Recognition sites for chemotactic repellents of Bacillus subtilis.

Repellents of Bacillus subtilis include many membrane-active compounds, such as uncouplers of oxidative phosphorylation, local anesthetics, chlorpromazine (a central nervous system depressant), and tetraphenylboron (a lipophilic anion). Normally, bacteria swim smoothly, and occasionally tumble, but addition of repellent causes all bacteria to tumble, then later resume original frequency of swimming and tumbling (adaptation). Bacteria adapted to repellent can then be tested to determine the minimum concentration (threshold) of the same or different repellents that causes tumbling. The results indicate that repellents act at (saturable) recognition sites, which differ for chemically different species. An implication is that uncouplers of oxidative phosphorylation affect cell properties by interaction at specific locations.     

A biochemical mechanism for bacterial chemotaxis

Peritrichous bacteria alternately swim and tumble (thrash about with little forward progress). By selective modulation of tumbling frequency, these bacteria carry out chemotaxis, which is migration to higher concentrations of attractant or lower concentrations of repellent. A model for chemotaxis is presented in which tumbling frequency is regulated by concentration of Ca²⁺ ion at the switch that controls tumbling and swimming. Attractants cause decreased levels of free cytoplasmic Ca²⁺ ion due to binding of Ca²⁺ ion by specific proteins. This Ca²⁺ ion is released when these proteins become methylated. An alternative model. involving a cytoplasmic metabolite “compound X”, is discussed.     

A model of excitation and adaptation in bacterial chemotaxis.

We present a model of the chemotactic mechanism of Escherichia coli that exhibits both initial excitation and eventual complete adaptation to any and all levels of stimulus ("exact" adaptation). In setting up the reaction network, we use only known interactions and experimentally determined cytosolic concentrations. Whenever possible, rate coefficients are first assigned experimentally measured values; second, we permit some variation in these rate coefficients by using a multiple-well optimization technique and incremental adjustment to obtain values that are sufficient to engender initial response to stimuli (excitation) and an eventual return of behavior to baseline (adaptation). The predictions of the model are similar to the observed behavior of wild-type bacteria in regard to the time scale of excitation in the presence of both attractant and repellent. The model predicts a weaker response to attractant than that observed experimentally, and the time scale of adaptation does not depend as strongly upon stimulant concentration as does that for wild-type bacteria. The mechanism responsible for long-term adaptation is local rather than global: on addition of a repellent or attractant, the receptor types not sensitive to that attractant or repellent do not change their average methylation level in the long term, although transient changes do occur. By carrying out a phenomenological simulation of bacterial chemotaxis, we find that the model is insufficiently sensitive to effect taxis in a gradient of attractant. However, by arbitrarily increasing the sensitivity of the motor to the tumble effector (phosphorylated CheY), we can obtain chemotactic behavior.     

Chemosensory and thermosensory excitation in adaptation-deficient mutants of Escherichia coli

Methyl-accepting chemotaxis protein-methyltransferase-deficient mutants, cheR mutants, of Escherichia coli showed a tumble response to repellents only at low temperatures, and the resultant tumbling lasted unless the condition was changed. The swimming pattern of the repellent-treated cells was different at different temperatures, indicating that the absolute temperature is a determinant of the tumbling frequency of those cells. The tumbling of those cells was also suppressed by the addition of attractants. Under a suitable repellent concentration, the tumbling frequency of the cells was found to be simply determined by the ligand occupancy of chemoreceptors for many attractants. In a methyl-accepting chemotaxis protein-methylesterase-deficient mutant, a cheB deletion mutant, the tumbling frequency was also determined by receptor occupancy of some attractants. These results indicate that in the adaptation-deficient mutants, sensory signals are produced in proportion to the amount of ligand-bound or of thermally altered receptors and transmitted to the flagellar motors without any modification. Thus, it is concluded that the adaptation system, namely, the methylation-demethylation system of methyl-accepting chemotaxis proteins, is not concerned with the step of chemosensory or thermosensory excitation. A simple model is proposed to explain how the swimming pattern of the adaptation-deficient mutants is determined.     

Tuning the responsiveness of a sensory receptor via covalent modification.

Down-regulation or adaptation of receptors is an essential part of the chemotaxis mechanism to sense gradients. Using localized mutagenesis it is shown that the covalent modification of the receptors makes a slight change in the binding constant (factor of 2) which is far too small to explain the adaptation. The modification does, however, alter the signaling dramatically, an increasing tumbling signal being correlated with increased covalent modification. Responses in the two extreme cases, namely, completely unmodified and completely modified receptor, occur at attractant concentrations separated by 2 orders of magnitude. Amidation of the regulatory glutamate residues causes essentially the same signaling change as methylation. Thus, adaptation in chemotaxis is due to modulation of the receptor's signaling properties, not its affinity for the chemoeffector.     

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

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

Effect of arsenate on chemotactic behavior of Escherichia coli.

Escherichia coli cells treated with arsenate cannot tumble. The relationship between cellular adenosine 5'-triphosphate (ATP) level and the ability to tumble has been studied. (i) Cells incubated with arsenate completely lost their tumbling ability, and the cellular ATP level was decreased to less than 0.3 nmol/mg of protein. (ii) Incubation with 10 mM arsenate-1 mM phosphate reduced the cellular ATP level to less than 0.25 nmol/mg of protein. However, the cells were still able to tumble. (iii) Tumbling of the arsenate-treated cells was completely recovered after addition of a slight amount of phosphate, although the ATP level was still as low as 0.2 nmol/mg of protein. (iv) The cellular ATP level of an arsenate-treated uncA mutant (Ca2+,Mg2+-adenosine triphosphatase defective) was lower than 0.1 nmol/mg of protein even after the addition of 5 5 mM phosphate. However, tumbling ability was almost completely restored upon addition of the phosphate.     

Effect of methionine on chemotaxis by Bacillus subtilis.

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

Roles of cheY and cheZ gene products in controlling flagellar rotation in bacterial chemotaxis of Escherichia coli.

To understand output control in bacterial chemotaxis, we varied the levels of expression of cellular cheY and cheZ genes and found that the overproduction of the corresponding proteins affected Escherichia coli swimming behavior. In the absence of other signal-transducing gene products, CheY overproduction made free-swimming cells tumble more frequently. A plot of the fraction of the population that are tumbling versus the CheY concentration was hyperbolic, with half of the population tumbling at 30 microM (25,000 copies per cell) CheY monomers in the cytosol. Overproduction of aspartate receptor (Tar) by 30-fold had a negligible effect on CheY-induced tumbling, so Tar does not sequester CheY. CheZ overproduction decreased tumbling in all tumbling mutants except certain flaAII(cheC) mutants. In the absence of other chemotaxis gene products, CheZ overproduction inhibited CheY-induced tumbling. Models for CheY as a tumbling signal and CheZ as a smooth-swimming signal to control flagellar rotation are discussed.     

Tumbling chemotaxis mutants of Escherichia coli: possible gene-dependent effect of methionine starvation.

Some mutants defective in chemotaxis show incessant tumbling behavior and are called tumbling mutants. Previously described tumbling mutations lie in two genes, cheB and cheZ (41.5 min on Escherichia coli map). Genetic analysis of various tumbling mutants, however, revealed that two more genetic loci, cheC (43 min) and cheE (99.2 min), could also mutate to produce tumbling mutants. The genetic map around cheC was revised: his flaP flaQ flaR flbD flaA (= cheC) flaE. flbD is a new gene. When cells were starved for methionine, the tumbling mutants changed their swimming behavior depending on the che gene mutated. cheZ mutants, like wild-type bacteria, ceased tumbling shortly after removal of methionine. The tumbling of cheB or cheE mutants was depressed after prolonged methionine starvation in the presence of a constant level of an attractant. cheC tumbling mutants appeared unique in that they did not cease tumbling even when cells were deprived of methionine. By contrast, arsenate treatment of the tumbling mutants resulted in smooth swimming of the cells in every case. These results suggest that two different processes are involved in regulation of tumbling; one requiring methionine and the other requiring some phosphorylated compound.     

Sensory adaptation and deadaptation by Bacillus subtilis.

Cells of Bacillus subtilis, when tethered by using antiflagellar antibody, rotate briefly counterclockwise (swimming behavior) or clockwise (tumbling behavior) when amino acids are added or removed, respectively. "Dissociation constants" for attractant-binding site interactions, calculated from duration of the rotational response to addition of amino acids, agreed with those calculated for their removal and with previous values calculated from sensitivity capillary assays. The ratio of adaptation times for addition versus removal of attractant averaged 1.7, which differs greatly from the value of 50 for Escherichia coli.     

Predicted Auxiliary Navigation Mechanism of Peritrichously Flagellated Chemotactic Bacteria

Chemotactic movement of Escherichia coli is one of the most thoroughly studied paradigms of simple behavior. Due to significant competitive advantage conferred by chemotaxis and to high evolution rates in bacteria, the chemotaxis system is expected to be strongly optimized. Bacteria follow gradients by performing temporal comparisons of chemoeffector concentrations along their runs, a strategy which is most efficient given their size and swimming speed. Concentration differences are detected by a sensory system and transmitted to modulate rotation of flagellar motors, decreasing the probability of a tumble and reorientation if the perceived concentration change during a run is positive. Such regulation of tumble probability is of itself sufficient to explain chemotactic drift of a population up the gradient, and is commonly assumed to be the only navigation mechanism of chemotactic E. coli. Here we use computer simulations to predict existence of an additional mechanism of gradient navigation in E. coli. Based on the experimentally observed dependence of cell tumbling angle on the number of switching motors, we suggest that not only the tumbling probability but also the degree of reorientation during a tumble depend on the swimming direction along the gradient. Although the difference in mean tumbling angles up and down the gradient predicted by our model is small, it results in a dramatic enhancement of the cellular drift velocity along the gradient. We thus demonstrate a new level of optimization in E. coli chemotaxis, which arises from the switching of several flagellar motors and a resulting fine tuning of tumbling angle. Similar strategy is likely to be used by other peritrichously flagellated bacteria, and indicates yet another level of evolutionary development of bacterial chemotaxis.     

Role of Methionine in Bacterial Chemotaxis

The effect of methionine starvation on the motility and behavior of normal and nonchemotactic mutants of Salmonella typhimurium was investigated. Methionine starvation eliminates tumbling in the wild type, but fails to do so in an uncoordinated (frequent tumbling) mutant. In the mutant, methionine starvation significantly extends the period of smooth motility that follows a sharp temporal increase of attractant. These results suggest that methionine metabolism is not tightly coupled to the generation of tumbles, but rather is necessary for the return of some tumble-regulating parameter to a steady-state level.     

Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol.

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.     

Excitatory signaling in bacterial probed by caged chemoeffectors.

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

Proton-motive force and the motile behavior of Bacillus subtilis

Changes in the proton-motive force cause a transient change in the motile behavior of Bacillus subtilis cells. Both an increase and a decrease in the proton-motive force cause transient tumbling. Simultaneous decrease of proton-motive force and increase of attractant concentration lessens the response toward the attractant. A simultaneous increase of proton-motive force and increase of attractant concentration prolonges the response toward attractant. A hypothesis explaining the various effects is given.Abbreviations KT medium potassium taxis medium - NaT medium sodium taxis medium - HT medium acidic taxis medium - OHT medium alkaline taxis medium - DNP 2,4-dinitrophenol     

Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase.

Strains of Escherichia coli lacking all cytoplasmic chemotaxis proteins except CheY swim smoothly under most conditions. However, they tumble when exposed to acetate. Acetate coenzyme A synthetase (EC 6.2.1.1) was thought to be essential for this response. New evidence suggests that the tumbling is mediated instead by acetate kinase (EC 2.7.2.1), which might phosphorylate CheY via acetyl phosphate. In strains that were wild type for chemotaxis, neither acetate coenzyme A synthetase, acetate kinase, nor phosphotransacetylase (EC 2.3.1.8) (and thus acetyl phosphate) was required for responses to aspartate, serine, or sugars sensed by the phosphotransferase system. Thus, acetate-induced tumbling does not appear to play an essential role in chemotaxis in wild-type cells.     

Chemotaxis in Escherichia coli proceeds efficiently from different initial tumble frequencies.

The relationships between the level of tumbling, tumble frequency, and chemotactic ability were tested by constructing two Escherichia coli strains with the same signaling apparatus but with different adapted levels of tumbling, above and below the level of wild-type E. coli. This was achieved by introducing two different aspartate receptor genes into E. coli: a wild-type (wt-tars) and a mutant (m-tars) Salmonella typhimurium receptor gene. These cells were compared with each other and with wild-type E. coli (containing the wild-type E. coli aspartate receptor gene, wt-tare). It was found that in spite of the differences in the adapted levels of tumbling, the three strains had essentially equal response times and chemotactic ability toward aspartate. This shows that the absolute level of the tumbling can be varied without impairing chemotaxis if the signal processing system is normal. It also appears that a largely smooth-swimming mutant may undergo chemotaxis by increasing tumbling frequency in negative gradients.     

Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologs.

We have characterized mutants in a novel gene of Bacillus subtilis, cheV, which encodes a protein homologous to both CheW and CheY. A null mutant in cheV is only slightly defective in capillary and tethered cell assays. However, a double mutant lacking both CheV and CheW has a strong tumble bias, does not respond to addition of attractant, and shows essentially no accumulation in capillary assays. Thus, CheV and CheW appear in part to be functionally redundant. A strain lacking CheW and expressing only the CheW domain of CheV is chemotactic, suggesting that the truncated CheV protein retains in vivo function. We speculate that CheV and CheW function together to couple CheA activation to methyl-accepting chemotaxis protein receptor status and that possible CheA-dependent phosphorylation of CheV contributes to adaptation.     

Orientation in two dimensions: chemosensory motile behaviour of Euplotes vannus

Most studies on chemosensory motile behaviour of protists apply to free-swimming species moving in 3-dimensional space. But many protists are associated with surfaces and this excludes the use of helical klinotaxis for orientation in chemical gradients. It is here shown that the predominantly surface-dwelling ciliated protozoon Euplotes vannus orients itself in chemical gradients by simple temporal gradient sensing (equivalent to the run and tumble mechanism described for bacteria) and they react only to a temporal decrease in attractant concentration. The motility of Euplotes can be described as a classical 2-dimensional random walk with Poisson distribution of run lengths punctuated by random changes in walking direction. The chemosensory motile behaviour allows cells that are distributed within about 1 cm² to accumulate at point sources of an attractant within 3–4 min.