RESUMEN
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 motor's 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.
Asunto(s)
Escherichia coli/fisiología , Flagelos/metabolismo , Locomoción/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas Bacterianas , Proteínas de Escherichia coli , Unión Proteica/fisiologíaRESUMEN
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 7.5â¯Å resolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P21, merged to 3.35â¯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.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , Oxidorreductasas/química , Dominios Proteicos , Sitios de Unión , Dominio Catalítico , Cristalografía , Cristalografía por Rayos X , Proteínas de Escherichia coli/metabolismo , Ligandos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Oxidorreductasas/metabolismo , RotaciónRESUMEN
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.
Asunto(s)
Complejo II de Transporte de Electrones/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Complejo II de Transporte de Electrones/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleótido/genéticaRESUMEN
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 ß4α4 loop, shifted toward an active state upon mutation. Removal of ß4α4 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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Acetatos/metabolismo , Acetilación , Quimiotaxis , Escherichia coli/química , Proteínas de Escherichia coli , Lisina/metabolismo , Espectrometría de Masas , Proteínas Quimiotácticas Aceptoras de Metilo , Simulación de Dinámica Molecular , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Thermotoga maritima/metabolismoRESUMEN
STUDY QUESTION: What is the behavioral mechanism underlying the response of human spermatozoa to a temperature gradient in thermotaxis? SUMMARY ANSWER: Human spermatozoa swim up a temperature gradient by modulating their speed and frequencies of hyperactivation events and turns. WHAT IS KNOWN ALREADY: Capacitated 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, DURATION: Human 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, METHODS: The 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 CHANCE: A 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 3-10 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 CAUTION: The 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 FINDINGS: This 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. STUDY FUNDING/COMPETING INTERESTS: The authors have no conflict of interest and no funding to declare.
Asunto(s)
Capacitación Espermática/fisiología , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Temperatura , Flagelos/fisiología , Voluntarios Sanos , Humanos , Masculino , Microscopía de Contraste de Fase , Microscopía por Video , ViscosidadRESUMEN
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 extracts-tri-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.
Asunto(s)
Opsinas , Retinaldehído , Espermatozoides , Vitamina A , Masculino , Animales , Espermatozoides/metabolismo , Espermatozoides/fisiología , Ratones , Opsinas/metabolismo , Humanos , Retinaldehído/metabolismo , Vitamina A/metabolismo , Taxia/fisiología , Motilidad Espermática/fisiología , IsomerismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Quimiotaxis , Microscopía por Crioelectrón , Flagelos , Salmonella typhimurium , Flagelos/ultraestructura , Flagelos/fisiología , Flagelos/metabolismo , Salmonella typhimurium/ultraestructura , Salmonella typhimurium/fisiología , Salmonella typhimurium/metabolismo , Salmonella typhimurium/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Rotación , Modelos Moleculares , Sitios de Unión , Torque , Conformación Proteica , Proteínas de la MembranaRESUMEN
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.
Asunto(s)
Escherichia coli/metabolismo , Flagelos/metabolismo , Genes de Cambio , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Escherichia coli/ultraestructura , Flagelos/enzimología , Flagelos/ultraestructura , Fumaratos/metabolismo , Eliminación de Gen , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Succinato Deshidrogenasa/aislamiento & purificación , Succinato Deshidrogenasa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis , Escherichia coli/fisiología , Proteínas de la Membrana/metabolismo , Acetilación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Histidina Quinasa , Lisina/genética , Lisina/metabolismo , Proteínas de la Membrana/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Datos de Secuencia MolecularRESUMEN
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, Ca(2+) concentration caused remarkable inhibition of the thermotactic response. The thermotactic response was also inhibited by each of the following: La(3+), a general blocker of Ca(2+) channels; U73122, an inhibitor of phospholipase C (PLC); and 2-aminoethoxy diphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate receptors (IP(3)R) 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 IP(3)R Ca(2+) channel, located on internal Ca(2+) stores, operates in sperm thermotaxis, and that the response is mediated by PLC and requires intracellular Ca(2+). They also suggest that the thermosensors for thermotaxis are not the currently known chemotaxis receptors.
Asunto(s)
Movimiento Celular/fisiología , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Espermatozoides/fisiología , Temperatura , Fosfolipasas de Tipo C/fisiología , Técnicas Biosensibles , Calcimicina/farmacología , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Movimiento Celular/efectos de los fármacos , Separación Celular/instrumentación , Separación Celular/métodos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ionóforos/farmacología , Masculino , Modelos Biológicos , Análisis de Semen/instrumentación , Análisis de Semen/métodos , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/fisiología , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
Asunto(s)
Rodopsina/metabolismo , Opsinas de Bastones/metabolismo , Transducción de Señal , Espermatozoides/citología , Espermatozoides/metabolismo , Taxia , Animales , Masculino , RatonesRESUMEN
BACKGROUND: A 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. METHODS: To 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. RESULTS: The 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. CONCLUSION: These 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.
Asunto(s)
Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Nucleótidos de Purina/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Aldehídos/farmacología , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Modelos Biológicos , Progesterona/farmacología , Programas Informáticos , Capacitación Espermática , Espermatozoides/fisiología , Estimulación Química , Factores de TiempoRESUMEN
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 chi2 tests of the very large sample of measured angles, where the critical chi2 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.
Asunto(s)
Quimiotaxis , Mamíferos/fisiología , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Animales , Factores Quimiotácticos/fisiología , Escherichia coli/metabolismo , Calor , Humanos , Masculino , Modelos Biológicos , Modelos Estadísticos , Oportunidad Relativa , Capacitación Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
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 FliG-one 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 (FliGcw). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdA-FliGcw 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 FliGcw. 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 FliGcw. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG0 between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.
Asunto(s)
Bacterias/metabolismo , Flagelos/metabolismo , Fumaratos/metabolismo , Rotación , Succinato Deshidrogenasa/metabolismo , Anaerobiosis , Animales , Proteínas Bacterianas/química , Bovinos , Colorantes Fluorescentes/metabolismo , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
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.
Asunto(s)
Quimiotaxis/fisiología , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Factores Quimiotácticos/química , Factores Quimiotácticos/metabolismo , Escherichia coli/fisiología , Femenino , Flagelos/fisiología , Humanos , Masculino , Modelos Biológicos , Modelos Moleculares , Oocitos/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Transducción de Señal , Espermatozoides/fisiologíaRESUMEN
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 delta acs 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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Quimiotaxis , Coenzima A Ligasas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Ciclo del Ácido Cítrico , Coenzima A Ligasas/genética , Proteínas de Escherichia coli/genética , Histidina Quinasa , Proteínas de la Membrana/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Peso MolecularRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Quimiotaxis , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Escherichia coli/fisiología , Proteínas de Escherichia coliRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Quimiotaxis , Escherichia coli/citología , Escherichia coli/fisiología , Acetilación/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Flagelos/efectos de los fármacos , Flagelos/metabolismo , Metilación/efectos de los fármacos , Movimiento/efectos de los fármacos , N-Metilaspartato/farmacologíaRESUMEN
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.