Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis

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

Negative chemotaxis in Dictyostelium and Polysphondylium

A method has been described to measure negative chemotaxis in the cellular slime molds directly and to purify the repellents. Conclusive evidence is given that negative chemotaxis exists in the cellular slime molds and that it occurs generally in Dictyostelium and Polysphondylium. Amoebae respond shortly after their exposure to repellents, which are secreted by vegetative and not by preaggregative cells. The amoebae are sensitive to repellents in both development stages and contain enzyme(s) to inactivate them. Cross reactions of different species indicate that there is more than one repellent, although it cannot be excluded that the variability in response depends on the balancing effect of attractants and repellents.     

Two mechanisms of chemotaxis inParamecium

Summary Paramecia show chemotaxis, that is, they accumulate in or disperse from the vicinity of chemicals. This study examines both the avoiding reactions (abrupt random changes of swimming direction) and velocities of normal and mutant paramecia in attractants and repellents and shows that the animals accumulate or disperse either by changing the frequency of avoiding reactions or by changing swimming velocity. Mutations or conditions that eliminate avoiding reactions abolish the chemotaxis response to chemicals that cause accumulation or dispersal by modulation of frequency of avoiding reactions but not the response to chemicals that cause chemotaxis by modulation of velocity.The current knowledge of the bioelectric control of the swimming behavior inParamecium and observations of mutants defective in bioelectric control and in chemotaxis are used to develop a hypothesis for membrane potential control of chemotaxis: attractants that require the avoiding reaction slightly hyperpolarize the membrane; repellents that require the avoiding reaction slightly depolarize the membrane; repellents that cause chemitaxis by modulation of velocity strongly hyperpolarize the membrane.I am grateful to D. Kusher and P. Foletta for their technical assistance, to C. Kung and E. Orias for support and discussion of this work, to H. Machemer and M. Levandowsky for stimulating discussions, and to B. Diehn for suggestion of the modified assay. This work was supported in part by Public Health Service Grant F32 NSO5587 to JVH and NSF GB-3164X and PHS GM-19406 to C. Kung.     

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.     

Genetic and biochemical properties of Escherichia coli mutants with defects in serine chemotaxis

In Escherichia coli, taxis to certain chemoeffectors is mediated through an intrinsic membrane protein called methyl-accepting chemotaxis protein I (MCP I), which is the product of the tsr gene. Mutants were selected that are defective in taxis toward all MCP I-mediated attractants (alpha-aminoisobutyrate, L-alanine, glycine, and L-serine) but are normal to MCP I-mediated repellents and to chemoeffectors mediated by other MCPs. The mutants could be divided into two classes based on their ability to respond to various concentrations of L-serine. Two MCP I-mediated L-serine systems appear to function in the wild type: one of high and one of lower affinity. The mutations responsible for the serine taxis defects map at about 99 min on the E. coli chromosome and are not complemented by episomes carrying mutations in the tsr gene; this suggests that they are defective in tsr function. Low concentrations of L-[14C]serine specifically bound to wild-type membranes with a Km of 5 microM; in contrast, there was greatly decreased binding to vesicles prepared from the new mutants or from the tsr mutant AW518. Binding of labeled serine to wild-type vesicles was inhibited by MCP I-mediated attractants, but not by MCP II-mediated attractants. The data suggest that MCP I may function as the L-serine chemoreceptor in E. coli.     

Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes

Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis. Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing. Two classes of mutations were found: nonsense and missense. The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein. Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation. Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling. These mutants will be useful in experiments designed to understand the mechanism of chemotaxis.     

Glycerol and ethylene glycol: members of a new class of repellents of Escherichia coli chemotaxis

By using the chemical-in-plug method, we found that glycerol and ethylene glycol caused negative chemotaxis in wild-type cells of Escherichia coli; the threshold concentration was about 10(-3) M for both chemicals. As with other known repellents, the addition of glycerol or ethylene glycol induced a brief tumble response in wild-type cells but not in generally nonchemotactic mutants. Experiments with mutants defective in various methyl-accepting chemotaxis proteins (MCPs) revealed that the presence of any one of three kinds of MCPs (MCP I, MCP II, or MCP III) was necessary to give a tumble response to these repellents. Consistently, it was found that the methylation-demethylation system of MCPs was involved in the adaptation of the cells to these repellents. The effect of glycerol or ethylene glycol was not enhanced by lowering the pH of the medium, and glycerol did not alter the membrane potential of the cells. All of these results suggest that glycerol and ethylene glycol are members of a new class of repellents which produce a tumble response in the cells by perturbing the MCPs in the membrane.     

Pausing of flagellar rotation is a component of bacterial motility and chemotaxis.

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

Motility and chemotaxis of filamentous cells of Escherichia coli

Filamentous cells of Escherichia coli can be produced by treatment with the antibiotic cephalexin, which blocks cell division but allows cell growth. To explore the effect of cell size on chemotactic activity, we studied the motility and chemotaxis of filamentous cells. The filaments, up to 50 times the length of normal E. coli organisms, were motile and had flagella along their entire lengths. Despite their increased size, the motility and chemotaxis of filaments were very similar to those properties of normal-sized cells. Unstimulated filaments of chemotactically normal bacteria ran and stopped repeatedly (while normal-sized bacteria run and tumble repeatedly). Filaments responded to attractants by prolonged running (like normal-sized bacteria) and to repellents by prolonged stopping (unlike normal-sized bacteria, which tumble), until adaptation restored unstimulated behavior (as occurs with normal-sized cells). Chemotaxis mutants that always ran when they were normal sized always ran when they were filament sized, and those mutants that always tumbled when they were normal sized always stopped when they were filament sized. Chemoreceptors in filaments were localized to regions both at the poles and at intervals along the filament. We suggest that the location of the chemoreceptors enables the chemotactic responses observed in filaments. The implications of this work with regard to the cytoplasmic diffusion of chemotaxis components in normal-sized and filamentous E. coli are discussed.     

Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis

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

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.     

Sensory adaptation mutants of E. coli.

The ability of E. coli to adapt to constant levels of attractant and repellent chemicals was studied by examining the patterns of flagellar movement in cells subjected to abrupt concentration changes. Wild-type bacteria exhibited transient responses to such stimuli, in support of previous findings. Nonchemotactic mutants of the cheX class responded to both attractants and repellents, but were unable to terminate these behavioral changes as long as the stimulating chemical was present. The sensory adaptation defect of cheX strains may be due to an inability to methylate several cytoplasmic membrane proteins that initiate changes in flagellar movement in response to chemoreceptor signals. Based on these results, possible mechanisms of stimulus transduction and sensory adaptation during chemotaxis are discussed.     

Relation between chemotaxis and consumption of amino acids in bacteria

Chemotaxis enables bacteria to navigate chemical gradients in their environment, accumulating toward high concentrations of attractants and avoiding high concentrations of repellents. Although finding nutrients is likely to be an important function of bacterial chemotaxis, not all characterized attractants are nutrients. Moreover, even for potential nutrients, the exact relation between the metabolic value of chemicals and their efficiency as chemoattractants has not been systematically explored. Here we compare the chemotactic response of amino acids with their use by bacteria for two well‐established models of chemotactic behavior, Escherichia coli and Bacillus subtilis. We demonstrate that in E. coli chemotaxis toward amino acids indeed strongly correlates with their utilization. However, no such correlation is observed for B. subtilis, suggesting that in this case, the amino acids are not followed because of their nutritional value but rather as environmental cues.     

Chemotactic response of Escherichia coli to chemically synthesized amino acids.

In Escherichia coli, seven of the commonly occurring amino acids are strong attractants: L-aspartate, L-serine, L-glutamate, L-alanine, L-asparagine, glycine, and L-cysteine, in order of decreasing effectiveness. The chemotactic response to each amino acid attractant is mediated by either methyl-accepting chemotaxis protein I or II, but not by both. Seven of the commonly occurring amino acids are repellents. This work was carried out with chemically synthesized amino acids.     

Selective modification of positive chemotaxis in the true slime mold Physarum polycephalum by ethylenediaminetetraacetic acid treatment.

The plasmodium of the true slime mold Physarum polycephalum was treated with EDTA or EGTA and the effect of the treatment on the chemotactic response was examined by measuring the chemotactic motive force with the double-chamber method. The results obtained were as follows: (1) The treatment of the plasmodium with 5 mM EDTA (pH 7.0, 20 min) did not give any significant effect on the protoplasmic streaming or motility. (2) The plasmodium treated with EDTA exhibited no chemotactic response to non-electrolyte attractants (D-glucose, D-galactose, D-mannose, and maltose) and negative chemotaxis to electrolyte attractants (cyclic AMP and NaH2PO4). (3) The EDTA treatment gave no effect on the chemotactic response to repellents (D-fructose, NaCl, CaCl2, MgCl2). (4) The EDTA-treated plasmodium exhibited changes in the membrane potential in response to both attractants and repellents as similar to the untreated plasmodium. (5) The treatment of the plasmodium with 5 mM EGTA (pH 7.0, 20 min) gave results similar to those obtained with the EDTA treatment. The results obtained suggested that EDTA (or EGTA) treatment did not affect the receptor sites but modified the transduction mechanism from reception into tactic movement.     

Complementation and characterization of chemotaxis mutants of Bacillus subtilis.

A set of chemotaxis mutants of Bacillus subtilis was complemented by using SP beta c2 transducing bacteriophage either containing cloned segments of DNA or derived from abnormal excision of SP beta c2 dl2::Tn917 inserted into the chemotaxis region. Representative mutants were characterized in capillary assays for chemotaxis toward four amino acids and mannitol and in tethered-cell experiments for addition and removal of two attractants and two repellents. Twenty complementation groups were identified, in addition to the cheR previously characterized. All were found to be defective in chemotaxis toward all chemoeffectors. They were assigned the names cheA through cheU. The large number of general chemotaxis genes in B. subtilis, in contrast to the six in Escherichia coli, suggests fundamental differences in the mechanism of chemotaxis in the two species.     

Influence of attractants and repellents on methyl group turnover on methyl-accepting chemotaxis proteins of Bacillus subtilis and role of CheW.

The ability of attractants and repellents to affect the turnover of methyl groups on the methyl-accepting chemotaxis proteins (MCPs) was examined for Bacillus subtilis. Attractants were found to cause an increase in the turnover of methyl groups esterified to the MCPs, while repellents caused a decrease. These reactions do not require CheW. However, a cheW null mutant exhibits enhanced turnover in unstimulated cells. Assuming that the turnover of methyl groups on the MCPs reflects a change in the activity of CheA, these results suggest that the activation of CheA via chemoeffector binding at the receptor does not require CheW.     

Participation of the chemotactic system in regulating cell division in Escherichia coli

The possible role of the chemotaxis system in regulating cell division of Escherichia coli was studied. Attractants increased the rate of division whereas repellents reduced it. Non-metabolisable attractants analogues were also effective in stimulating cell division. Fucose, a non-metabolisable analogue of galactose, increased the rate of division by 20-25%. Co2+ at concentrations which had no effect on the tar-mutant division suppressed the division of the wild type. Likewise, indole at concentrations which did not influence the division of the tsr-mutant, suppressed the division of the wild type.     

Sites of deamidation and methylation in Tsr, a bacterial chemotaxis sensory transducer

The sensory transducer proteins in bacterial chemotaxis undergo two covalent modifications, deamidation and reversible methylation, in response to attractants and repellents. Oligonucleotide-directed mutagenesis was used to alter putative methylation and deamidation sites in one of the transducers to further define these sites and their role in chemotaxis. The mutations, in combination with peptide maps and Edman analysis, have clarified the sites of covalent modification in Tsr. Tsr contains six specific glutamates and glutamines that serve as methyl-accepting sites. An arginine-containing tryptic peptide (R1) has two sites, one at glutamate 493 and a newly located site at glutamate 502. A lysine-containing peptide (K1) has four methyl-accepting sites. Two of the lysine peptide sites are glutamates and can accept methyl groups without deamidation. The other two sites are glutamines and two methyl-accepting sites are created by two distinct deamidations. Both deamidations can occur on the same polypeptide chain. Single glutamate mutants have shown that one deamidation (at glutamine 311) proceeds rapidly, while the other deamidation (at glutamine 297) has a half-life of approximately 60 min under our experimental conditions.