The short form of CheA couples chemoreception to CheA phosphorylation.

Escherichia coli cells express two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in chemotactic signal transduction. As a histidine protein kinase, it first autophosphorylates at amino acid His-48; then, it phosphorylates two other chemotaxis proteins, CheY and CheB. The short form, CheAS, lacks the amino-terminal 97 amino acids of CheAL and, therefore, does not contain the site of autophosphorylation. However, it does retain a functional kinase domain. As a consequence, CheAS can mediate transphosphorylation of kinase-deficient CheAL variants. Here we demonstrate in vitro that CheAS also can mediate transphosphorylation of a CheAL variant that lacks the C-terminal segment, a portion of the protein which is thought to interact with CheW and the chemoreceptors. The presence of CheW and the chemoreceptor Tsr enhances this activity and results in modulation of the transphosphorylation rate in response to the Tsr ligand, L-serine. Because CheAS can mediate this activity, it can restore chemotactic ability to Escherichia coli cells that express this truncated CheAL variant.     

Tandem translation starts in the cheA locus of Escherichia coli.

The cheA locus of Escherichia coli encodes two protein products, CheAL and CheAS. The nucleotide sequences of the wild-type cheA locus and of two nonsense alleles confirmed that both proteins are translated in the same reading frame from different start points. These start sites were located on the coding sequence by direct determination of the amino-terminal sequences of the two CheA proteins. Both starts are flanked by inverted repeats that may play a role in regulating the relative expression rates of the CheA proteins through alternative mRNA secondary structures.     

pH dependence of CheA autophosphorylation in Escherichia coli.

Chemotaxis by cells of Escherichia coli and Salmonella typhimurium depends upon the ability of chemoreceptors called transducers to communicate with switch components of flagellar motors to modulate swimming behavior. This communication requires an excitatory pathway composed of the cytoplasmic signal transduction proteins, CheAL, CheAS, CheW, CheY, and CheZ. Of these, the autokinase CheAL is most central. Modifications or mutations that affect the rate at which CheAL autophosphorylates result in profound chemotactic defects. Here we demonstrate that pH can affect CheAL autokinase activity in vitro. This activity exhibits a bell-shaped dependence upon pH within the range 6.5 to 10.0, consistent with the notion that two proton dissociation events affect CheAL autophosphorylation kinetics: one characterized by a pKa of about 8.1 and another exhibiting a pKa of about 8.9. These in vitro results predict a decrease in the rate of CheAL autophosphorylation in response to a reduction in intracellular pH, a decrease that should cause increased counterclockwise flagellar rotation. We observed such a response in vivo for cells containing a partially reconstituted chemotaxis system. Benzoate (10 mM, pH 7.0), a weak acid that when undissociated readily traverses the cytoplasmic membrane, causes a reduction of cytoplasmic pH from 7.6 to 7.3. In response to this reduction, cells expressing CheAL, CheAS, and CheY, but not transducers, exhibited a small but reproducible increase in the fraction of time that they spun their flagellar motors counterclockwise. The added presence of CheW and the transducers Tar and Trg resulted in a more dramatic response. The significance of our in vitro results, their relationships to regulation of swimming behavior, and the mechanisms by which transducers might affect the pH dependence of CheA autokinase activity are discussed.     

Coexpression of the long and short forms of CheA, the chemotaxis histidine kinase, by members of the family Enterobacteriaceae.

CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species.     

Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli.

CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli. CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation. Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells. To find out why, we examined the effects of a purified P1 fragment, CheA[1-149], on CheA-related signaling activities in vitro and devised in vivo assays for those same activities. Although readily phosphorylated by CheA[260-537], the CheA catalytic domain, CheA[1-149], was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center. CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA[1-149], suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect. In agreement with these findings, a 40-fold excess of CheA[1-149] fragments did not impair the CheA autophosphorylation reaction. CheA[1-149] did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules. An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site. The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells. However, an excess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragments have both transphosphorylation and phosphotransfer capability in vivo.     

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

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.     

Characterization of the chemotaxis protein CheW from Rhodobacter sphaeroides and its effect on the behaviour of Escherichia coli

In contrast to the situation in enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. A chemotaxis operon has been identified containing homologues of the enteric cheA , cheW , cheR genes and two homologues of the cheY gene. However, mutations in these genes have only minor effects on chemotaxis. In enteric species, CheW transmits sensory information from the chemoreceptors to the histidine protein kinase, CheA. Expression of R. sphaeroides cheW in Escherichia coli showed concentration-dependent inhibition of wild-type behaviour, increasing counter-clockwise rotation and thus smooth swimming — a phenotype also seen when E. coli cheW is overexpressed in E. coli . In contrast, overexpression of R. sphaeroides cheW in wild-type R. sphaeroides inhibited motility completely, the equivalent of inducing tumbly motility in E. coli . Expression of R. sphaeroides cheW in an E. coli Δ cheW chemotaxis mutant complemented this mutation, confirming that CheW is involved in chemosensory signal transduction. However, unlike E. coli Δ cheW mutants, in-frame deletion of R. sphaeroides cheW did not affect either swimming behaviour or chemotaxis to weak organic acids, although the responses to sugars were enhanced. Therefore, although CheW may act as a signal-transduction protein in R. sphaeroides , it may have an unusual role in controlling the rotation of the flagellar motor. Furthermore, the ability of a Δ cheW mutant to swim normally and show wild-type responses to weak acids supports the existence of additional chemosensory signal-transduction pathways.     

Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium.

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.     

Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.     

Proteomic mapping of a suppressor of non-chemotactic cheW mutants reveals that Helicobacter pylori contains a new chemotaxis protein

Bacterial chemotaxis is a colonization factor for the ulcer-causing pathogen Helicobacter pylori. H. pylori contains genes encoding the chemotaxis signalling proteins CheW, CheA and CheY; CheW couples chemoreceptors to the CheA kinase and is essential for chemotaxis. While characterizing a cheW mutant, we isolated a spontaneous, chemotactic variant (Che+). We determined that this phenotype was caused by a genetic change unlinked to the original cheW mutation. To locate the underlying Che+ mutation, we compared total protein profiles of the non-chemotactic mutant (cheW) with those from the cheW Che+ variant by two-dimensional differential in-gel electrophoresis. One protein was found only in the cheW Che+ variant. This protein was identified by MS/MS as HP0170, a hypothetical protein with no known function. DNA sequencing verified that hp0170 was mutated in the cheW Che+ suppressor, and deletion of this open reading frame in the cheW background nearly recapitulated the Che+ suppressor phenotype. Using hidden Markov models, we found that HP0170 is a remote homologue of E. coli CheZ. CheZ interacts with phosphorylated CheY and stimulates its autodephosphorylation. CheZ was not predicted to be present in epsilon-proteobacteria. We found that chemotaxis in the cheW Che+ suppressor depended on both cheY and cheA. We hypothesize that a small amount of phosphorylated CheY is generated via CheA in the cheW mutant, and this amount is sufficient to affect flagellar rotation when HP0170 is removed. Our results suggest that HP0170 is a remote homologue of CheZ, and that CheZ homologues are found in a broader range of bacteria than previously supposed.     

The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses.

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.     

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.     

Expression of the gltP gene of Escherichia coli in a glutamate transport-deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli , chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides , chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn 5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue γ-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H⁺-linked glutamate carrier of E. coli , was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides .     

Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response

Pseudomonas aeruginosa, a gamma-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.     

Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.     

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.