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1.
One of the processes by which CheY, the excitatory response regulator of chemotaxis in Escherichia coli, can be activated to generate clockwise flagellar rotation is by acetyl-CoA synthetase (Acs)-mediated acetylation. Deletion of Acs results in defective chemotaxis, indicating the involvement of Acs-mediated acetylation in chemotaxis. To investigate whether Acs is the sole acetylating agent of CheY, we purified the latter from a delta acs mutant. Mass spectrometry analysis revealed that this protein is partially acetylated in spite of the absence of Acs, suggesting that CheY can be post-translationally acetylated in vivo by additional means. Using [14C]AcCoA in the absence of Acs, we demonstrated that one of these means is autoacetylation, with AcCoA serving as an acetyl donor and with a rate similar to that of Acs-mediated acetylation. Biochemical characterization of autoacetylated CheY and mass spectrometry analysis of its tryptic digests revealed that its acetylated lysine residues are those found in CheY acetylated by Acs, but the acetylation-level distribution among the acetylation sites was different. Like CheY acetylated by Acs, autoacetylated CheY could be deacetylated by Acs. Also similarly to the case of Acs-mediated acetylation, the phosphodonors of CheY, CheA and acetyl phosphate, each inhibited the autoacetylation of CheY, whereas the phosphatase of CheY, CheZ, enhanced it. A reduced AcCoA level interfered with chemotaxis to repellents, suggesting that CheY autoacetylation may be involved in chemotaxis of E. coli. Interestingly, this interference was restricted to repellent addition and was not observed with attractant removal, thus endorsing our earlier suggestion that the signaling pathway triggered by repellent addition is not identical to that triggered by attractant removal.  相似文献   

2.
Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.  相似文献   

3.
CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.  相似文献   

4.
Spirochetes have a unique cell structure: These bacteria have internal periplasmic flagella subterminally attached at each cell end. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis is poorly understood. In other bacteria, modulation of flagellar rotation is essential for chemotaxis, and phosphorylation-dephosphorylation of the response regulator CheY plays a key role in regulating this rotary motion. The genome of the Lyme disease spirochete Borrelia burgdorferi contains multiple homologues of chemotaxis genes, including three copies of cheY, referred to as cheY1, cheY2, and cheY3. To investigate the function of these genes, we targeted them separately or in combination by allelic exchange mutagenesis. Whereas wild-type cells ran, paused (flexed), and reversed, cells of all single, double, and triple mutants that contained an inactivated cheY3 gene constantly ran. Capillary tube chemotaxis assays indicated that only those strains with a mutation in cheY3 were deficient in chemotaxis, and cheY3 complementation restored chemotactic ability. In vitro phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by CheA2 than by CheA1, and the CheY3-P intermediate generated was considerably more stable than the CheY-P proteins found in most other bacteria. The results point toward CheY3 being the key response regulator essential for chemotaxis in B. burgdorferi. In addition, the stability of CheY3-P may be critical for coordination of the rotation of the periplasmic flagella.  相似文献   

5.
Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.  相似文献   

6.
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.  相似文献   

7.
Agrobacterium tumefaciens has a chemtaxis operon, which includes orf1, orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, and orf10. In-frame deletions of cheY1 and cheY2 were constructed and the chemosensory behavior of the mutants was examined on swarm plates and in a chemotaxis assay toward acetosyringone. The cheY2 mutant (C1/delY2) showed impaired chemotactic capabilities in both swarming and chemotaxis assays. The effect of lacking CheY1 on chemotaxis is less severe than that of CheY2, under the conditions studied.  相似文献   

8.
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.  相似文献   

9.
The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.  相似文献   

10.
11.
CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression of fliG mutations. Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.  相似文献   

12.
Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air–liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.  相似文献   

13.
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.  相似文献   

14.
15.
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.  相似文献   

16.
The silent information regulator (Sir2) family proteins are NAD+‐dependent deacetylases. Although a few substrates have been identified, functions of the bacteria Sir2‐like protein (CobB) still remain unclear. Here the role of CobB on Escherichia coli chemotaxis was investigated. We used Western blotting and mass spectrometry to show that the response regulator CheY is a substrate of CobB. Surface plasmon resonance (SPR) indicated that acetylation affects the interaction between CheY and the flagellar switch protein FliM. The presence of intact flagella in knockout strains ΔcobB, Δacs, Δ(cobB) Δ(acs), Δ(cheA) Δ(cheZ), Δ(cheA) Δ(cheZ) Δ(cobB) and Δ(cheA) Δ(cheZ) Δ(acs) was confirmed by electron microscopy. Genetic analysis of these knockout strains showed that: (i) the ΔcobB mutant exhibited reduced responses to chemotactic stimuli in chemotactic assays, whereas the Δacs mutant was indistinguishable from the parental strain, (ii) CheY from the ΔcobB mutant showed a higher level of acetylation, indicating that CobB can mediate the deacetylation of CheY in vivo, and (iii) deletion of cobB reversed the phenotype of Δ(cheA) Δ(cheZ). Our findings suggest that CobB regulates E. coli chemotaxis by deacetylating CheY. Thus a new function of bacterial cobB was identified and also new insights of regulation of bacterial chemotaxis were provided.  相似文献   

17.
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.  相似文献   

18.
The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome. In order to verify the function of these proteins, the putative cheY genes were cloned into pQE31 vector and then expressed, respectively, in wild-type Escherichia coli strain RP437 and cheY defective strain RP5232. The results showed that all the five cheYs could restore the swarming of RP5232 strain to some extend. Overexpression of CheYs in RP437 showed inhibited swarming of RP437. To investigate the mechanism of chemotaxis signaling in L. interrogans strain Lai, certain aspartates (Asp-53, Asp-61, Asp-70, Asp-62, and Asp-66 for L. interrogans strain Lai CheY1, CheY2, CheY3, CheY4, and CheY5, respectively) were mutated. Expression of these mutated cheYs manifested neither restoration of the swarming ability of RP5232 nor inhibition on swarming ability of RP437. Multiple amino acid sequence alignment predicted ternary structures and the result of mutation experiment suggested that these conserved aspartate residues of L. interrogans were analogous to that in E. coli CheY in function and structure. So, L. interrogans and E. coli may have similar mechanisms of activation of the chemotaxis phosphorelay pathway, but there are differences in their control by signal terminator.  相似文献   

19.
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.  相似文献   

20.
CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis. In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons. We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense. We analyzed the functions of divergent CheW and CheY proteins from A. brasilense by heterologous expression in E. coli wild-type and mutant strains. Both proteins were able to specifically inhibit chemotaxis of a wild-type E. coli strain; however, only CheW from A. brasilense was able to restore signal transduction in a corresponding mutant of E. coli. Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins. Multiple, but conservative, substitutions were found in CheW homologs. No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important. Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway. Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.  相似文献   

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