The Role of Adaptation in Bacterial Speed Races

Evolution of biological sensory systems is driven by the need for efficient responses to environmental stimuli. A paradigm among prokaryotes is the chemotaxis system, which allows bacteria to navigate gradients of chemoattractants by biasing their run-and-tumble motion. A notable feature of chemotaxis is adaptation: after the application of a step stimulus, the bacterial running time relaxes to its pre-stimulus level. The response to the amino acid aspartate is precisely adapted whilst the response to serine is not, in spite of the same pathway processing the signals preferentially sensed by the two receptors Tar and Tsr, respectively. While the chemotaxis pathway in E. coli is well characterized, the role of adaptation, its functional significance and the ecological conditions where chemotaxis is selected, are largely unknown. Here, we investigate the role of adaptation in the climbing of gradients by E. coli. We first present theoretical arguments that highlight the mechanisms that control the efficiency of the chemotactic up-gradient motion. We discuss then the limitations of linear response theory, which motivate our subsequent experimental investigation of E. coli speed races in gradients of aspartate, serine and combinations thereof. By using microfluidic techniques, we engineer controlled gradients and demonstrate that bacterial fronts progress faster in equal-magnitude gradients of serine than aspartate. The effect is observed over an extended range of concentrations and is not due to differences in swimming velocities. We then show that adding a constant background of serine to gradients of aspartate breaks the adaptation to aspartate, which results in a sped-up progression of the fronts and directly illustrate the role of adaptation in chemotactic gradient-climbing.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Chemotaxis of Pseudomonas aeruginosa: involvement of methylation.

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.     

Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source.

The regulation of amino acid chemotaxis by nitrogen was investigated in the gram-negative bacterium Pseudomonas aeruginosa. The quantitative capillary tube technique was used to measure chemotactic responses of bacteria to spatial gradients of amino acids and other attractants. Chemotaxis toward serine, arginine, and alpha-aminoisobutyrate was sharply dependent on the form in which nitrogen was presented to the bacteria. Bacteria grown on mineral salts-succinate with potassium nitrate gave responses to amino acids that were 2 to 3 times those of cells grown on ammonium sulfate and 10 to 20 times those of cells grown in mineral salts-succinate with Casamino Acids as the nitrogen source. A combination of ammonium sulfate and glutamate was as effective as Casamino Acids in depressing serine taxis. The threshold concentration for alpha-aminoisobutyrate taxis was consistently lower in nitrate-grown bacteria than in ammonia-grown bacteria. Responsiveness to sodium succinate, however, was not subject to regulation by nitrogen, and glucose chemotaxis was inhibited, rather than enhanced, in nitrate-grown bacteria. These results indicate that chemotaxis of P. aeruginosa toward amino acids is subject to regulation by nitrogen and that this regulation probably is expressed at the level of the chemoreceptors or transducers.     

Amino acid efflux in response to chemotactic and osmotic signals in Bacillus subtilis.

We observed a large efflux of nonvolatile radioactivity from Bacillus subtilis in response to the addition of 31 mM butyrate or the withdrawal of 0.1 M aspartate in a flow assay. The major nonvolatile components effluxed were methionine, proline, histidine, and lysine. In studies of the release of volatile radioactivity in chemotaxis by B. subtilis cells that had been labeled with [3H]methionine, the breakdown of methionine to methanethiol can contribute substantially to the volatile radioactivity in fractions following addition of 0.1 M aspartate. However, methanol was confirmed to be released after aspartate addition and, in lesser quantities, after aspartate withdrawal. Methanol and methanethiol were positively identified by derivitization with 3,5-dinitro-benzoylchloride. Amino acid efflux but not methanol release was observed in response to 0.1 M aspartate stimulation of a cheR mutant of B. subtilis that lacks the chemotaxis methylesterase. The amino acid efflux could be reproduced by withdrawal of 0.1 M NaCl, 0.2 M sucrose, or 0.2 M xylitol and is probably the result of changes in osmolarity. Chemotaxis to 10 mM alanine or 10 mM proline resulted in methanol release but not efflux of amino acids. In behavioral studies, B. subtilis tumbled for 16 to 18 s in response to a 200 mosM upshift and for 14 s after a 20 mosM downshift in osmolarity when the bacteria were in perfusion buffer (40 mosM). The pattern of methanol release was similar to that observed in chemotaxis. This is consistent with osmotaxis in B. subtilis away from an increase or decrease in the osmolarity of the incubation medium. The release of methanol suggests that osmotaxis is correlated with methylation of a methyl-accepting chemotaxis protein.     

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.     

Histidine protein kinases: key signal transducers outside the animal kingdom

Histidine protein kinases (HPKs) are a large family of signal-transduction enzymes that autophosphorylate on a conserved histidine residue. HPKs form two-component signaling systems together with their downstream target proteins, the response regulators, which have a conserved aspartate in a so-called 'receiver domain' that is phosphorylated by the HPK. Two-component signal transduction is prevalent in bacteria and is also widely used by eukaryotes outside the animal kingdom. The typical HPK is a transmembrane receptor with an amino-terminal extracellular sensing domain and a carboxy-terminal cytosolic signaling domain; most, if not all, HPKs function as dimers. They show little similarity to protein kinases that phosphorylate serine, threonine or tyrosine residues, but may share a distant evolutionary relationship with these enzymes. In excess of a thousand known genes encode HPKs, which are important for multiple functions in bacteria, including chemotaxis and quorum sensing, and in eukaryotes, including hormone-dependent developmental processes. The proteins divide into at least 11 subfamilies, only one of which is present in eukaryotes, suggesting that lateral gene transfer gave rise to two-component signaling in these organisms.     

Chemotaxis of Salmonella typhimurium to amino acids and some sugars.

Patterns of chemotaxis by Salmonella typhimurium strain LT-2 to l-amino acids and to several sugars were quantitated by the Adler capillary procedure. Competition experiments indicated that LT-2 possesses three predominant receptors, or interacting sets of receptors, for amino acids. These were termed the aspartate, serine, and alanine classes, respectively. Studies with strains carrying point and deletion mutations affecting components of the phosphoenolpyruvate: glycose phosphotransferase system (PTS) made unlikely a role in primary reception of d-glucose by the three soluble PTS components, namely HPr, enzyme I, and factor III. A ptsG mutant defective in membrane-bound enzyme IIB' of the high-affinity glucose transport system was shown to exhibit normal chemotaxis providing pleiotropic effects of the mutation were eliminated by its genotypic combination with other pts mutations or, phenotypically, by addition of cyclic AMP and substrate. A correlation was demonstrated between chemotaxis to glucose and activity of the low-affinity glucose transport complex, membrane-bound enzymes IIB:IIA, and an enzyme IIB:IIA mutant was shown to have a preponderant defect in chemotaxis to glucose and mannose. Of four systems capable of galactose transport, only the beta-methylgalactoside transport system was implicated in chemotaxis to galactose. Some properties of a mutant possibly defective in processing of signals for chemotaxis to sugars is described.     

Imprecision of Adaptation in Escherichia coli Chemotaxis

Adaptability is an essential property of many sensory systems, enabling maintenance of a sensitive response over a range of background stimulus levels. In bacterial chemotaxis, adaptation to the preset level of pathway activity is achieved through an integral feedback mechanism based on activity-dependent methylation of chemoreceptors. It has been argued that this architecture ensures precise and robust adaptation regardless of the ambient ligand concentration, making perfect adaptation a celebrated property of the chemotaxis system. However, possible deviations from such ideal adaptive behavior and its consequences for chemotaxis have not been explored in detail. Here we show that the chemotaxis pathway in Escherichia coli shows increasingly imprecise adaptation to higher concentrations of attractants, with a clear correlation between the time of adaptation to a step-like stimulus and the extent of imprecision. Our analysis suggests that this imprecision results from a gradual saturation of receptor methylation sites at high levels of stimulation, which prevents full recovery of the pathway activity by violating the conditions required for precise adaptation. We further use computer simulations to show that limited imprecision of adaptation has little effect on the rate of chemotactic drift of a bacterial population in gradients, but hinders precise accumulation at the peak of the gradient. Finally, we show that for two major chemoeffectors, serine and cysteine, failure of adaptation at concentrations above 1 mM might prevent bacteria from accumulating at toxic concentrations of these amino acids.     

In vivo and in vitro chemotactic methylation in Bacillus subtilis

Two doublets of Bacillus subtilis membrane proteins with molecular weights of 69,000 and 71,000 and of 30,000 and 30,800, were labeled by C3H3 transfer in the absence of protein synthesis. In addition, there was intense methylation of several low-molecular-weight substances. Both doublets were missing in a chemotaxis mutant. The equivalent proteins in Escherichia coli and Salmonella typhimurium are believed to be the methyl-accepting chemotaxis proteins. The higher-molecular-weight doublet bands were increased in degree of methylation upon addition of attractant to the bacteria. A methyltransferase from B. subtilis that methylates the wild-type membrane significantly better than the mutant membrane, using S-adenosylmethionine, has been partly purified. The methylated product was alkali labile and is probably a gamma-glutamyl methyl ester, as in E. coli and S. typhimurium. Ca2+ ion inhibited the methyltransferase, with a Ki of about 80 nM. Analysis of the in vitro methylation product showed labeling of the 69,000-dalton methyl-accepting chemotaxis protein and a low-molecular-weight protein, using wild-type membrane. Labeling of the low-molecular-weight protein but not of the 69,000 dalton protein was observed when the mutant membrane was used. The chemotaxis mutant tumbled much longer than the wild type when diluted away from attractant.     

Dictyostelium chemotaxis: essential Ras activation and accessory signalling pathways for amplification

Central to chemotaxis is the molecular mechanism by which cells exhibit directed movement in shallow gradients of a chemoattractant. We used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signalling module providing activation of Ras at the leading edge, which is sufficient for chemotaxis. The signalling enzymes PI3K, TorC2, PLA2 and sGC are not required for Ras activation and chemotaxis to folate or to steep gradients of cAMP, but they provide a memory of direction and improved orientation of the cell, which together increase the sensitivity about 150-fold for chemotaxis in shallow cAMP gradients.     

Ligand-specific activation of Escherichia coli chemoreceptor transmethylation

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.     

Characterization of Halobacterium halobium mutants defective in taxis.

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.     

Chemotactic-like response of Escherichia coli cells lacking the known chemotaxis machinery but containing overexpressed CheY

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(-5) M; the response was inverted at > 10(-4) M). The temporal response was partial and delayed, as was the response of a control wild-type strain having a high CheY level. alpha-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.     

Role of methylation in aerotaxis in Bacillus subtilis.

Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly. A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen. Adaptation to a step increase in oxygen concentration was impaired when B. subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins. There was a transient increase in methanol release when wild-type B. subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration. The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins. This indicated that methylation is involved in aerotaxis in B. subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent.     

Membrane receptors for aspartate and serine in bacterial chemotaxis.

High affinity binding sites for serine and aspartate have been characterized in membranes from Salmonella typhimurium and Escherichia coli. Greater than 80% of these sites have been identified as chemotaxis receptors. Mutants lacking binding sites for these amino acids have been shown to have corresponding defects in taxis. The substrate specificity of each of the receptors in Salmonella is very high; most analogs of serine and aspartate do not bind to these receptor sites and do not affect chemotaxis. The transport of these amino acids is apparently not related to chemotaxis. At least 2500 serine receptors and 1200 aspartate receptors with dissociation constants of about 5 microM are present in the membrane fraction of logarithmically growing cells.     

An Unorthodox Sensory Adaptation Site in the Escherichia coli Serine Chemoreceptor

The serine chemoreceptor of Escherichia coli contains four canonical methylation sites for sensory adaptation that lie near intersubunit helix interfaces of the Tsr homodimer. An unexplored fifth methylation site, E502, lies at an intrasubunit helix interface closest to the HAMP domain that controls input-output signaling in methyl-accepting chemotaxis proteins. We analyzed, with in vivo Förster resonance energy transfer (FRET) kinase assays, the serine thresholds and response cooperativities of Tsr receptors with different mutationally imposed modifications at sites 1 to 4 and/or at site 5. Tsr variants carrying E or Q at residue 502, in combination with unmodifiable D and N replacements at adaptation sites 1 to 4, underwent both methylation and demethylation/deamidation, although detection of the latter modifications required elevated intracellular levels of CheB. These Tsr variants could not mediate a chemotactic response to serine spatial gradients, demonstrating that adaptational modifications at E502 alone are not sufficient for Tsr function. Moreover, E502 is not critical for Tsr function, because only two amino acid replacements at this residue abrogated serine chemotaxis: Tsr-E502P had extreme kinase-off output and Tsr-E502I had extreme kinase-on output. These large threshold shifts are probably due to the unique HAMP-proximal location of methylation site 5. However, a methylation-mimicking glutamine at any Tsr modification site raised the serine response threshold, suggesting that all sites influence signaling by the same general mechanism, presumably through changes in packing stability of the methylation helix bundle. These findings are consistent with control of input-output signaling in Tsr through dynamic interplay of the structural stabilities of the HAMP and methylation bundles.     

S-adenosylmethionine may not be essential for signal transduction during bacterial chemotaxis.

We previously showed that a mutant strain of Salmonella typhimurium completely deficient in both the chemoreceptor methylating (CheR) and demethylating (CheB) enzymes can still exhibit chemotaxis to aspartate and other attractants (J. Stock, A. Borczuk, F. Chiou, and J. E. B. Burchenal, Proc. Natl. Acad. Sci. USA 82:8364-8368, 1985). We used this cheR cheB mutant to examine the possibility of an additional requirement for S-adenosylmethionine in chemotaxis besides its role in chemoreceptor methylation. A metE mutation was transduced into a cheR cheB double mutant, and the cells were starved for methionine. Despite the fact that intracellular S-adenosylmethionine dropped from approximately 100 microM to less than 0.2 microM, chemotaxis was largely unaffected. In contrast, a corresponding cheR+ cheB+ metE mutant completely lost its chemotaxis ability after being starved for methionine. We conclude from this observation that the primary requirement for S-adenosylmethionine during bacterial chemotaxis is in the methylation of receptor proteins.     

How Do Bacteria Avoid High Oxygen Concentrations?

Bacteria, such as Escherichia coli and Azospirillum brasilense, avoid microenvironments with elevated oxygen concentrations, not by sensing reactive oxygen derivatives, but by sensing a metabolic down-shift that results from elevated oxygen levels. A novel protein, Aer, and the chemotaxis serine receptor, Tsr, have recently been identified as transducers for aerotaxis which monitor internal energy levels in the bacteria.     

Adaptive-Control Model for Neutrophil Orientation in the Direction of Chemical Gradients

Neutrophils have a remarkable ability to detect the direction of chemoattractant gradients and move directionally in response to bacterial infections and tissue injuries. For their role in health and disease, neutrophils have been extensively studied, and many of the molecules involved in the signaling mechanisms of gradient detection and chemotaxis have been identified. However, the cellular-scale mechanisms of gradient sensing and directional neutrophil migration have been more elusive, and existent models provide only limited insight into these processes. Here, we propose a what we believe is a novel adaptive-control model for the initiation of cell polarization in response to gradients. In this model, the neutrophils first sample the environment by extending protrusions in random directions and subsequently adapt their sensitivity depending on localized, temporal changes in stimulation levels. Our results suggest that microtubules may play a critical role in integrating all the sensing events from the cellular periphery through their redistribution inside the neutrophils, and may also be involved in modulating local signaling. An unexpected finding was that model neutrophils exhibit significant randomness in timing and directionality of activation, comparable to our experimental observations in microfluidic devices. Moreover, their responses are robust against alterations of the rate and amplitude of the signaling reactions, and for a broad range in chemoattractant concentrations and spatial gradients.