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Keywords:

  • acetylation;
  • bacterial chemotaxis;
  • CheY ;
  • phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

CheY, the response regulator of the chemotaxis system in Escherichia coli, can be regulated by two covalent modifications – phosphorylation and acetylation. Both covalent modifications are involved in chemotaxis, but the mechanism and role of the acetylation are still obscure. While acetylation was shown to repress the binding of CheY to its target proteins, the effect of acetylation on the ability of CheY to undergo autophosphorylate with AcP is not fully investigated. To obtain more information on the function of this acetylation, we successfully expressed and purified CheY protein with a 6 × His-tag on the C-terminus. Subsequently, acetylated CheY (AcCheY) was obtained with AcCoA as the acetyl donor, and the acetylation level of AcCheY was confirmed by Western blotting and then mass spectrometry. Using tryptophan fluorescence intensity measurements as a monitor of phosphorylation, we showed that acetylation reduces the ability of CheY to undergo autophosphorylation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Chemotaxis is a mechanism by which bacteria respond to changes in the chemical compositions of their environment, approaching attractants, and avoiding repellents. CheY, the excitatory response regulator of bacterial chemotaxis, transduces the chemotactic signal from the receptor supramolecular complex, located at the cell pole, to the switch elements in the flagellar–motor supramolecular complexes, which are randomly distributed around the cell (Silversmith & Bourret, 1999; Eisenbach, 2004; Sourjik, 2004). In Escherichia coli, the activity of CheY can be modulated by two covalent modifications: phosphorylation and acetylation (Bren & Eisenbach, 2000; Baker et al., 2006). CheY autophosphorylates on a conserved aspartyl residue, using its phosphorylated cognate sensor kinase (CheA) as the phosphoryl donor. CheY can also be phosphorylated directly by small phosphodonors such as acetyl phosphate (AcP) (Wolfe, 2005). The kinetics of CheY phosphorylation by small molecules has generally been analyzed by measuring a phosphorylation-associated quenching of intrinsic fluorescence of the single tryptophan residue in CheY, Trp58, which is located adjacent to the site of aspartate phosphorylation, Asp57 (Lukat et al., 1992). This approach was used to measure the rate of CheY phosphorylation by AcP (Silversmith et al., 1997). The phosphorylated form, CheY-P, binds to the switch protein FliM much better than does the nonphosphorylated form at the base of the flagellar motor, which then increases the probability of shifting the direction of flagellar rotation from the default direction, counterclockwise (CCW), to clockwise (CW) (Sagi et al., 2003; Wadhams & Armitage, 2004). Phosphorylated CheY dephosphorylates spontaneously, an activity enhanced by the phosphatase CheZ. This dephosphorylation reduces the binding of CheY to the switch (Blat & Eisenbach, 1996; Bren et al., 1996; Zhao et al., 2002).

Acetylation is another covalent modification that activates CheY to generate clockwise rotation (Barak et al., 1992; Ramakrishnan et al., 1998; Eisenbach, 2004). CheY is acetylated at six lysine residues – lysines 91, 92, 109, 119, 122, and 126, all clustered at the C-terminus of the protein and localized on the surface that interacts with CheA, CheZ, and FliM (Shukla et al., 1998; Welch et al., 1998; McEvoy et al., 1999). Two mechanisms of CheY acetylation have been revealed: AcCoA synthetase (Acs)–catalyzed acetylation using acetate as the acetyl donor (Barak et al., 2004), and autoacetylation using acetyl-CoA (AcCoA) as the acetyl donor (Barak et al., 2006). A third mechanism, acetylation by a currently unknown acetyltransferase, has been suggested (Yan et al., 2008; Li et al., 2010). Two deacetylation mechanisms have been reported: one that depends on Acs, which mediates reversible CheY acetylation (Barak et al., 2004), and a second that depends on the sirtuin CobB. It is likely that the CobB-dependent mechanism predominates in vivo, as defect in cobB leads to defective chemotaxis (Yan et al., 2008; Hu et al., 2010; Li et al., 2010).

In 2004, Barak & Eisenbach reported that two covalent modifications of CheY, phosphorylation and acetylation, are not mutually exclusive but rather affect each other. Recently, the coregulation of CheY was proved to be true in vivo (Li et al., 2010). Furthermore, surface plasmon resonance (SPR) analysis clearly demonstrated that the acetylation represses the binding of CheY to CheA, CheA, and FliM (Li et al., 2010; Liarzi et al., 2010). However, the effect of acetylation on the ability of CheY to undergo autophosphorylate with AcP is not fully investigated. In this work, we successfully expressed and purified CheY protein with a 6 × His-tag on the C-terminus. Subsequently, CheY was acetylated with AcCoA as the acetyl donor, and the acetylation level of AcCheY was determined by Western blotting and then mass spectrometry. To determine whether the acetylation affect CheY autophosphorylation, fluorescence assays were performed. The results are presented herein.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Bacterial strains, growth conditions, and plasmids

Escherichia coli DH5α was used as the host for cloning. The E. coli W3110 CheY protein was expressed using E. coli BL21 (λDE3) strain. Plasmids pET20b (Novagen, Darmstadt, Germany) was used to construct vectors for the expression of E. coli W3110 CheY protein. The medium used was Luria–Bertani (LB) medium. Ampicillin (Ap) (100 mg mL−1) was added if needed. All chemicals were purchased from Sigma (St. Louis) unless otherwise specified.

Construction of protein expression vectors

The chey gene was amplified from E. coli W3110 genomic DNA. The nucleotide sequence of forward and reverse primers for the reaction was 5′-CGCGCATATG (NdeI) GCGGATAAAGAACTTAAATT-3′ and 5′-ATATCTCGAG (XhoI) CATGCCCAGTTTCTCAAAGAT-3′, respectively. The polymerase chain reaction (PCR) product (390 bp) was cloned into the NdeI and (XhoI) sites of pET20b to generate pET20b-chey. The recombinant expression vector was confirmed by restriction enzyme digestion and DNA sequencing, and then introduced into E. coli BL21 (λDE3) by CaCl2 transformation, and the antibiotic-resistant transformants were selected for expression experiment.

Protein overexpression and purification

Escherichia coli BL21(λDE3)/pET20b-chey was grown in 5 mL LB medium, and then, the overnight culture was transferred into 500 mL fresh LB medium containing Ap at 37 °C in shaking flasks to OD600 nm of 0.4. The temperature was adjusted to 18 °C, and the expression was induced by the addition of isopropyl-b-d-thiogalactoside (IPTG) to 0.4 mM. The cells were grown for an additional 10 h at 18 °C, harvested by centrifugation, and then resuspended in binding buffer [20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 5 mM imidazole]. The cell suspension was then sonicated on ice at the intensity of 3 s burst at 200 W with a 5-s cooling period between each burst with an ultrasonic cell disruptor (VCX750; Ningbo Scientz Biotechnology, Ningbo, China). The lysate was centrifuged (12 000 g for 30 min at 4 °C), and supernatant was applied to an affinity Ni2+ column pre-equilibrated with the binding buffer. According to the manufacturer's protocol (Novagen), the column was washed with binding buffer, followed by washing buffer [20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 60 mM imidazole]. The histidine-tagged protein was eluted with elution buffer [20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 200 mM imidazole]. The purity of eluted protein was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the peak fractions containing CheY were pooled and concentrated by ultrafiltration using a 10 000 molecular weight cutoff concentrator (Millipore, Billerica, MA) in storing buffer [50 mM Tris-HCl (pH 8.0)].

Autoacetylation assay

To obtain acetylated CheY, we incubated purified CheY (20 μM) with acetyl-CoA (80 μM, unless otherwise indicated) and 50 mM Tris-HCl buffer (pH 8.0) for 20 h at 35 °C, then separated AcCheY from the low-molecular-mass components by ultrafiltration (Barak et al., 2006; Li et al., 2011). The acetylation level of AcCheY was determined by Western blotting and then mass spectrometry.

Western blot analysis

For Western blot analysis, the samples were separated by SDS–PAGE and then transferred semidry on a Bio-Rad SD device (Bio-Rad Laboratories, Hercules, CA) for 20 min at 15 V to a poly (vinylidene difluoride) membrane. The membrane was blocked overnight at 4 °C in 1 × TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) containing 5% nonfat dry milk (NFDM). Primary rabbit anti-acetylated lysine polyclonal antibody (Cell Signaling Technology, Danvers, MA) diluted 1 : 1000 in TBST/0.5% NFDM was used. After incubation at room temperature for 2 h, the blot was washed with TBST and incubated with 1 : 3000 dilution of alkaline phosphatase-conjugated goat anti-rabbit antibody (Boster Bio-Technology, Wuhan, China) for 1 h at room temperature. The color development was achieved by reacting with chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT).

LC–MS/MS analysis

Prepared digested peptides were analyzed with a Finnigan Surveyor HPLC system coupled online with a LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanospray source. Briefly, the peptide mixtures were loaded onto a C18 column (100 mm i.d., 10 cm long, 5 mm resin from Michrom Bioresources, Auburn, CA) using an autosampler. Peptides were eluted with a 0–35% gradient (Buffer A, 0.1% formic acid and 5% ACN; Buffer B, 0.1% formic acid and 95% ACN) over 80 min and detected online in LTQ-Orbitrap mass spectrometer using a data-dependent TOP10 method (Haas et al., 2006).

Fluorescence assays

Fluorescence measurements were made on a Perkin–Elmer LS55 spectrofluorimeter, and UV WinLab software was used to operate the instrument and analyze data. CheY phosphorylation was monitored by measuring changes in the fluorescence of Trp58, known to be strongly reduced upon CheY phosphorylation (Lukat et al., 1992). The tryptophan fluorescence intensity was measured at an excitation wavelength of 285 nm and an emission wavelength of 348 nm with slit widths of 4 and 7 nm for excitation and emission, respectively. For all reactions, the concentrations for CheY and AcCheY were 10 μM in a buffer containing 50 mM Tris-HCl, pH 8.0, and 10 mM MgCl2. All experiments were carried out at room temperature.

For equilibrium titrations with AcP, the progress of the phosphorylation reactions was plotted as the total phosphodonor concentration in the cuvette vs. ΔI/I0, where ΔI is the cumulative change in tryptophan fluorescence intensity as a result of addition of the given concentration of phosphodonor and I0 is the initial fluorescence intensity of CheY in the absence of phosphodonor. Further analysis of the equilibrium fluorescence titrations was carried out by the method of Lukat et al. (1992).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Protein expression and purification

The chey gene was cloned into the expression vector pET20b, and the recombinant CheY with a 6 × His-tag at the C-terminus was expressed in E. coli. SDS–PAGE analysis of the bacterial transformants revealed that IPTG induced a dominant protein, migrating at 14 kDa. This dominant protein was absent in cells growing in the absence of IPTG (Fig. 1a).

image

Figure 1. SDS–PAGE analysis of expression and purification of CheY protein. (a) SDS–PAGE of CheY expression. Lane 1, the molecular mass marker; lane 2, the whole protein extracts of Escherichia coli BL21(λDE3)/pET20b-cheY without IPTG induction; lane 3, the whole protein extracts of E. coli BL21 (λDE3)/pET20b-cheY induced by IPTG. (b) SDS–PAGE of purified CheY protein. Lane 1, the molecular mass marker; lanes 2 and 3, the purified CheY protein, 14 kDa.

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Ni–NTA His-binding resin affinity chromatography was used to purify the soluble recombinant CheY. SDS–PAGE results (Fig. 1b) showed that a CheY protein fraction was eluted with 200 mM imidazole, and a pure band migrated slightly faster than the 14.4 kDa protein standard, and was consistent with the estimated mass of CheY (14 kDa). Then, the protein was concentrated, and imidazole was fully removed by ultrafiltration with 50 mM Tris-HCl (pH 8.0). Finally, c. 4 mg of the CheY protein was obtained from 500 mL bacterial culture.

CheY autoacetylation

It is reported that when CheY is purified from cells, its acetylation level drops to a very large extent (Yan et al., 2008). Therefore, to achieve a sufficiently high acetylation level of CheY, purified CheY was incubated with AcCoA in vitro. We performed Western blotting analysis to test the acetylation level of CheY using an anti-acetylated lysine antibody. As shown in Fig. 2, the addition of acetyl groups to CheY was shown to be dependent on the concentration of AcCoA.

image

Figure 2. CheY autoacetylation. The acetylation levels of proteins were determined by Western blot using specific anti-acetyl lysine antibody. (a) SDS–PAGE of samples. (b) Western blot of the gel in (a). Lane 1, CheY; lane 2, CheY + 80 μM AcCoA; lane 3, CheY + 160 μM AcCoA; lane 4, the molecular mass marker. The concentration of CheY was 20 μM. The reaction was carried out for 20 h at 35 °C.

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To examine whether the Western blotting analysis indeed reflects autoacetylation of CheY, AcCheY was prepared, enzymatically digested, and analyzed by LC–MS in a LTQ-Orbitrap mass spectrometer. The lysine-acetylated peptide can be conformed as they have a mass increment of 42 Da (Larsen et al., 2006). The acetylated peptide in AcCheY was shown to be KENIIAAAQAGASGYVVK(109)PFTAATLEEK.L and had a molecular mass of 2918.55, +42 Da heavier than the equivalent peptide in CheY (2876.53). The fragment ion signals reflect the amino acid sequence as read from either the N-terminal (b-ion series) or the C-terminal (y-ion series) direction. The acetylated lysine residues can be identified based on the observation of a mass difference of 170 Da between the adjacent b-ions or y-ions (Trelle & Jensen, 2008). As can be seen in Fig. 3, the boxed b(18)++ and y(11)++ ions have a mass of 85 Da greater than the boxed b(17)++ and y(10)++ ions, respectively. This indicates the presence of an acetyl group on lysine 109 in CheY. In addition, the acetylation of N-terminal residue lysine 91 was also observed in AcCheY (data not shown). More detailed data are available in Supporting Information, Tables S1 and S2. Thus, these mass spectrometry results confirm that AcCheY was obtained by CheY autoacetylation with AcCoA as the acetyl donor.

image

Figure 3. LC–MS/MS analysis confirms that lysine 109 is acetylated in AcCheY. The sequence of the tryptic peptide of AcCheY containing lysine 109 is shown with lysine 109 boxed. The b- and y-ion series are shown above and below the sequences, respectively. The boxed b(18)++ and y(11)++ ions have a mass of 85 Da greater than the boxed b(17)++ and y(10)++ ions, respectively. This indicates the presence of an acetyl group on lysine 109.

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Influence of acetylation on the ability of CheY autophosphorylation

In 2004, Barak and Eisenbach found that phosphorylation and acetylation of CheY affect with each other: CheA inhibits acetylation, whereas CheZ enhances it. Conversely, Acs enhances phosphorylation and acetate inhibits this enhancement (Barak & Eisenbach, 2004). Furthermore, acetylation was shown to repress the binding of CheY to its interaction partners (Liarzi et al., 2010; Li et al., 2011). Therefore, we chose to test the direct role of the acetylation in the ability of CheY to autophosphorylate.

To achieve this, we incubated AcP with CheY and AcCheY, respectively, at 25 °C. The ability of CheY phosphorylation was measured in this study by following the fluorescence of the only tryptophan residue of CheY. This residue, Trp58, is located adjacently to the phosphorylation site of CheY, Asp57, and its fluorescence is quenched upon phosphorylation (Blat et al., 1998).

As shown in Fig. 4a, CheY exhibited similar fluorescence emission spectrum with AcCheY in the absence of AcP. As expected, the maximum fluorescence intensity of AcCheY reduced in the presence of 20 mM AcP at an emission wavelength of 348 nm. In comparison, the decreasing fluorescence intensity of CheY was more significant than AcCheY. Furthermore, equilibrium fluorescence titration of both CheY and AcCheY with AcP gave different results (Fig. 4b). The degree of fluorescence quench with saturating AcP concentrations was different for the two proteins; AcCheY quenched to about 20% less than CheY. Analysis of these data rendered different Km values: 1.3 ± 0.2 mM and 5.0 ± 0.1 mM for CheY and AcCheY, respectively. Thus, the phosphodonor AcP was able to phosphorylate CheY at a concentration about fourfold lower than AcCheY. These results therefore suggest that acetylation reduces the ability of CheY to undergo autophosphorylate with AcP as the donor.

image

Figure 4. Autophosphorylation of CheY and AcCheY with AcP as phosphodonor. The concentrations for CheY and AcCheY were 10 μM. All experiments were carried out at room temperature (25 °C). Experiments were replicated three times, and representative results are shown. (a) Fluorescence emission spectrum of CheY and AcCheY. The tryptophan fluorescence intensity was measured at an excitation wavelength of 285 nm. The concentration of AcP was 20 mM. (b) Equilibrium phosphorylation. The relative decrease in fluorescence intensity (expressed as ΔI/I0) upon sequential addition of AcP to a single sample of CheY is shown. Data were expressed as mean ± SD of three independent experiments. (□) CheY; (●) AcCheY.

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Linkage between phosphorylation and acetylation is not specific to CheY. It is also shown in histones and eukaryotic transcription factors. Several studies demonstrated various kinds of relationships between acetylation and phosphorylation, including inhibition of acetylation by phosphorylation (Lambert et al., 1998; Kumar et al., 2001; Edmondson et al., 2002) and acetylation of histones inhibited H2A phosphorylation (Zhang et al., 2004). In the case of CheY, phosphorylating agent CheA inhibits CheY autoacetylation (Barak & Eisenbach, 2004). Acetylation could repress the binding of CheY to its target proteins. Therefore, Liarzi et al. (2010) proposed that both phosphorylation and acetylation determine CheY's ability to bind to its target proteins, thus providing two levels of regulation, fast and slow, respectively. Acetylation would be a way to make chemotaxis less active so that it will consume less energy.

In this study, tryptophan fluorescence intensity analysis, a sensitive technique for measuring phosphorylation, clearly demonstrates that the acetylation of CheY reduces its ability to undergo autophosphorylation. This observation may be the consequence of one or both of the following hypothetical possibilities. (1) The acetyl groups on CheY may affect the structure of the protein. As shown in Fig. 5, Lys109 is known to form a salt bridge with the phosphorylation site, Asp57 (Lukat et al., 1991). The acetylation of Lys109 may lead to a conformational change in the protein due to the missing salt bridge, and this change may interfere the phosphorylation of Asp57. (2) Based on the kinetic characterization of CheY mutant protein, K109R shows decreased phosphorylation rate relative to that of wild-type CheY. Thus, the Lys 109 is important for the phosphotransfer to CheY (Silversmith et al., 1997). This notion is consistent with the observation that AcCheY had a weaker phosphorylation rate than CheY.

image

Figure 5. Cartoon diagram of the structure of CheY protein (Protein Data Bank code 1CYE). Six lysine residues – K 91, 92, 109, 119, 122, and 126 – are shown in cyan. Broken black lines highlight salt bridge between K109 and D57.

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Together, our data show a new function of acetylation in CheY autophosphorylation by tryptophan fluorescence intensity measurement. These results may provide more insights into the role of acetylation in bacterial chemotaxis. Exactly how CheY acetylation interfere the ability of CheY to undergo autophosphorylation, however, is the subject of future studies.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

This work was supported by the National Natural Science Foundation of China (Grant No. 31070049 and 31260027).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results and discussion
  6. Acknowledgement
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
fml12224-sup-0001-TableS1-S2.docWord document105KTable S1.

Mass spectrometry confirms Lysine-91 is acetylated on AcCheY.

Table S2. Mass spectrometry confirms Lysine-109 is acetylated on AcCheY.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.