as indole, Ni²⁺ and Co²⁺ increased the clockwise bias. These observations raise the possibility that, at least when the conventional signal transduction components are missing, a non-conventional chemotactic signal transduction pathway might be functional in E. coli. Potential molecular mechanisms are discussed.

The acrosome reaction (AR), an essential step for achieving mammalian fertilization, was recently introduced as a means of clinical evaluation of male fertility. However, most of the available techniques for acrosomal status assessment (except those employing electron microscopy) do not define whether the measurements represent partial or complete AR. We, therefore, performed a crossover investigation of the types of inducers and probes required for detecting partial or complete AR in human spermatozoa. The acrosomal status before and after stimulation with four AR inducers was evaluated after incubation for 3 h in capacitating conditions. We used a fluorescence-activated cell sorter with fluorescein isothiocyanate-conjugated monoclonal antibody CD46 (FITC-CD46) targeting the inner acrosomal membrane for detecting a complete AR, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) targeting the acrosomal content for detection of both partial and complete AR. Without stimulation or following stimulation with progesterone, follicular fluid (FF) or phorbol myristate ester (PMA), the AR could be detected with FITC-PSA but not with FITC-CD46. Following stimulation with the calcium ionophore A23187, the AR could be detected by both FITC-PSA and FITC-CD46. These results suggest that spontaneous AR as well as AR induced by progesterone, PMA and FF are partial. In contrast, the AR induced by A23187 is total, i.e. both partial and complete. These findings are valuable for both research and clinical purposes and are a step towards an international agreement on a standard test for human sperm AR, for which there is an urgent need.

Bacterial chemotaxis is the most studied model system for signaling by the widely spread family of two-component regulatory systems. It is controlled by changes in the phosphorylation level of the chemotactic response regulator, CheY, mediated by a histidine kinase (CheA) and a specific phosphatase (CheZ). While it is known that CheA activity is regulated, via the receptors, by chemotactic stimuli, the input that may regulate CheY dephosphorylation by CheZ has not been found. We measured, by using stopped-flow fluorometry, the kinetics of CheZ-mediated dephosphorylation of CheY. The onset of dephosphorylation was delayed by ~50 ms after mixing phosphorylated CheY (CheY~P) with CheZ, and a distinct overshoot was observed in the approach to the new steady state of CheY~P. The delay and overshoot were not observed in a hyperactive mutant CheZ protein (CheZ54RC) that does not support chemotaxis in vivo and appears to be constitutively active. CheZ activity was cooperative with respect to CheY~P, with a Hill-coefficient of 2.5. The observed delayed modulation of CheZ activity and its cooperativity suggest that the phosphatase activity is regulated at the level of CheY~P-CheZ interaction. This novel kind of interplay between a response regulator and its phosphatase may be involved in signal tuning and in adaptation to chemotactic signals.

Switching flagellar rotation from one direction to another is an essential part of bacterial chemotaxis. Fumarate has been shown to possess the capacity to restore to flagella of cytoplasm-free, CheY-containing bacterial envelopes the ability to switch directions and to increase the probability of reversal in intact cells. Neither the target of fumarate action nor the mechanism of function is known. To distinguish between the two potential targets of fumarate, the response regulator CheY and the flagellar switch-motor complex, we compared flagellar rotation between isogenic strains that lacked CheY and had either low or high levels of fumarate. The difference in the fumarate levels was due to a deletion of the genes encoding the enzymes that synthesize and metabolize fumarate; succinate dehydrogenase and fumarase, respectively. The strains were in a gutted background (i.e. a background deleted for the cytoplasmic chemotaxis proteins and some of the receptors), and switching was achieved by carrying out the measurements at 2.5°C, where it has been demonstrated that gutted cells switch spontaneously. The flagellar rotation of the strain with the highest level of fumarate was the most clockwise-biased and had the highest reversal frequency, indicating that fumarate is effective even in the absence of CheY. Fumarate reduced the free energy difference of the counterclockwise-to-clockwise transition and had no appreciable effect on the activation energy of this transition. Similar observations were made at room temperature, provided that intracellular CheY was present. In a wild-type background, both mutants made rings on semisolid agar typical of normal chemotaxis. Taken together, the results suggest that the target of fumarate is the switch-motor complex, that fumarate acts by increasing the probability of the clockwise state, and that a fumarate level as low as that found in succinate dehydrogenase mutants is sufficient for normal chemotaxis.

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counter-clockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.

Impressive progress has been made in understanding the mechanism of bacterial chemotaxis and function of the flagellar motor, but how the direction of rotation is reversed by the 'flagellar switch' - a central step in chemotaxis - remains obscure and calls for new experimental approaches.

A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY ~ P) and the switch of the flagellar motor, located at its base. The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise. The docking site of CheY ~ P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17 fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products. The CheY-binding ability of each of the FliM mutant proteins was determined by chemical crosslinking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM1OEG, bound CheY ~ P to a much lesser extent than did wild-type FliM. CheY ~ P-binding of the other mutant proteins was similar to wild-type FliM. To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FliM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY ~ P. It also competed with full-length FliM on CheY ~ P. These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding.

The acrosome reaction (AR) - an essential step in mammalian fertilization - can occur, according to the consensus, only in capacitated spermatozoa. In apparent contrast, recent reports have demonstrated that human spermatozoa incubated in vitro in an albumin-free medium and therefore believed to be non-capacitated do undergo the AR. With the aim of determining unequivocally whether or not capacitation is required for the AR and whether albumin is essential for capacitation, we compared the potential to undergo partial and complete AR (induced by phorbol myristate ester or by the Ca²⁺ ionophore A23187) between human spermatozoa incubated in a capacitating medium, albumin-free medium, and non-capacitating medium. The results clearly demonstrate that capacitation is, after all, a prerequisite for both partial and complete AR. Albumin, on the other hand, is essential only for acquiring the capacity to undergo complete, not partial AR.

Escherichia coli strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins. With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response. Thus, a response was observed when one of these enzymes was missing but not when both were absent.

Chemotaxis in bacteria is controlled by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein CheY. When phosphorylated, CheY binds to FliM, which is one of the proteins that constitute the 'gear box' (or 'switch') of the flagellar motor. Consequently, the motor shifts from the default direction of rotation, counterclockwise, to clockwise rotation. This biased rotation is terminated when CheY is dephosphorylated either spontaneously or, faster, by a specific phosphatase, CheZ. Logically, one might expect CheZ to act directly on FliM- bound CheY. However, here we provide direct biochemical evidence that, in contrast to this expectation, phosphorylated CheY (Chey~P), bound to FliM, is protected from dephosphorylation by CheZ. The complex between CheY~P and Flim was trapped by cross-linking with dimethylsuberimidate, and its susceptibility to CheZ was measured. CheY~P complexed with FliM, unlike free CheY~P, was not dephosphorylated by CheZ. However, it did undergo spontaneous dephosphorylation. Nonspecific cross-linked CheY dimers, measured as a control, were dephosphorylated by CheZ. No significant binding between CheZ and any of the switch proteins was detected. It is concluded that, in the termination mechanism of signal transduction in bacterial chemotaxis, CheZ acts only on free CheY~P. We suggest that CheZ affects switch-bound CheY~P by shifting the equilibrium between hound and free CheY~P.

CheZ is the phosphatase of the chemotactic response regulator, CheY. There are three conserved domains on CheZ. Here we determined the function of the C-terminal domain (residues 202-214). A truncated form of CheZ, missing residues 202-214, hardly bound to the phosphorylated form of CheY. Conversely, a peptide composed of the last 19 amino acid residues of CheZ (residues 196-214), generated by tryptic digestion, bound specifically to the phosphorylated form of CheY. This was demonstrated by both fluorescence depolarization of the peptide (labeled with fluorescein)and cross-linking. It is concluded that the conserved C-terminus of CheZ functions as a CheY- binding domain.

CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis. The mechanism by which the activity of CheZ is regulated is not known. We used cheZ mutants of Salmonella typhimurium, which had been isolated by Sockett et al. (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V. M., and Macnab, R. M. (1992) J. Bacteriol. 174, 795-806), for cloning the mutant cheZ genes, overexpressing and purifying their products. We then measured the phosphatase activity, binding to CheY and to phosphorylated CheY (CheY∼P), and CheY∼P-dependent oligomerization of the mutant CheZ proteins. While all the mutant proteins were defective in their phosphatase activity, they bound to CheY and CheY∼P as well as wild-type CheZ. However, unlike wild-type CheZ, all the four mutant proteins failed to oligomerize upon interaction with CheY∼P. On the basis of these and earlier results it is suggested that (i) oligomerization is required for the phosphatase activity of CheZ, (ii) the region defined by residues 141-145 plays an important role in mediating CheZ oligomerization and CheY∼P dephosphorylation but is not necessary for the binding to CheY∼P, (iii) the oligomerization and hence the phosphatase activity are regulated by the level of CheY∼P, and (iv) this regulation plays a role in the adaptation to chemotactic stimuli.

Earlier studies have suggested that CheZ, the phosphatase of the signaling protein CheY in bacterial chemotaxis, may be in an oligomeric state both when bound to phosphorylated CheY (CheY∼P) (Blat, Y., and Eisenbach, M. (1994) Biochemistry 33, 902-906) or free (Stock, A., and Stock, J. B. (1987) J. BacteriOl. 169, 3301-3311). The purpose of the current study was to determine the oligomeric state of free CheZ and to investigate whether it changes upon binding to CheY∼P. By using either one of two different sets of cross-linking agents, free CheZ was found to be a dimer. The formation of the dimer was specific, as it was prevented by SDS which does not interfere with cross-linking mediated by random collisions. The dimeric form of CheZ was confirmed by sedimentation analysis, a cross-linking-free technique. In the presence of CheY∼P (but not in the presence of non-phosphorylated CheY), a high molecular size cross-linked complex (90-200 kDa) was formed, in which the CheZ:CheY ratio was 2:1. The size of the oligomeric complex was estimated by fluorescence depolarization to be 4-5-fold larger than the dimer, suggesting that its size is in the order of 200 kDa. These results indicate that CheZ oligomerizes upon interaction with CheY∼P. This phosphorylation-dependent oligomerization may be a mechanism for regulating CheZ activity.

Bacterial chemotaxis, which has been extensively studied for three decades, is the most prominent model system for signal transduction in bacteria. Chemotaxis is achieved by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein, CheY. This protein is activated by a stimulus-dependent phosphorylation mediated by an autophosphorylatable kinase (CheA) whose activity is controlled by chemoreceptors. Upon phosphorylation, CheY dissociates from its kinase, binds to the switch at the base of the flagellar motor, and changes the motor rotation from the default direction (counter-clockwise) to clockwise. Phosphorylation may also be involved in terminating the response. Phosphorylated CheY binds to the phosphatase CheZ and modulates its oligomeric state and thereby its dephosphorylating activity. Thus CheY phosphorylation appears to be involved in controlling both the excitation and adaptation mechanisms of bacterial chemotaxis. Additional control sites might be involved in bacterial chemotaxis, e.g. lateral control at the receptor level, control at the motor level, or control by metabolites that link central metabolism with chemotaxis.

Fumarate restores to flagella of cytoplasm-free, CheY-containing envelopes of Escherichia coil and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemotaxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.

In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was ≃13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed. Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm cheroot axis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.

The flagellar motor of Escherichia coli (E. coli) is driven by a proton-motive force (PMF), hence it was of interest to determine whether the motor is symmetrical in the sense that it can be rotated by any polarity of PMF. For this purpose the cells had to be deenergized first. Conventional deenergization procedures caused irreversible loss of motility, presumably due to ATP-dependent degradative processes. However, E. coli cells deenergized by incubation with arsenate manifested a slow, reversible depletion of PMF. In this procedure there was a sufficiently long time window, during which a considerable proportion of the cells lost their motility and could be made to rotate again by an artificially-imposed PMF. The motors of these cells rotated in response to any PMF polarity, but positive and negative polarities rotated different sub-populations of cells and the direction was almost exclusively counterclockwise. The reason for the unidirectionality of the rotation was not the intervention of the chemotaxis system. A number of potential reasons are suggested. One is the arsenate effect on the motor function found previously [Margolin, Y., Barak, R. and Eisenbach, M. (1994) J. Bacteriol. 176, 5547-5549]. A possible interaction between arsenate and the motor is discussed.

Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, Δacs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild- type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (≤10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (≥25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A- dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs.

The purpose of this work was to study the changes in rotation rate of the bacterial motor and to try to discriminate between various sources of these changes with the aim of understanding the mechanism of force generation better. To this end Escherichia coli cells were tethered and videotaped with brief stroboscopic light flashes. The records were scanned by means of a computerized motion analysis system, yielding cell size, radius of rotation, and accumulated angle of rotation as functions of time for each cell selected. In conformity with previous studies, fluctuations in the rotation rate of the flagellar motor were invariably found. Employing an exclusively counterclockwise rotating mutant ("gutted" RP1091 strain) and using power spectral density, autocorrelation and residual mean square angle analysis, we found that a simple superposition of rotational diffusion on a steady rotary motion is insufficient to describe the observed rotation. We observed two additional rotational components, one fluctuating (0.040.6 s) and one oscillating (0.87 s). However, the effective rotational diffusion coefficient obtained after taking these two components into account generally exceeded that calculated from external friction by two orders of magnitude. This is consistent with a model incorporating association and dissociation of force-generating units.

When Salmonella typhimurium cells were allowed to swarm on either a minimal or complex semisolid medium, patterns of cell aggregates were formed (depending on the thickness of the medium). No patterns were observed with nonchemotactic mutants. The patterns in a minimal medium were not formed by a mutant in the aspartate receptor for chemotaxis (Tar) or by wild-type cells in the presence of α-methyl-D,L-aspartate (an aspartate analog), thus resembling the patterns observed earlier in Escherichia coli (E. O. Budrene and H. C. Berg, Nature [London] 349:630-633, 1991) and S. typhimurium (E. O. Budrene and H. C. Berg, Abstracts of Conference II on Bacterial Locomotion and Signal Transduction, 1993). Distinctively, the patterns in a complex medium had a different morphology and, more importantly, were Tar independent. Furthermore, mutations in any one of the genes encoding the methyl-accepting chemotaxis receptors (tsr, tar, trg, or tcp) did not prevent the pattern formation. Addition of saturating concentrations of the ligands of these receptors to wild-type cells did not prevent the pattern formation as well. A tar tsr tcp triple mutant also formed the patterns. Similar results (no negative effect on pattern formation) were obtained with a ptsI mutant (defective in chemotaxis mediated by the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system [PTS]) and with addition of mannitol (a PTS ligand) to wild-type cells. It therefore appears that at least two different pathways are involved in the patterns formed by S. typhimurium: Tar dependent and Tar independent. Like the Tar-dependent patterns observed by Budrene and Berg, the Tar-independent patterns could be triggered by H₂O₂, suggesting that both pathways of pattern formation may be triggered by oxidative stress.

Sperm capacitation is an essential process in fertilization. It apparently involves a large number of processes, the common denominator of which is that they donate to sperm the potential to undergo the acrosome reaction, i.e., to release proteolytic enzymes enabling sperm penetration through the egg coat. Although the phenomenon of capacitation has been known for more than 40 years, it is far from understood, and consequently, there is, as yet, no operational definition of it. The lack of an assay to identify capacitated spermatozoa is both the cause and the effect of this situation. Here we critically review the major changes that are thought to occur during sperm capacitation, and assess their potential use as markers for the identification of capacitated spermatozoa.

CheY is the response regulator of bacterial chemotaxis. Previously, we showed that CheY binds to the flagellar switch protein FliM and that this binding is increased upon phosphorylation of CheY [Welch, M., Oosawa, ?., Aizawa, S.-I., & Eisenbach, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 87878791], Here, we demonstrate that it is the phosphorylated conformation of CheY, rather than the phosphate group itself, that is recognized and bound by FliM. We found that subsequent to the phosphorylation of CheY, Mg²⁺ was not required for the binding of CheY to FliM. However, phosphorylation of CheY did cause a change in the coordination properties of Mg²⁺ in the acid pocket of the protein. This change in the coordination of Mg²⁺ required the presence of the absolutely conserved residue Lysl09. When Lysl09 was substituted by arginine, the resulting CheY protein was unable to adopt an active conformation upon phosphorylation, and the protein was not bound by FliM. Surprisingly, the CheY13DK mutant protein, which is active in vivo but cannot be phosphorylated in vitro, exhibited only a low level of FliM binding activity, suggesting that its ability to cause clockwise rotation in the cell is not due to a constitutively high level of FliM binding. On the basis of these findings, we propose a mechanism for CheY activation by phosphorylation.

Bacterial chemotaxis is accomplished by regulating the direction of flagellar rotation. The primary target of the control appears to be CheY, a diffusible clockwise-signal molecule which interacts with the switch at the base of the flagellar motor and causes clockwise rotation. The regulatory mechanism appears to be phosphorylation/dephosphorylation of CheY. Here we demonstrate that CheZ, which accelerates the dephosphorylation of CheY, binds to CheY (immobilized on CNBr-activated Sepharose beads), that the binding to phosphorylated CheY is higher by over 2 orders of magnitude than the binding to nonphosphorylated CheY, and that the binding to both the phosphorylated and nonphosphorylated forms of CheY is significantly higher in the presence of Mg²⁺. We also show that the mutant proteins CheY 13DK, CheY57DE, and CheY 109KR bind CheZ to the same extent as wild-type CheY. The extent of the binding of these mutant proteins was not, however, increased in the presence of acetyl phosphate, the phosphorylating agent. The results indicate that neither a conformation which has a clockwise-causing activity in vivo nor phosphorylation is sufficient, alone, for maximal binding of CheZ to CheY and that Mg²⁺ is required for the binding of these proteins as well as for the phosphorylation and dephosphorylation of CheY.

Recent studies have indicated that human spermatozoa respond to follicular fluid by attraction to chemotactic factor(s) in the fluid, accompanied by enhancement of motility and ultimately hyperactivation. In this study, we quantified the sperm response. We exposed spermatozoa to a gradient of a chemotactically active fraction of follicular fluid (denoted as 'the attractant') and separated the spermatozoa that accumulated in the attractant and those that did not. We thus obtained two subpopulations: one enriched with chemotactically responsive spermatozoa, and one deficient in such spermatozoa. The fraction of the responsive spermatozoa out of the total sperm population was 2-12% at any measured time point. With time, the responsive spermatozoa lost their ability to be attracted, while such activity was gradually acquired by the subpopulation originally deficient in responsive spermatozoa. These results indicate that the identity of responsive spermatozoa is continuously changing. If the in vitro results are representative of the physiological conditions in vivo, they imply that the role of sperm chemotaxis combined with enhanced motility may be to select capacitated spermatozoa and bring them to the egg. Such a mechanism may, over an extended period of time, increase the prospect that an egg will meet capacitated spermatozoa as soon as it ovulates.

Human spermatozoa accumulate in vitro in diluted follicular fluids obtained from follicles from which the eggs have been fertilized. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of follicular fluid, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that the sperm accumulation in follicular fluid was the result of both sperm chemotaxis and chemokinesis and eventually hyperactivation-like motility. We determined the optimal conditions for sperm accumulation, which involved sperm preincubation (possibly to induce sperm capacitation) and proper dilution of follicular fluid. In all the assays, the net accumulation was low, probably reflecting the chemotactic responsiveness of only a small fraction of the sperm population at any given time. We partially fractionated follicular fluid in a Centricon microconcentrator (Amicon, Danvers, MA) and by acetone precipitation, and found that at least one of the chemotactic factors is a small (< 10-kDa) molecule that is probably nonhydrophobic. This is the first time that sperm chemotaxis and chemokinesis in response to a follicular factor(s) in mammals has been established and has been distinguished from other processes that might cause sperm accumulation. The physiological significance of these findings is discussed.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellularly) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.

Here we report that atrial natriuretic peptide (ANP), a known activator of particulate guanylate cyclase, induces attraction and swimming speed enhancement of human spermatozoa in vitro. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of ANP, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that spermatozoa followed the gradient of ANP and accumulated in it. Speed enhancement was detected in the presence of ANP without a gradient. These observations suggest either that an ANP-like substance is the physiological attractant for human spermatozoa, or, more likely, that ANP directly affects guanylate cyclase in a manner similar to that caused by the physiological attractant.

Regulation of the direction of flagellar rotation is central to the mechanism of bacterial chemotaxis. The transitions between counterclockwise and clockwise rotation are controlled by a "switch complex" composed of three proteins (FliG, FliM, and FliN) and located at the base of the flagellar motor. The mechanism of function of the switch is unknown. Here we demonstrate that the diffusible clockwise-signal molecule, the CheY protein, binds to the switch, that the primary docking site is FliM, that the extent of CheY binding to FliM is dependent upon the phosphorylation level of CheY, and that it is unaffected by the other two switch proteins. This study provides a biochemical demonstration of binding of a signal molecule to the bacterial switch and demonstrates directly that phosphorylation regulates the activity of this molecule.

Phosphorylation of the chemotaxis protein CheY by its kinase CheA appears to play a central role in the process of signal transduction in bacterial chemotaxis. It is presumed that the role is activation of CheY which results in clockwise (CW) flagellar rotation. The aim of this study was to determine whether this activity of CheY indeed depends on the protein being phosphorylated. Since the phosphorylation of CheY can be detected only in vitro, we studied the ability of CheY to cause CW rotation in an in vitro system, consisting of cytoplasm-free envelopes of Salmonella typhimurium or Escherichia coli having functional flagella. Envelopes containing just buffer rotated only counterclockwise. Inclusion of CheY caused 14% of the rotating envelopes to go CW. This fraction of CW-rotating envelopes was not altered when the phosphate potential in the envelopes was lowered by inclusion of ADP together with CheY in them, indicating that CheY has a certain degree of activity even without being phosphorylated. Attempts to increase the activity of CheY in the envelopes by phosphorylation were not successful. However, when CheY was inserted into partially-lysed cells (semienvelopes) under phosphorylating conditions, the number of CW-rotating cells increased 3-fold. This corresponds to more than a 100-fold increase in the activity of a single CheY molecule upon phosphorylation. It is concluded that nonphosphorylated CheY can interact with the flagellar switch and cause CW rotation, but that this activity is increased by at least 2 orders of magnitude by phosphorylation. This increase in activity requires additional cytoplasmic constituents, the identity of which is not yet known.Errata:Page 1822. In column 2, line 11, 20 mg/mL should read20 ug/mL.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [¹⁴C]acetate under similar conditions, CheY became acetylated: [1-¹⁴C]acetate was as effective as [2-¹⁴C] acetate in labeling CheY, and ACS-dependent labeling of CheY by [α-³²P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.

Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged.

Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pili on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A^- S⁺ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A⁺ S^- mutants, i.e., strains capable of movement by the A- motility system only, and (iii) a retardation of streaming speed in the wild- type strain (A⁺ S⁺). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pili. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.

In creatures with external fertilization, e.g., metazoa, it is well established that there is precontact sperm-egg communication in the form of chemotaxis. An intriguing question is whether also in mammals, where fertilization is internal and the need for precontact sperm-egg communication is not self-evident, such a process occurs and what its physiological significance may be. Here we review the evidence related to such a process in mammals, evidence which suggests that sperm attraction to the ovulated egg may indeed occur. On the basis of the available data we propose a hypothesis, according to which a sperm population is heterogeneous with respect to its physiological state; some spermatozoa are at a physiological state ready for fertilizing an egg, while others are premature or overmature. According to the hypothesis this is a dynamic state; the population of fertilizing spermatozoa gradually loses its potency and, at the same time, other spermatozoa mature and acquire fertilizing ability. After ovulation, only the fertilizing spermatozoa are attracted to the egg, while the rest are either repelled or inhibited and thus prevented from reaching the egg. The potential significance of sperm-egg communication is discussed.

Spermatozoa normally encounter the egg at the fertilization site (in the Fallopian tube) within 24 hr after ovulation. A considerable fraction of the spermatozoa ejaculated into the female reproductive tract of mammals remains motionless in storage sites until ovulation, when the spermatozoa resume maximal motility and reach the fertilization site within minutes. The nature of the signal for sperm movement is not known, but one possible mechanism is attraction of spermatozoa to a factor(s) released from the egg. We have obtained evidence in favor of such a possibility by showing that human spermatozoa accumulate in follicular fluid in vitro. This accumulation into follicular fluid was higher by 30-260% than that observed with buffer alone and was highly significant (P < 10^-8). Not all of the follicular fluids caused sperm accumulation; however, there was a remarkably strong correlation (P < 0.0001) between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized. These findings suggest that attraction may be a key event in the fertilization process and may give an insight into the mechanism underlying early egg-sperm communication.

Wild-type Escherichia coli and Salmonella typhimurium cells, tethered to glass by their flagella, rotate with brief intermittent pauses, the prevalence of which is decreased by attractants and increased by repellents. By attaching latex beads to filaments of a S. typhimurium mutant having straight rather than helical flagella, it was established that the flagella on free cells also pause intermittently. Pausing is therefore an intrinsic feature of the motor and not an artifact associated with tethering. In tethered cells of wild-type strains and non-chemotactic mutants defective in transducers, chemotaxis proteins, or the flagellar switch, both the classical response to chemotactic stimuli (change in direction of rotation from counterclockwise to clockwise or vice versa), and the pausing response to such stimuli, were linked together. No separate signal for pausing was found. In comparing different strains under different stimulation conditions, it was found that cells that never reversed seldom if ever paused, while cells that reversed frequently paused frequently. It is suggested that pausing is the result of futile switching events. A modified description of tumbling and chemotaxis is provided in which pausing, as well as reversal, has a role. Suppression of reversals and pauses by attractant stimuli commonly resulted in an increase in the speed of counterclockwise rotation; this may be because of suppression of pauses or reversals that are too brief to be detected. The clockwise rotation rate of unstimulated cells, which commonly was faster than their counterclockwise rate, was not further increased by repellent stimuli. The rotation rate of any given cell under any given condition was found to fluctuate on all time-scales measured. The study also revealed that some of the common repellents of E. coli and S. typhimurium slow down or stop the motor; these effects are not mediated by the chemotaxis machinery or intracellular pH.

Bacteria swim by rotating their flagella, the rotation being due to a motor located at the base of each flagellum. In this paper the correlation between motor function and mode of swimming is reviewed, with special emphasis on recent data that indicate that the motor is a threestate device. Novel findings with regard to the motor function and bioenergetics are surveyed, and mechanisms are proposed to account for these findings.

A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system. This study addressed this question. The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles. Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO₄ decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP was sufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined. The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO₄) were retained in these cells. In a control experiment, the response of the attractant maltose, whose receptor is periplasmic, was lost. This indicates that these repellents are not sensed by periplasmic receptors. In view of this finding and the involvement of the MCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.

An in vitro approach to study bacterial motility and chemotaxis is described. The approach is based on a preparation of flagellated cell envelopes. The envelopes are prepared from bacteria by a penicillin treatment and subsequent osmotic lysis. When the envelopes are energized, their flagella rotate. The direction of rotation in wild type envelopes is counterclockwise. Inclusion of the CheY protein within the envelopes may restore clockwise rotation. The advantages and disadvantages of this approach are pointed out. 1988 Deutsche Botanische Gesellschaft/German Botanical Society

When bacterial cells are tethered to glass by their flagella, many of them spin. On the basis of experiments with tethered cells it has generally been thought that the motor which drives the flagellum is a two-state device, existing in either a counterclockwise or a clockwise state. Here we show that a third state of the motor is that of pausing, the duration and frequency of which are affected by chemotactic stimuli. We have recorded on video tape the rotation of tethered Escherichia coli and Salmonella typhimurium cells and analyzed the recordings frame by frame and in slow motion. Most wild-type cells paused intermittently. The addition of repellents caused an increase in the frequency and duration of the pauses. The addition of attractants sharply reduced the number of pauses. A chemotaxis mutant which lacks a large part of the chemotaxis machinery owing to a deletion of the genes from cheA to cheZ did not pause at all and did not respond to repellents by pausing. A tumbly mutant of S. typhimurium responded to repellents by smooth swimming and to attractants by tumbling. When tethered, these cells exhibited a normal rotational response but an inverse pausing response to chemotactic stimuli: the frequency of pauses decreased in response to repellents and increased in response to attractants. It is suggested that (i) pausing is an integral part of bacterial motility and chemotaxis, (ii) pausing is independent of the direction of flagellar rotation, and (iii) pausing may be one of the causes of tumbling.

When cells of the bacterium Salmonella typhimurium are incubated with penicillin and lysed in a dilute buffer, flagellated cytoplasm-free envelopes are formed. When the envelopes are tethered to glass by their flagella and then energized, some of them spin. The direction of rotation of wild-type envelopes is exclusively counterclockwise (CCW). We perturbed this system by including in the lysis medium (and hence in the envelopes) the chemotaxis protein CheY. As a result, some of the envelopes rotated exclusively clockwise (CW). The fraction of envelopes that did so increased with the concentration of CheY; at a concentration of 48 μM (pH 8), all functional envelopes spun CW. The fraction also increased with the pH of the lysis medium in the range 6.6-8.4. The results were the same in the presence of absence of intracellular Ca²⁺. Reconstituted envelopes failed to respond to chemotactic stimuli. None of them changed the direction of their rotation. However, when the intracellular pH was lowered to 6.6 or below, envelopes that spun CW stopped rotating, while envelopes that spun CCW continued to rotate. This pnenomenon was reversible. We conclude that CheY per se, without any additional free cytoplasmic mediators, interacts with a switch at the base of the flagellum to cause CW rotation.

To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis. The potential was clamped by valinomycin, a K+ -specific ionophore, in the presence of K+. Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions. It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential.

We have studied the interaction between flagellated cell envelopes from Escherichia coli and liposomes. Oligolamellar liposomes of ca. 0.45-ttm diameter, composed of azolectin, phosphatidylserine, and cholesterol at a molar ratio of 7:1:2, were prepared by freezing and thawing and subsequent extrusion through polycarbonate filters. These liposomes exhibited high entrapment capacity and low leak-iness. Liposome-cell envelope interaction was monitored flow cytometrically in a fluorescence-activated cell sorter with a fluorescent aqueous space marker and by a filtration assay with radiolabels for the lipid phase and the liposomal aqueous space. Maximal association of liposomes with the envelopes was observed in both assays after ca. 25 min at 30 °C. After such period of time, it seems that up to 200 liposomes (depending on the liposome to envelope ratio) were associated with a single cell envelope, as calculated from the radiotracer studies. Fluorometric measurements of the transfer of liposomal contents and the intermixing of membrane lipids indicated that at least 20% of the envelope-associated liposomes had delivered their content into the envelopes, possibly by fusion. Electron microscopic observations confirmed the transfer of liposome-encapsulated ferritin molecules into the cell envelopes. Our data suggest that liposomal carriers might be employed to deliver cytoplasmic, chemotaxis-related macromolecules into bacterial cell envelopes.

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli, and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable.

Galactose and other chemotactic attractants have been shown to trigger an apparent hyperpolarization in Escherichia coli (Eisenbach, M., 1982, Biochemistry, 21:68186825). The probe used to measure membrane potential in that study, tetraphenylphosphonium (TPP+), may respond also to surface-charge changes in the membrane. The distinction between true changes in membrane potential and changes in the surface charge of the membrane is crucial for the study of this phenomenon in bacterial chemotaxis. To distinguish between these parameters, we compared the response to galactose with different techniques: K+ distribution in the presence of valinomycin (measured with a K+-selective electrode), TPP+ distribution (measured with a TPP+-selective electrode) at different ionic strengths, absorbance changes of bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V), and fluorescence changes of three probes with different mechanisms of response. All the techniques revealed stimulation by galactose of transient hyperpolarization, of comparable magnitude. This indicates the involvement of ion currents rather than alterations of local surface properties.

The molecular nature of signal transduction in bacterial chemotaxis is virtually unknown. If the signal transduction is electrical in nature, an externally applied electric field should affect chemotactic behavior. We therefore studied the effect of an electromagnetically induced electric field on macroscopic assays of chemotaxis and motility of Escherichia coli. The electric field had opposing effects on these phenomena: it doubled motility, but inhibited chemotaxis by 70%. Controls for viability, for electrophoretic effects, and for other parameters that may affect chemotaxis, showed that this inhibition was specific for chemotaxis. These observations suggest that an electrical process may be involved in the chemotaxis machinery of E. coli. However, other interactions of the electric field with one or more of the membrane components of the chemotaxis machinery cannot be excluded.

Attractants, in the presence of respiration and ATPase inhibitors, stimulate a hyperpolarization in Escherichia coli [Eisenbach, M. (1982) Biochemistry 21, 68186825]. In order to examine whether this hyperpolarization is correlated with Chemotaxis, the effect of the attractant D-galactose and its analogues on the membrane potential of wild-type E. coli strains and some of their mutants was studied. The main observations were the following: (i) Wild-type cells became hyperpolarized by either galactose or its nonmetabolizable analogues, d-fucose and l-sorbose. (ii) A mutant defective in galactose metabolism became hyperpolarized by galactose. (iii) Inhibiting the galactose permease system did not prevent the hyperpolarization, rather it facilitated the observation of the hyperpolarization. (iv) Mutants unable to transport galactose via the methyl β-galactoside (Mgl) transport system but having normal Chemotaxis to galactose became normally hyperpolarized by d-fucose. (v) Mutants which cannot bind galactose were not hyperpolarized by galactose. (vi) The hyperpolarization in flaI mutants, in which the whole chemotaxis machinery is repressed, was reduced to 1215% of the hyperpolarization in the parent strains. (vii) Nonattractant sugars did not stimulate hyperpolarization. It is concluded that the hyperpolarization is the consequence of neither galactose metabolism nor transport but rather is correlated with galactose taxis.

We studied the adsorption of phage χ to various behavioral mutants (che mutants) of Escherichia coli having different swimming modes. Bacteriophage χ infects only bacteria with active flagella, and it was therefore of interest to examine whether the mode of swimming has an effect on the susceptibility of the bacteria to the phage. Neither the mode of swimming (smooth swimming or tumbling) nor the direction of flagellar rotation affected the degree of χ adsorption to the bacterial cells. Furthermore, the tumbling frequency, the rotation speed (tethered cells of all of the strains examined had the same average speed of rotation), the time proportion of rotation, and the reversal frequency were not important in determining susceptibility to χ. The only variable that influenced χ adsorption was the fraction of the population whose flagella rotated incessantly. A direct, linear correlation was found between χ adsorption and the fraction of unceasing rotation in each population. It seems, therefore, that an individual bacterium whose flagella pause periodically and briefly during rotation is not susceptible to irreversible adsorption of the phage. Pausing of rotation thus seems to be a new feature of motility that is prevalent especially in che mutants. It is concluded that irreversible χ adsorption can serve as a quantitative assay only for incessant flagellar rotation of E. coli.

The molecular nature of signal transduction in bacterial chemotaxis is virtually unknown. If the signal transduction is electrical in nature, an externally applied electric field should affect chemotactic behavior. We therefore studied the effect of an electromagnetically induced electric field on macroscopic assays of chemotaxis and motility of Escherichia coli. The electric field had opposing effects on these phenomena: it doubled motility, but inhibited chemotaxis by 70%. Controls for viability, for electrophoretic effects, and for other parameters that may affect chemotaxis, showed that this inhibition was specific for chemotaxis. These observations suggest that an electrical process may be involved in the chemotaxis machinery of E. coli. However, other interactions of the electric field with one or more of the membrane components of the chemotaxis machinery cannot be excluded.

Changes in membrane potential of Escherichia coli in response to addition of chemoattractants have been studied by several groups, but their observations and conclusions disagree [e.g., Szmelcman and Adler [Szmelcman, S., & Adler, J. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 43874391] and Snyder et al. [Snyder, M. A., Stock, J. B., & Koshland, D. E., Jr. (1981) J. Mol. Biol. 149, 241257]]. This study was undertaken to resolve the differences in these reports. The discrepancies were probably the consequence of differences in the energy level of the bacteria, caused by differences in the availability of oxygen. In the presence of oxygen the relatively small changes in membrane potential that may be correlated with Chemotaxis could be masked and compensated for by the changes in membrane potential caused by respiration and the related electrogenic transport processes. In the present study the contribution of the respiratory and related systems was reduced by using electron transport inhibitors such as KCN or amytal or 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO). Membrane potential was then monitored by tetraphenylphosphonium distribution in response to the addition of a chemoeffector stimulus. Addition of the chemoattractant galactose caused an increase in the membrane potential only if respiration was inhibited. This hyperpolarization was not caused by ATP hydrolysis since N,N-dicyclohexylcarbodiimide (DCCD), an ATPase inhibitor, did not prevent it but rather increased it. The inhibitors used did not abolish the motility or the chemotactic response of the bacteria. Nine other attractants, metabolizable and nonmetabolizable chemicals, were tested under these conditions. All caused hyperpolarization, independent of the receptor identity or the Chemotaxis focusing system with which they interact. The significance of these results and of earlier works in light of the present observations is discussed. This study neither proves nor disproves the possible correlation between the observed hyperpolarization and the process of Chemotaxis. It describes the conditions that enable the detection of these changes in membrane potential.

Our aim was to isolate from bacteria a flagellated, subcellular system whose content could be changed at will. Because the control of bacterial chemotaxis resides in the direction of rotation of the flagella, such a system would be ideal for the study of this control mechanism. By incubating bacteria with penicillin and then lysing them osmotically, we were able to isolate cell envelopes from Escherichia coli and Salmonella typhimurium. These envelopes have the same sidedness and similar shape and dimensions as the original bacteria; they are practically free of cytoplasm; they are osmotically sensitive, having intact the cytoplasmic membrane and at least part of the cell wall; and they have flagella. This preparation was used to find out what is required to restore flagellar rotation, which had been lost during osmotic lysis. By visualizing the image of individual flagella with high intensity light microscopy or by tethering the cell envelopes, we found that adding artificial electron donors as an energy source is enough to restore rotation. This seems to indicate that no cytoplasmic components are required and that the proton electrochemical potential is indeed the driving force for flagellar rotation. However, the rotation was almost entirely counterclockwise, while in intact bacteria the flagella rotate in both directions. This may indicate that a cytoplasmic component is required to allow clockwise rotation. The significance of these results for the study of chemotaxis is discussed.

We carried out spectral studies on the interaction between purple membrane fragments (isolated from Halobacterium halobium) and a series of different solvents, classified quantitatively according to their solubility parameters δ_d, δ_p, δ_h. These represent the contribution of dispersion forces, polar forces, and hydrogen bonding, respectively, to the cohesive energy density of the solvent. Purple membrane fragments, kept in the dark, were suspended in each of the solvents as well as in binary mixtures of solvents, and the spectrum of the resulting suspension was recorded in the wavelength region 250-700 nm. The interaction of each solvent with the membrane fragments can be represented by a point on either a ternary diagram, where each of the three axes represents one of the solubility parameters, or a binary diagram, where one of the two axes is a combination of two of the solubility parameters ( δ v= δ d 2+ δ p 2 or δ p 2+ δ h 2). In the former type of solvent map the contribution of each of the parameters is distinct but only their relative contributions are expressed. In the latter the absolute values of δ_i are considered. In each of these modes of presentation an inner closed region is observed. The solvents inside its borders interact with bacteriorhodopsin with a resultant spectral change. Mixtures of solvents fit the maps according to their calculated δ values. Thus, a mixture of an apolar solvent with a highly polar solvent interacts with bacteriorhodopsin, even though each of these solvents alone does not.

Using the solubility parameter mapping technique (Eisenbach, M., Caplan, S.R. and Tanny, G. (1979) Biochim. Biophys. Acta 554, 269-280) we studied spectroscopically the mode of interaction between the purple membrane of Halobacterium halobium and pure organic solvents or solvent mixtures. Although the interacting solvents formed a well-defined closed region in the interaction maps, mapping the modes of interaction did not reveal a closed region for each spectrally classifiable type. A suggested interpretation for this is that interaction with the purple membrane chromophore requires that a solvent (or solvent mixture) possess apolar groups in order to obtain access to the chromophore, together with a polar character and hydrogen-bonding capacity. The mode of interaction, however, is dependent on the specificity of the reactive group of the solvent for retinal, and this has nothing to do with membrane properties. We also examined the influence of the duration of the interaction and of illumination. Some solvents appeared to react more sluggishly than others, but no generalization in terms of the solubility parameter mapping was found, probably because the map describes thermodynamic rather than kinetic phenomena. The only effect of illumination was to enhance the reaction of some of these solvents. It did not change the solubility parameters of purple membrane.

The kinetics of light-induced acidification and of the subsequent dark-induced alkalization in suspensions of sub-bacterial particles of Halobacterium halobium may be expressed as the sum of two exponentials, indicating two processes (Eisenbach, M., Bakker, E.P., Korenstein, R. and Caplan, S.R. (1976) FEBS Lett. 71, 228-232). We studied the effects of carbonyl cyanide p-trifluoromethoxy phenylhydrazone, nigericin, gramicidin D, valinomycin, and monactin on the extents and the rate constants of the two processes. The various ionophores affected the two processes differently and in general the slower process was more sensitive to their presence. Valinomycin and monactin had relatively minor effects, apparently due to the high ionic strength of the suspension. When an artificial membrane potential was created in the dark, the light-induced acidification was preceded by a transient alkalization as is usually observed in intact cells. These results are discussed in the light of a suggested model accounting for the two processes (Caplan, S.R., Eisenbach, M., Cooper, S., Garty, H., Klemperer, G. and Bakker, E.P. (1977) in Bioenergetics of Membranes (Packer, L., Papageorgiou, G.C. and Trebst, A., eds.), pp. 101-114, Elsevier/North-Holland Biomedical Press, Amsterdam), taking into account the different selectivities of the ionophores applied.

A simple technique for electrophoresis of particles is presented. The technique is based on running charged particles in a vertical tube along a sucrose gradient (20-50%). Purple membrane fragments from Halobacterium halobium were used to demonstrate the method. The migration of the fragments was linear with time in the region of 20 to 40% sucrose. Electrophoresis of purple membrane fragments under illumination, darkness, or darkness interrupted by short periods of illumination showed that at pH 4.5 the dark-adapted form of bacteriorhodopsin is less negative than its light-adapted form. At pH 6.5 and 8.5 no difference between these forms could be detected.

This chapter focuses on the proton pump, attempts to construct a self-consistent picture of its mode of action, and assesses recent experimental findings in terms of its function. It discusses that purple-patched bacteria belong to the family of extreme halophiles, Halobacteria, and usually grows aerobically in concentrated brine. The development of the purple color, for which illumination is necessary, occurs only when the bacteria are grown essentially in the absence of air. The most thoroughly studied halophilic purple bacterium is halobacterium halobium. A detailed discussion of the modus Vivendi and culture of these bacteria has been discussed. The bacteria owe their purple hue to the presence of a rhodopsinlike membrane protein, bacteriorhodopsin (bR). This protein constitutes the light-driven proton pump.

The polar lipids of the purple membrane were exchanged for different phosphatidylcholine species. The resulting complexes had the same protein to lipid-phosphorus ratio as the natural membrane, but only about 0.5-1.0 mole of original lipid was still present per mole of bacteriorhodopsin. In such complexes the bacteriorhodopsin photocycle is slowed down 10-20 times, but the strong protein-protein interaction is not abolished. Due to the slow rate of the photocycle we were able to measure in the light the ratio between net proton release and net accumulation of the last intermediate of the photocycle, the unprotonated M412. This ratio was not constant and equal to 1.0, as expected for a single deprotonation reaction, but varied with pH from 1.5 to 0.4. The variable ratio suggests that light-induced conformational changes occur in the nonchromophore part of the protein, which shift the pKa values of unidentified groups so as to cause binding or release of additional protons. A similar conclusion was drawn from experiments on the kinetics of proton transfer by bacteriorhodopsin in subbacterial particles of Halobacterium halobium and in reconstituted bacteriorhodopsin proteoliposomes. However, in this case light-induced association and dissociation of additional protons occurs simultaneously on different sides of the membrane.

Illumination of a suspension of subbacterial particles from Halobacterium halobium or of bacteriorhodopsin-containing proteoliposomes led to acidification or al-kalinization, respectively, of the suspending medium. Both processes were reversible upon turning the light off. Each of the \u201con\u201d and \u201coff\u201d reactions in either of the preparations fitted kinetically a sum of two exponentials, the first phase being faster than the second. The kinetics were the same whether measured electrometrically with a pH electrode or fluorime-trically with the impermeant pH indicator fluorescein iso-thiocyanate-dextran. The action spectrum of each of the phases overlapped the absorption spectrum of bacteriorhodopsin. The extents of both phases were too large to be accounted for stoichiometrically by the amount of the retinylidene-lysine Schiff base that was deprotonated. The two phases were compared with respect to the effects of altering pH, temperature, and the concentration of a permeant cation. The extent of the slow hase increased considerably either with decreasing the pH or with increasing the temperature or concentration of triphenylmethylphosphonium ion (TPMP+). The extent of the rapid phase, on the other hand, increased only slightly with decreasing pH, decreased with increasing temperature (in the \u201con\u201d reaction only), and was unaffected by TPMP+. Furthermore, the increased acidification subsequent to the addition of TPMP+ to subbacterial particles during the steady state in the light followed monophasic first-order kinetics, with a rate constant typical of the slow phase. Various interpretations of the observed kinetics are considered. The interpretation which seems to be in accord with the experimental results is that the slow phase represents net proton transport across the membrane, and the rapid phase represents a proton dissociation-association reaction.

The light-driven energy-converting system of Halobacterium halobium is the simplest biological energy converter known so far; consequently this bacterium has become a prime object of research in the field of bioenergetics. Experimental data on light-induced ion transport published recently are summarized and explained on the basis of this system.

Light-induced Na⁺ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na⁺ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na⁺ efflux, the inhibition being complete at low light intensity. The Na⁺ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na⁺ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na⁺ transport. Proton influx in the dark followed the artificial formation of a sodium gradient ([Na⁺]_in > [Na⁺]_out). These results may be explained by a Na⁺/H⁺ antiport mechanism. The fluxes of Na⁺ and H⁺ were of comparable magnitude, but the initial rate of Cl^- efflux in the same experiment was one-third of the initial rate of Na⁺ efflux. Consequently Cl^- is not regarded as a participant in the Na⁺ efflux mechanism.

In the presence of KCN and a saturating concentration of antimycin the reduction of the btype cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:b_A the active form which was reduced in the rapid phase, and b_s the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, named \u201cY\u201d, located between cytochromes b and c₁. Pretreatment with ascorbate plus N,N,N¹,N¹tetramethylpphenylenediamine transforms all the btype cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name \u201cdynamic control mechanism\u201d was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107116]. Increasing concentrations of antimycin (02 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of \u201cY\u201d and thus permits the observation of the biphasic phenomenon, but that it is not essential for the operation of this dynamic control mechanism. Substituting antimycin with 2heptyl4hydroxyquinoline Noxide (HpHOQnO) as an inhibitor, did not change significantly the pattern of the kinetics; i.e. the reduction was still biphasic. Omission of KCN from the reaction mixture or pretreatment with ascorbate plus tetramethylphenylenediamine did not affect the kinetics of the reduction (in contrast to the experiments with antimycin as inhibitor). From these phenomena it is concluded that the inhibition sites of antimycin and HpHOQnO are not identical. Antimycin is located on the substrate side of \u201cY\u201d and HpHOQnO is located on its oxygen side. This assumption is supported by measuring the effect of an oxidant, added in the presence of antimycin or HpHOQnO, on the rate of the reduction of cytochrome b. After aerobic reduction of submitochondrial particles by succinate in the presence of KCN, either antimycin or HpHOQnO was added, followed by ferricyanide. With antimycin, a rapid reduction was observed after the addition of the oxidant, followed by a slow reaction. In the presence of HpHOQnO, the addition of ferricyanide did not accelerate the reduction. On the contrary it induced a rapid oxidation of cytochrome b followed by a slow monophasic reduction. If the order of the additions was reversed, i.e. first K₃Fe(CN)₆ and then either antimycin or HpHOQnO, a biphasic reduction was observed in either case. It is suggested, that a redox equilibrium exists between \u201cY\u201d and cytochrome c₁, and that this equilibrium is sensitive to HpHOQnO but not to antimycin.

In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic firstorder kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of cytochromes c + c₁ and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a falloff in the rate of cytochrome b reduction. The biphasic reduction of cytochrome b was observed over a wide temperature range (0 30°C), with succinate of NADH as electron donors and with phosphorylating particles or coupled ratheart mitochondria. With ratheart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide ptrifluoromethoxyphenylhydrazone or oligomycin. In both the rapid and the slow phases, the rate of reduction of cytochrome b561 was equal to that of b565. Thus both cytochromes b561 and b565 were affected by the mechanism which determined the reductionrate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by the experimental conditions. When antimycin was used as the only inhibitor, 96% of the btype cytochromes were reduced in the rapid phase. If the c and atype cytochromes were first reduced by ascorbate and tetramethylpphenylenediamine in the presence of KCN and antimycin, all the btype cytochromes were fully reduced at the slowrate. With succinate, the rate of the rapid phase depended on the activation level of the succinicdehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH₂) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH₂ or durohydroquinone. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.

The lightinduced absorbance changes of electron transport carriers were measured in intact (class I) chloroplasts which catalyzed oxygen evolution with phosphoglyceric acid or CO₂ as electron acceptors. In these chloroplasts the reduction of cytochrome b₅₆₃, induced by light at 720 nm, increased in the presence of ADP or in the presence of uncouplers such as gramicidin and NH₄Cl. In the presence of ADP there was a decrease in the lightinduced reduction of C550 and in the oxidation of cytochrome f. The effect of ADP was augmented when phosphoglyceric acid was used as an electron acceptor in class I chloroplasts. A decrease in the photoreduction of C550 was also observed in the presence of the uncoupler gramicidin in class I and class II, with benzylviologen as an electron acceptor, and in class II chloroplasts with ferricyanide. The uncoupler gramicidin caused an increase in the lightinduced reduction of cytochrome b₅₆₉. Based on these effects of ADP or uncoupler on the steady state of the lightinduced redox level of the carriers, we proposed sites along the electron transport chain in which the rate of electron flow is controlled by the energy conversion system.

In acid aqueous solutions, ferric ions and NADH were found to react rapidly to form a blue charge-transfer complex with a stoichiometry of Fe³⁺(NADH)₂. This complex is unstable and decomposes in a redox reaction with Fe²⁺ and NAD⁺ as products. The formation of the complex was studied under similar conditions by the stopped-flow technique and found to be a two-step reaction (see eq 1 and 2 of text). The rate constants of these two reactions were calculated from both kinetics and equilibrium studies. Within experimental error the results obtained by the two methods were the same (k₁ = (5.0 ± 0.2) × 10³ m¹ sec¹, k¹ = 0.6 ± 0.1 sec¹; k₂ = (1.30 ± 0.05) × 10₃ M¹ sec¹, k₂ = 0.03 ± 0.01 sec¹). The relatively small magnitude of k₂ is the explanation for the complete conversion of Fe³⁺ into the Fe³⁺(NADH)₂ complex. The two complexes have similar absorption spectra with a maximum at 540 nm, except that the extinction coefficient of the Fe³⁺(NADH) is lower than that of Fe³⁺(NADH)₂. The properties of the Fe³⁺(NADH) complex are discussed.