Conditional inversion of the thermoresponse in Escherichia coli.

Mutants in Escherichia coli having defects in one of the methyl-accepting chemotaxis proteins, Tsr protein, which is the chemoreceptor and transducer for L-serine, showed a reduced but similar type of thermoresponse compared with wild-type strains; the cells showed smooth swimming upon temperature increase and tumbling upon temperature decrease. However, when the mutant cells were adapted to attractants such as L-aspartate and maltose, which are specific to another methyl-accepting chemotaxis protein, Tar protein, the direction of the thermoresponse was found to be inverted; a temperature increase induced tumbling and a temperature decrease induced smooth swimming. Consistent with this, the mutant cells showed inverted changes in the methylation level of Tar protein upon temperature changes. Wild-type strains but not Tar protein-deficient mutants exhibited the inverted thermoresponse when the cells were simultaneously adapted to L-aspartate and L-serine, indicating that Tar protein has a key role in the inversion of the thermoresponse. Thus, besides Tsr protein, Tar protein has a certain role in thermoreception. A simple model for thermoreception and inversion of the thermoresponse is also discussed.     

Requirement of the cheB function for sensory adaptation in Escherichia coli.

The chemotactic behavior of Escherichia coli mutants defective in cheB function, which is required to remove methyl esters from methyl-accepting chemotaxis proteins, was investigated by subjecting swimming or antibody-tethered cells to various attractant chemicals. Two cheB point mutants, one missense and one nonsense, exhibited stimulus response times much longer than did the wild type, but they eventually returned to the prestimulus swimming pattern, indicating that they were not completely defective in sensory adaptation. In contrast, strains deleted for the cheB function showed no evidence of adaptation ability after stimulation. The crucial difference between these strains appeared to be the residual level of cheB-dependent methylesterase activity they contained. Both point mutants showed detectable levels of methanol evolution due to turnover of methyl groups on methyl-accepting chemotaxis protein molecules, whereas the cheB deletion mutant did not. In addition, it was possible to incorporate the methyl label into the methyl-accepting chemotaxis proteins of the point mutants but not into those of the cheB deletion strain. These findings indicate that cheB function is essential for sensory adaptation in Escherichia coli.     

Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria.

Bacteria migrate away from an acid pH and from a number of chemicals, including organic acids such as acetate; the basis for detection of these environmental cues has not been demonstrated. Membrane-permeant weak acids caused prolonged tumbling when added to Salmonella sp. or Escherichia coli cells at pH 5.5. Tethered Salmonella cells went from a prestimulus behavior of 14% clockwise rotation to 80% clockwise rotation when 40 mM acetate was added and remained this way for more than 30 min. A low external pH in the absence of weak acid did not markedly affect steady-state tumbling frequency. Among the weak acids tested, the rank for acidity (salicylate greater than benzoate greater than acetate greater than 5,5-dimethyl-2,4-oxazolidinedione) was the same as the rank for the ability to collapse the transmembrane pH gradient and to cause tumbling. At pH 7.0, the tumbling responses caused by the weak acids were much briefer. Indole, a non-weak-acid repellent, did not cause prolonged tumbling at low pH. Two chemotaxis mutants (a Salmonella mutant defective in the chemotaxis methylesterase and an E. coli mutant defective in the methyl-accepting protein in MCP I) showed inverse responses of enhanced counterclockwise rotation in the first 1 min after acetate addition. The latter mutant had been found previously to be defective in the sensing of gradients of extracellular pH and (at neutral pH) of acetate. We conclude (i) that taxes away from acid pH and membrane-permeant weak acids are both mediated by a pH-sensitive component located either in the cytoplasm or on the cytoplasmic side of the membrane, rather than by an external receptor (as in the case of the attractants), and (ii) that both of these taxes involve components of the chemotaxis methylation system, at least in the early phase of the response.     

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.     

A methyl-accepting protein involved in multiple-sugar chemotaxis by Cellulomonas gelida.

Tethered-cell and capillary assays indicated that L-methionine is required by Cellulomonas gelida for its normal cell motility pattern and chemotaxis and that S-adenosylmethionine is involved in sugar chemotaxis by this cellulolytic bacterium. In addition, in vivo methylation assays showed that several proteins were methylated in the absence of protein synthesis. The incorporated methyl groups were alkali sensitive. Of special interest was the observation that the methylation level of a 51,000-Mr protein increased two- to fivefold upon addition of various sugar attractants and decreased after the removal of the attractants. The increase was less pronounced in mutants defective in sugar chemotaxis and appeared to be specifically involved with sugar chemotaxis. Furthermore, cell fractionation and in vitro methylation assays demonstrated that the 51,000-Mr protein is located in the cytoplasmic membrane. These results suggest that a specific methyl-accepting chemotaxis protein is involved in multiple-sugar chemotaxis by C gelida. During chemotaxis, the changes of methylesterase activity in C gelida cells were similar to those in Escherichia coli RP437 cells, as determined by a continuous-flow assay for methanol evolution. Thus, the mechanism of methyl-accepting chemotaxis protein-mediated chemotaxis of the gram-positive C. gelida appears to be similar to that of the gram-negative E. coli rather than to that of other gram-positive bacteria, such as Bacillus subtilis.     

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.     

Role of CheB and CheR in the complex chemotactic and aerotactic pathway of Azospirillum brasilense

It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.     

Aberrant regulation of methylesterase activity in cheD chemotaxis mutants of Escherichia coli

The adaptation process in several cheD chemotaxis mutants, which carry defects in tsr, the serine transducer gene, was examined. cheD mutants are smooth swimming and generally nonchemotactic; the defect is dominant to the wild-type tsr gene (J. S. Parkinson, J. Bacteriol. 142:953-961, 1980). All classes of methyl-accepting chemotaxis proteins synthesized in unstimulated cheD strains are overmethylated relative to the wild type. We found that the steady-state rate of demethylation in cheD mutants was low; this may explain their overmethylated phenotype. In addition, all cheD mutants showed diminished responsiveness of methylesterase activity to attractant and repellent stimuli transduced by either the Tsr or Tar protein, and they did not adapt. These results suggest that the dominant nature of the cheD mutations is manifested as a general defect in the regulation of demethylation. Some of these altered properties of methylesterase activity in cheD mutants were exhibited in wild-type cells that were treated with saturating concentrations of serine. The mutant Tsr protein thus seems to be locked into a signaling mode that suppresses tumbling and inhibits methylesterase activity in a global fashion. We found that the Tar and mutant Tsr proteins synthesized in cheD strains were methylated and deamidated at the correct sites and that the mutations were not located in the methylated peptides. Thus, the signaling properties of the transducers may be controlled at sites distinct from the methyl-accepting sites.     

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.     

Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis

The Bacillus subtilis gene encoding CheB, which is homologous to Escherichia coli CheY, the regulator of flagellar rotation, has been cloned and sequenced. It has been verified, using a phage T7 expression system, by showing that a small protein, the same size as E. coli CheY, is actually made from this DNA. Despite the fact that the two proteins are 36% identical, with many highly conserved residues, they appear to play different roles. Unlike CheY null mutants, which swim smoothly, CheB null mutants tumble incessantly. However, a CheB point mutant swims smoothly, even in the presence of a plasmid bearing cheB, which restores the null mutants to wild type. Expression of CheB in wild type B. subtilis makes the cells exhibit more tumbling. Since both absence of CheB and presence of high levels of CheB cause tumbling, CheB appears to be required, in certain circumstances, for both smooth swimming and tumbling. Expression in wild type E. coli makes the cells smooth swimmers and strongly inhibits chemotaxis.     

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.     

Identification and characterization of the chemotactic transducer in Pseudomonas aeruginosa PAO1 for positive chemotaxis to trichloroethylene

Pseudomonas aeruginosa PAO1 is repelled by trichloroethylene (TCE), and the methyl-accepting chemotaxis proteins PctA, PctB, and PctC serve as the major chemoreceptors for negative chemotaxis to TCE. In this study, we found that the pctABC triple mutant of P. aeruginosa PAO1 was attracted by TCE. Chemotaxis assays of a set of mutants containing deletions in 26 potential mcp genes revealed that mcpA (PA0180) is the chemoreceptor for positive chemotaxis to TCE. McpA also detects tetrachloroethylene and dichloroethylene isomers as attractants.     

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.     

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants.

Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.     

Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA , cheW , and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild-type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is over-expressed in E. coli . The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing. The MCP-dependent pathway, appears conserved. In R. sphaeroides this pathway may mediate responses to sugars, while responses to organic acids may in involve a second system, possibly using the second CheY protein identified in this study.     

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.     

The Helicobacter pylori chemotaxis receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa

The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40-/-)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.     

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.     

A methyl-accepting protein is involved in benzoate taxis in Pseudomonas putida.

Pseudomonas putida is attracted to at least two groups of aromatic acids: a benzoate group and a benzoylformate group. Members of the benzoate group of chemoattractants stimulated the methylation of a P. putida polypeptide with an apparent molecular weight of 60,000 in sodium dodecyl sulfate-polyacrylamide gels. This polypeptide is presumed to be a methyl-accepting chemotaxis protein for several reasons: its molecular weight is similar to the molecular weights of Escherichia coli methyl-accepting chemotaxis proteins, the amount of time required to attain maximal methylation correlated with the time needed for behavioral adaptation of P. putida cells to benzoate, and methylation was stimulated by benzoate only in cells induced for chemotaxis to benzoate. Also, a mutant specifically defective in benzoate taxis failed to show any stimulation of methylation upon addition of benzoate. Benzoylformate did not stimulate protein methylation in cells induced for benzoylformate chemotaxis, suggesting that sensory input from this second group of aromatic-acid attractants is processed through a different kind of chemosensory pathway. The chemotactic responses of P. putida cells to benzoate and benzoylformate were not sensitive to external pH over a range (6.2 to 7.7) which would vary the protonated forms of these weak acids by a factor of about 30. This indicates that detection of cytoplasmic pH is not the basis for aromatic-acid taxis in P. putida.