A simple expression for quantifying bacterial chemotaxis using capillary assay data: application to the analysis of enhanced chemotactic responses from growth-limited cultures.

An individual cell-based mathematical model of Rivero et al. provides a framework for determining values of the chemotactic sensitivity coefficient chi 0, an intrinsic cell population parameter that characterizes the chemotactic response of bacterial populations. This coefficient can theoretically relate the swimming behavior of individual cells to the resulting migration of a bacterial population. When this model is applied to the commonly used capillary assay, an approximate solution can be obtained for a particular range of chemotactic strengths yielding a very simple analytical expression for estimating the value of chi 0, [formula: see text] from measurements of cell accumulation in the capillary, N, when attractant uptake is negligible. A0 and A infinity are the dimensionless attractant concentrations initially present at the mouth of the capillary and far into the capillary, respectively, which are scaled by Kd, the effective dissociation constant for receptor-attractant binding. D is the attractant diffusivity, and mu is the cell random motility coefficient. NRM is the cell accumulation in the capillary in the absence of an attractant gradient, from which mu can be determined independently as mu = (pi/4t)(NRM/pi r2bc)2, with r the capillary tube radius and bc the bacterial density initially in the chamber. When attractant uptake is significant, a slightly more involved procedure requiring a simple numerical integration becomes necessary. As an example, we apply this approach to quantitatively characterize, in terms of the chemotactic sensitivity coefficient chi 0, data from Terracciano indicating enhanced chemotactic responses of Escherichia coli to galactose when cultured under growth-limiting galactose levels in a chemostat.     

Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays

A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis. The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values. We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope. We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions. Chemotaxis of E. coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay. Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range. Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient. These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth. Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data.     

The Range of Attractant Concentrations for Bacterial Chemotaxis and the Threshold and Size of Response over This Range : Weber law and related phenomena

Attractant was added to a suspension of bacteria (the background concentration of attractant) and then these bacteria were exposed to a yet higher concentration of attractant in a capillary. Chemotaxis was measured by determining how many bacteria accumulated in the capillary. The response range for chemotaxis lies between the threshold concentration and the saturating concentration. The breadth of this range is different for attractants detected by different chemoreceptors. Attractants detected by the same chemoreceptor can have their response ranges in widely different places. Over the center of the response range (on a logarithmic scale), bacteria give similar sized responses to similar fractional increases of concentration, i.e. they respond to ratios of attractant concentration, but the response peaks at the center of the range. The size of the response is different for attractants detected by different chemoreceptors. For a detectable response, a smaller increase in attractant concentration is needed for attractants detected by some chemoreceptors than for attractants detected by others. Although the data are inadequate, it appears that the Weber law may be observed over a wide range of concentrations for some attractants but not for others. In the Appendix we aim to explain some of these results in terms of the interaction of an attractant with its chemoreceptor according to the law of mass action.     

A high-throughput capillary assay for bacterial chemotaxis

We present a high-throughput capillary assay in order to characterize the chemotactic response of the E. coli bacterium. We measure the number of organisms attracted into an array of 96 capillary tubes containing the attractant L-aspartate. The effect of bacterial concentration on the chemotactic response is reported. Such high-throughput assay can be used to characterize bacterial chemotaxis function of a wide range of biochemical parameters.     

The role of methylation in chemotaxis. An explanation of outstanding anomalies

The role of methylation in chemotaxis is understood generally, but several anomalies exist which bring into question the timing of methylation relative to sensing. A double mutant bacterium, deficient in both methyltransferase and methylesterase (Tr-Es-) is capable of chemotaxis even though the respective single mutants (Tr- and Es-) are not. This Tr-Es- mutant will accumulate in capillaries containing aspartic acid but not in capillaries containing serine despite the fact that both the aspartate and serine receptors are part of the methylation-dependent pathway. To understand these anomalies, a combination of theoretical analyses and experimental studies was performed. A mathematical analysis of the gradients of aspartate and serine in the capillary assay shows that outside the capillary the gradients are shallow, but just inside the mouth of the capillary they are very steep. Also, when the number of bacteria accumulated in the capillary is at a maximum, the range of attractant concentrations in the steep gradient just inside the mouth of the capillary is optimal for response and partial adaptation by the Tr-Es- mutant. We postulate that random motion brings the Tr-Es- mutant into the capillary, where it is able to move up the steep gradient. The difference in timing of the responses to serine and aspartate explains why the Tr-Es- mutant accumulates in aspartate- but not in serine-containing capillaries. A simple diffusion-capture model incorporating these concepts can account for experimental values of the number of Tr-Es- bacteria accumulating in the capillary. These studies provide a rational explanation for all of the apparent anomalies and lead to the conclusion that methylation/demethylation plays a crucial role in sensing as well as setting the zero point of the receptor.     

Capillary electrophoresis-based profiling and quantitation of total salicylic acid and related phenolics for analysis of early signaling in Arabidopsis disease resistance

A capillary electrophoresis-based method for quantitation of total salicylic acid levels in Arabidopsis leaves was developed. Direct comparison to previous high-performance liquid chromatography (HPLC)-based measurements showed similar levels of salicylic acid. Simultaneous quantitation of trans-cinnamic acid, benzoic acid, sinapic acid, and an internal recovery standard was achieved. A rapid, streamlined protocol with requirements for plant tissue reduced relative to those of HPLC-based protocols is presented. Complicated, multiparameter experiments were thus possible despite the labor-intensive nature of inoculating plants with bacterial pathogens. As an example of this sort of experiment, detailed time course studies of total salicylic acid accumulation by wild-type Arabidopsis and two lines with mutations affecting salicylic acid accumulation in response to either of two avirulent bacterial strains were performed. Accumulation in the first 12h was biphasic. The first phase was partially SID2 and NDR1 dependent with both bacterial strains. The second phase was largely independent of both genes with bacteria carrying avrB, but dependent upon both genes with bacteria carrying avrRpt2. Virulent bacteria did not elicit salicylic acid accumulation at these time points. Application of this method to various Arabidopsis pathosystems and the wealth of available disease resistance signaling mutants will refine knowledge of disease resistance and associated signal transduction.     

Identification of specific chemoattractants and genetic complementation of a Borrelia burgdorferi chemotaxis mutant: flow cytometry-based capillary tube chemotaxis assay

Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.     

A Mathematical Model for Bacterial Chemotaxis

A differential equation describing the chemotactic migration of a bacterial population in a fixed exponential gradient of attractant has been integrated using the appropriate boundary conditions. The solution predicts an initial bacterial accumulation at the concentration “knee” with the final distribution of bacteria approaching a time-independent state. Specific additional experiments to obtain further data for a rigorous test of the theory are suggested.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Salmonella chemoreceptors McpB and McpC mediate a repellent response to L-cystine: a potential mechanism to avoid oxidative conditions

Chemoreceptors McpB and McpC in Salmonella enterica have been reported to promote chemotaxis in LB motility-plate assays. Of the chemicals tested as potential effectors of these receptors, the only response was towards L-cysteine and its oxidized form, L-cystine. Although enhanced radial migration in plates suggested positive chemotaxis to both amino acids, capillary assays failed to show an attractant response to either, in cells expressing only these two chemoreceptors. In vivo fluorescence resonance energy transfer (FRET) measurements of kinase activity revealed that in wild-type bacteria, cysteine and cystine are chemoeffectors of opposing sign, the reduced form being a chemoattractant and the oxidized form a repellent. The attractant response to cysteine was mediated primarily by Tsr, as reported earlier for Escherichia coli. The repellent response to cystine was mediated by McpB/C. Adaptive recovery upon cystine exposure required the methyl-transferase/-esterase pair, CheR/CheB, but restoration of kinase activity was never complete (i.e. imperfect adaptation). We provide a plausible explanation for the attractant-like responses to both cystine and cysteine in motility plates, and speculate that the opposing signs of response to this redox pair might afford Salmonella a mechanism to gauge and avoid oxidative environments.     

Aerotaxis and chemotaxis ofAzospirillum brasilense: A note

Azospirillum brasilense was attracted to capillaries containing either phosphate buffer, distilled water, or saline. The number of bacteria in these capillaries was 3–4×10⁴, after 1 h of incubation. In the presence of phosphate buffer + attractants, the number of cells accumulated in the capillary increased only to 5×10⁴–1.1×10⁵ cells. It was not possible, therefore, to measure chemotaxis inA. brasilense as distinct from aerotaxis by the capillary method. Chemotaxis was observed in semi-solid agar plates and was determined by a growth band oriented towards the attractant. Positive chemotactic response was obtained with peptone, tryptone, yeast extract, amino acids, organic acids, arabinose and galactose.     

A material-balance approach for modeling bacterial chemotaxis to a consumable substrate in the capillary assay

A mathematical model was developed to simulate the movement of chemotactic bacteria into and within a capillary tube containing a metabolizable chemoattractant. The model was based on a material balance that accounts for the transport of bacteria and chemoattractant as well as consumption of the substrate throughout the capillary assay system. By solving the model with a numerical method, it was possible to predict the concentration of substrate and bacteria at points within the capillary and throughout the chamber. The model was tested for its ability to simulate the results of capillary assay experiments performed with Pseudomonas putida G7 and one of its chemoattractants, naphthalene, under conditions wherein naphthalene consumption was expected to affect the flux of bacteria into the capillary. While variations in the chemotactic responses were evident among different experiments, the model could simulate the accumulation of cells in the capillary using previously determined parameters, including the chemotactic sensitivity and random motility coefficients, chi(0) and mu. In particular, model predictions were consistent with the experimental observation that the chemotactic response in the capillary is diminished under conditions wherein consumption would be expected to be significant.     

Aerotactic response of Azospirillum brasilense.

Five strains of Azospirillum brasilense and two of Azospirillum spp., from Israel, responded to self-created and preformed oxygen gradients by forming aerotactic bands in capillary tubes and actively moving toward a specific zone with low dissolved oxygen. Increasing the oxygen concentration in capillaries containing phosphate buffer increased the number of attracted bacteria and decreased band velocity. High O2 concentrations and H2O2 temporarily repulsed the bacteria, causing the formation of a bacterial arc around the capillary mouth. There was no band formation under anaerobic conditions, although the bacteria remained highly motile. Exogenous energy sources were unnecessary for aerotaxis in Azospirillum spp. The addition of oxidizable substrates to the capillary slightly enhanced aerotaxis, possibly by accelerating O2 consumption. Aerotactic band formation was affected by pH, bacterial concentration and age, incubation time, and respiratory inhibitors, but not by the lack of combined nitrogen in the growth medium. It is proposed that aerotaxis plays a role in the capacity of Azospirillum spp. to reach an environment suitable for N2 fixation.     

Enhancement of chemotaxis in Spirochaeta aurantia grown under conditions of nutrient limitation.

Spirochaeta aurantia M1 cells were grown in a chemostat under conditions of energy and carbon source limitation. The chemotactic responses of the chemostat-grown cells were compared with those of S. aurantia cells grown in batch culture in the presence of excess energy and carbon source. Chemotactic responses were measured by determining the number of cells that entered a capillary tube containing a solution of attractant. S. aurantia cells grown in the chemostat under energy and carbon source limitation exhibited enhanced chemotactic responses and detected lower concentrations of attractant, as compared with cells grown in batch culture. The chemotactic response toward an attractant was specifically enhanced when that attractant was the growth-limiting energy and carbon source. The medium used contained either D-glucose or D-xylose as the sole energy and carbon source. Cells had the greatest chemotactic response toward glucose when grown at a dilution rate (D) of 0.045 h-1 under glucose limitation and toward xylose when grown at D = 0.06 h-1 under xylose limitation. When cells were grown under glucose limitation (D = 0.045 h-1), they sensed concentrations of attractant (glucose) ca. 1,000 times lower than those sensed by batch-grown cells. A similar enhancement of sensing ability (toward xylose) was observed in cells grown under xylose limitation. The results indicated that S. aurantia cells are able to regulate their chemosensory system in response to nutrient limitation. Maximum enhancement of chemotaxis occurs in cells growing at very low concentrations of energy and carbon source. Most likely, this property provides the spirochetes with competitive advantages when the availability of nutrients becomes severely limited in their habitats.     

Studies of bacterial chemotaxis in defined concentration gradients. A model for chemotaxis toward L-serine

The details of the chemotactic response of Salmonella typhimurium to gradients of L-serine have been examined in some detail. Two relatively macroscopic techniques have been employed to measure the bacterial response. These include measurements of the average velocity as the bacterial population moves toward attractants, and measurement of the upward-to-downward flux ratio, R, in the stable preformed attractant gradients. The dependence of the average velocity on gradient appears to be hyperbolic in nature, while the flux ratio depends linearly on the gradient. These data suggest a microscopic model for the dependence of bacterial behavior on the serine gradient. The model involves a linear dependence of the mean lifetime of a bacterial trajectory on the gradient for those bacteria moving toward higher attractant concentration. Those moving toward low concentrations of attractant do not change the mean duration of their trajectories, or the speed at which a given bacterium swims through the solution. This model generates the observed dependences of the average velocity and flux ratio on gradient. Interpretation of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient. The concentration dependence of the chemotactic response to serine is more complicated. It suggests that more than one receptor of serine may be involved in determining chemotactic behavior to this attractant.     

Identification of Specific Chemoattractants and Genetic Complementation of a Borrelia burgdorferi Chemotaxis Mutant: Flow Cytometry-Based Capillary Tube Chemotaxis Assay

Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.     

The chemotactic behavior of computer-based surrogate bacteria

BACKGROUND: Chemotaxis is the process by which organisms migrate toward nutrients and favorable environments and away from toxins and unfavorable environments. In many species of bacteria, this occurs when extracellular signals are detected by transmembrane receptors and relayed to flagellar motors, which control the cell's swimming behavior. RESULTS: We used a molecularly detailed reaction-kinetics model of the chemotaxis pathway in Escherichia coli coupled to a graphical display based on known swimming parameters to simulate the responses of bacteria to 2D gradients of attractants. The program gives the correct phenotype of over 60 mutants in which chemotaxis-pathway components are deleted or overexpressed and accurately reproduces the responses to pulses and step increases of attractant. In order to match the known sensitivity of bacteria to low concentrations of attractant, we had to introduce a set of "infectivity" reactions based on cooperative interactions between neighboring chemotaxis receptors in the membrane. In order to match the impulse response to a brief stimulus and to achieve an effective accumulation in a gradient, we also had to increase the activities of the adaptational enzymes CheR and CheB at least an order of magnitude greater than published values. Our simulations reveal that cells develop characteristic levels of receptor methylation and swimming behavior at different positions along a gradient. They also predict a distinctive "volcano" profile in some gradients, with peaks of cell density at intermediate concentrations of attractant. CONCLUSIONS: Our results display the potential use of computer-based bacteria as experimental objects for exploring subtleties of chemotactic behavior.     

Chemotaxis by Pseudomonas syringae pv. tomato

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl₂ in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23°C, which is 5°C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 × 10⁶ CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.     

毛细管电泳在细菌分离分析中的应用

介绍了近年来毛细管电泳技术在细菌分离分析方面的研究进展。毛细管电泳以细菌表面的特征信息为分离的基础,可以快速鉴定相应的菌株,可以对微生物进行快速定量,可以反映细菌特殊时期的生理特征,也可以研究微生物与分子之间的相互作用。同时应用该技术可分离分析自然界不能纯培养的微生物。因而毛细管电泳分离与检测细菌方法的建立及其应用在分离科学和微生物学方面都有很大的实际意义。     

The effect of amino acids on the motile behavior of Bacillus subtilis

Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays. The stimulation of velocity did not occur via the receptors for chemotaxis. Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant. After methionine starvation B. subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria. Addition of methionine to starved cells restored the response toward attractant. Revertants of B. subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant.Abbreviation O.D.₆₀₀ optical density measured at 600 nm