Biochemical Study of Multiple CheY Response Regulators of the Chemotactic Pathway of Rhodobacter sphaeroides

Abstract

The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac³²P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.

The paradigm of the bacterial chemotactic response is the signal transduction pathway found in enteric bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium. This system regulates the direction of flagellar rotation through the interaction of the phosphorylated response regulator, CheY-P, with the switch protein FliM, which is a key process in bacterial chemotaxis (for reviews, see references 1, 7, 12, and 26). When most of the flagella of a cell rotate in the counterclockwise direction, they coalesce in a bundle that pushes the cell body in a linear trajectory called run. When flagella reverse the sense of rotation to clockwise, the bundle loses stability, and the uncoordinated motion of the filaments produces a tumble that reorients the cell. Therefore, the frequency of tumble determines the overall swimming direction of the cell. Mutational and genome analyses have identified multiple homologues of the signal transduction proteins in several bacterial species, i.e., Agrobacterium tumefaciens, Rhodobacter sphaeroides, and Sinorhizobium meliloti. S. meliloti has two CheY homologues: CheY2 modulates flagellar rotation, whereas CheY1 does not. CheA phosphorylates both CheY1 and CheY2 and, when CheA becomes inactive, CheY1 acts as the signal terminator by dephosphorylating CheY2, replacing the phosphatase CheZ and acting as a phosphate sink (23).

R. sphaeroides is a facultative non-sulfur-photosynthetic bacterium that belongs to the α-subgroup of the proteobacteria. It is able to grow photoheterotrophically and chemoheterotrophically, to utilize a wide range of organic acids for growth, and to fix nitrogen and CO₂. Unlike E. coli and S. enterica, R. sphaeroides has a single subpolar flagellum that propels the cell by CW rotation stopping randomly producing reorientation of the bacterium. During the stop periods, the filament retracts into a coiled form (3). It has been suggested that the slow motion of the filament in this conformation helps reorient the cell (4). Multiple homologues of the chemotactic genes have been found in R. sphaeroides, including six cheY, four cheA and cheW, three cheR, and two cheB copies. The gene encoding the phosphatase cheZ has not been identified (11).

Bacterial strains, plasmids, and oligonucleotides are listed in Table 1. Luria broth was prepared as previously described (5). All chemicals used were reagent grade. R. sphaeroides cell cultures were grown in Sistrom's solid or liquid medium (21) under continuous illumination at 30°C for photoheterotrophic growth or in the dark with strong shaking. Recombinant DNA techniques were carried out by standard procedures (5). Reverse transcription-PCR (RT-PCR) was performed with specific primers that were synthesized by Sigma-Genosys.

TABLE 1.

Bacterial strains, plasmids, and oligonucleotides

Strains, plasmids, and oligonucleotides	Relevant characteristicsa	Source or reference
Strains
E. coli
JM103	hsdR4 Δ(lac-pro) F′ traD36 proAB laclqZΔM15	5
JM109	hsdR17 Δ(lac-pro) F′ traD36 proAB laclqZΔM15	5
XL1-Blue	supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac F′ [proAB+laclqlacZΔM15 Tn10(Tetr)]	5
M15(pREP4)	thi lac ara+gal mtl F′ recA+uvr+lon+; pREP4 plasmid Kanr	Qiagen
R. sphaeroides WS8	Wild type; spontaneous Nalr	22
Plasmids
pQE30	Expression vector; Apr, N-terminal His6 tag	Qiagen
pQE60	Expression vector; Apr, C-terminal His6 tag	Qiagen
pRSM	fliM cloned into the NcoI/BglII sites of pQE60	This work
pRSMΔ13	fliMΔ13 cloned into the NcoI/BglII sites of pQE60	This work
pRSMΔ20	fliMΔ20 cloned into the NcoI/BglII sites of pQE60	This work
pRSY1	cheY1 cloned into the NcoI/BamHI sites of pQE60	This work
pRSY2	cheY2 cloned into the SacI/PstI sites of pQE30	This work
pRSY3	cheY3 cloned into the BamHI/PstI sites of pQE30	This work
pRSY4	cheY4 cloned into the NcoI/BamHI sites of pQE60	This work
pRSY5	cheY5 cloned into the BamHI/PstI sites of pQE30	This work
pRSY6	cheY6 cloned into the BamHI/PstI sites of pQE30	This work
Oligonucleotidesb
cheY1 fw	5′-CATGCCATGGCGCCGCTGACCGTTCTTGCC-3′	This work
cheY1 rev	5′-CTCACGGACTGCTCGGATGAAGCC-3′	This work
cheY2 fw	5′-CCGAGCTCGTGGTCGATGACATGTCGACCAGC-3′	This work
cheY2 rev	5′-AAAACTGCAGTCATAGAGCGCCTACGACAGC-3′	This work
cheY3 fw	5′-CGCGGATCCAGCAGGACGGTTCTCGCCGTG-3′	This work
cheY3 rev	5′-AAAACTGCAGTCATCCGAGCACCTTCTTGAT-3′	This work
cheY4 fw	5′-CATGCCATGGCGACGAAAACCGTCCTCGCA-3′	This work
cheY4 rev	5′-CGCGGATCCGGCCAAGAAGCTTCTTCATCAC-3′	This work
cheY5 fw	5′-CGCGGATCCAGCAAGACGATCCTCGCGGTGGAT-3′	This work
cheY5 rev	5′-AAAACTGCAGTCAGGCGCCGGCCACCTTCTTGAC-3′	This work
cheY6 fw	5′-GCGGGATCCCCCTACAATGTCATGATC-3′	This work
cheY6 rev	5′-AAAACTGCAGTCAGGCGGCCATCAGCGT-3′	This work

^aAp^r, ampicillin resistance; Kan^r, kanamycin resistance; Nal^r, nalidixic acid resistance.

^bfw, forward; rev, reverse.

Multiple alignment of the R. sphaeroides CheY proteins (CheY_Rs) with CheY from S. enterica (CheY_St) shows extensive overall homology through the complete protein sequence. Conserved patterns of α-helix, β-strand, and random coil were predicted from the sequence by using the PSIPRED program (Fig. 1). This protein is a doubly wound α/β protein with a central five-stranded parallel β-sheet surrounded by five α-helices (25, 29). The phosphorylation site in CheY_St is Asp57 (19), which is located in the solvent-exposed loop between β3 and α3. This residue lies adjacent to other acidic residues, Asp12 and Asp13, which are involved in coordination of the Mg²⁺ ion required for phosphoryl transfer and dephosphorylation (9, 10, 15). Two residues, Thr87 and Lys109, complete the conserved cluster surrounding the active site of the regulatory domain and are thought to participate in the phosphorylation-induced conformational change in CheY (2, 9, 32). The six CheY_Rs proteins show these highly conserved amino acid residues that are present in Salmonella CheY. The phosphorylation site Asp57, in addition to Asp12, Asp13, and Lys109, is conserved in the six R. sphaeroides CheY proteins. Thr87 is the exception, given that it is present only in CheY1, whereas CheY2, CheY3, CheY4, and CheY5 contain Ser that conserves the neutral character of the Thr residue, as well as an OH group as a lateral chain. Apparently, CheY6 contains nonpolar Val in this position (Fig. 1), although it could be possible that the preceding Ser is out of register in our alignment.

FIG. 1.

Multiple alignment of the six CheY proteins from R. sphaeroides and CheY from S. enterica serovar Typhimurium. Sequence alignment was performed by using PILEUP (GCG-Wisconsin Package, version 9.1). Secondary structure prediction was carried out by using the program PSIPRED (8, 14). Identical residues are shaded in tones of black. Asterisks indicate relevant residues for the function of CheY_St.

Analysis of expression of the multiple cheY genes under two different metabolic conditions.

The expression of the mRNAs of the six cheY genes found in R. sphaeroides was addressed by using RT-PCR, and the results are shown in Fig. 2. Cells were grown either heterotrophically or photoheterotrophically, and total RNA was isolated by using standard reported procedures (5). Specific primers for each one of the cheY genes were tested to determine their expression (Table 1). Figure 2 shows that they are similarly expressed under the two growth conditions. Nevertheless, the expression of cheY5 under heterotrophic conditions is notably reduced, suggesting that its expression could be regulated by the prevailing environmental conditions. If no posttranscriptional control of the expression of these proteins exists, it can be assumed that the six proteins are present in the cytoplasm simultaneously, especially under phototrophic conditions. This could imply that a fine-tuning regulation of the chemotactic response in R. sphaeroides requires the simultaneous presence of all of the chemotactic response regulators.

FIG. 2.

RT-PCR of the six R. sphaeroides cheY transcripts. Total RNA was isolated from WS8 wild-type cells. One-fifth of each of the PCRs was loaded onto a 7.5% polyacrylamide gel. Lanes 1 to 6, show the amplification products of cheY1-6, respectively, and the expected sizes of these products are for 207 bp for cheY1, 354 bp for cheY2, 360 bp for cheY3, 360 bp for cheY4, 363 bp for cheY5, and 399 bp for cheY6. Lane 7 shows a control, with the oligonucleotides for amplification of cheY6, lacking avian myeloblastosis virus reverse transcriptase in the reaction.

Purification and characterization of the CheY_Rs proteins.

The cheY genes were amplified by PCR and cloned into pQE60 expression vector, which attaches a His₆ tag at the C terminus of the protein (CheY1 and CheY4) or pQE30, which attaches a His₆ tag at the N terminus of the protein (CheY2, CheY3, CheY5, and CheY6). These proteins were expressed in either JM103, XL1-Blue or M15(pREP4) strains (Table 1) and purified according to standard procedures (Qiagen, Inc., Valencia, Calif.). His-tagged CheY proteins were purified to homogeneity by Ni-nitrilotriacetic acid affinity chromatography. The six CheY proteins migrate as a single band of molecular mass of ∼14,400 Da (data not shown). The structural integrity of purified CheY_Rs proteins was assessed by analyzing their secondary structure by means of far UV circular dicroism (CD) spectra. Scans from 190 to 260 nm were performed on the purified CheY proteins. The CD spectra show that all CheY proteins maintained their structural integrity during purification and contain the secondary structure motifs that characterize these response regulators. It should be noted that CheY2 was the only protein recovered from inclusion bodies after denaturation and refolding. As a result of the refolding procedures, this protein shows a slight shift toward far UV wavelengths, which indicates that a fraction of refolded CheY2 acquires a different structural conformation (data not shown).

Effect of small-molecule phosphodonor AcP on the six CheY proteins of R. sphaeroides.

We tested the ability of the CheY_Rs proteins to become phosphorylated by the small-molecule phosphodonor acetyl phosphate (AcP). Since four of the six CheY_Rs proteins contain a tryptophan residue (Trp106) that can be used to report conformational changes, we determined intrinsic fluorescence changes due to phosphate binding to the conserved aspartic residue, Asp57. Figure 3 shows that fluorescence intensity decreases upon the addition of 25 mM AcP to the reaction medium in three of the four CheY proteins that contain Trp106 (CheY3, CheY4, and CheY5). CheY1 that also contains Trp106 behaves as CheY2 or CheY6 that contain phenylalanine or valine residues, respectively, in the same position. Since in CheY_Ec the substitution Tyr106Trp renders a tumbly phenotype (31), it would be interesting to determine whether Trp106 in CheY_Rs favors switching. Since fluorescence quenching of CheY3, CheY4, and CheY5 is dependent on the presence of Mg²⁺ (data not shown), this assay seems useful for detecting phosphorylation or changes associated with phosphorylation for at least these three CheY proteins. In addition, we determined in vitro phosphorylation by Ac³²P of the CheY_Rs proteins according to procedures previously reported (27). The reactions were stopped after 1 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phosphorimaging. Radioactive labeling of five of the six CheY proteins was observed, indicating that AcP is indeed a phosphodonor for the CheY_Rs proteins (Fig. 4). As mentioned above, CheY2 showed an altered CD spectrum, due possibly to the fact that during renaturation from inclusion bodies the native α/β structures were partially lost, abolishing the ability to become phosphorylated by Ac³²P. A lower level of ³²P labeling of CheY3 and CheY6 was observed. This can be explained given that CheY3 has a lower affinity for AcP (data not shown) and CheY6 has a lower dephosphorylation half-time (17).

FIG. 3.

Intrinsic fluorescence of the R. sphaeroides CheY proteins. Purified CheY proteins were mixed with 25 mM AcP never exceeding 4% of the initial volume, and kinetic determinations were performed as described previously (13). Given that each CheY displays a different fluorescence intensity, emission was normalized for each protein to an arbitrary intensity of 1.0.

FIG. 4.

CheY phosphorylation by AcP. A phosphorimage of the six CheY proteins incubated with Ac³²P for 1 min is shown. The reaction was stopped by the addition of 5 μl of 5× sample buffer for SDS-PAGE. Ac³²P was synthesized as described previously (24).

Phospho-CheY binding to the switch protein FliM.

We tested whether these response regulators could interact with purified FliM_Rs. For this purpose, the coding region of fliM_Rs was amplified by PCR, cloned in pQE60, and expressed in E. coli JM109. Inclusion bodies were pelleted by centrifugation and solubilized in a buffer containing 5.5 M guanidine-HCl. His-tagged FliM was purified by Ni-nitrilotriacetic acid affinity chromatography under denaturing conditions and refolded by dilution in a buffer containing 100 mM Tris-HCl (pH 7.9), 500 mM NaCl, 2 M urea, 2.5 mM reduced and 0.5 mM oxidized glutathione, and 20% glycerol, followed by dialysis against TNG buffer (50 mM Tris-HCl [pH 7.9], 150 mM NaCl, 10% glycerol). Purified CheY proteins were immobilized on CNBr-activated Sepharose beads CL-4B (Pharmacia, Uppsala, Sweden) as previously described (30). Control beads contained covalently bound bovine serum albumin. CheY beads were incubated with FliM_Rs in the presence or absence of AcP (25 mM) and MgCl₂ (5 mM) and subjected to SDS-PAGE and immunoblotting with polyclonal rabbit antibodies raised against FliM_Rs (the present study). Immunoblotting with anti-His antibodies confirmed that all of the experiments were performed with comparable amounts of CheY (data not shown). Figure 5 shows that the six CheY proteins bind FliM in the absence of AcP and that the addition of this phosphodonor results in a two- to threefold increase in FliM binding. This result indicates that the contact surface of CheY that interacts with FliM is well conserved in all of the CheY proteins. To explain the apparent contradiction between the absence of labeling of CheY2 by Ac³²P (see Fig. 4) and the effect of AcP on FliM binding, we consider that covalent binding of CheY2 to CNBr-activated Sepharose beads possibly stabilizes a conformation that allows this protein to become phosphorylated by AcP. The specificity of binding of the different CheY proteins was addressed by using two deletion mutants lacking the first 13 or 20 residues of the N-terminal region of FliM_Rs (16). This region has been identified in FliM_St and FliM from Bacillus subtilis as the binding site of the response regulator CheY (6, 27, 28). Both fliMΔ13 and fliMΔ20 were cloned into pQE60, purified as for wild-type FliM_Rs, and subjected to the binding assay described above. We found that deletion of the N-terminal 13 or 20 amino acids of FliM reduces its binding capacity significantly. It should also be noted that anti-FliM_Rs antibodies recognize the two deletion mutants with equal efficiency (Fig. 5).

FIG. 5.

Binding of CheY and phospho-CheY to FliM. The six CheY proteins immobilized to Sepharose beads were assayed for binding to FliM, FliMΔ13, and FliMΔ20 in the presence or absence of AcP. Control lane shows nonspecific binding of FliM to bovine serum albumin-Sepharose beads. Immunoblots were probed with anti-FliM_Rs antibodies. The added purified FliM, FliMΔ13, and FliMΔ20 proteins are shown for reference.

Binding is the first step to control flagellar rotation. Studies carried out in E. coli indicated that switching is altered without modification of the binding affinity of CheY for FliM. From these studies Tyr106 has been identified as a relevant residue for signaling (31, 32). As mentioned above, CheY1, CheY3, CheY4, and CheY5 contain Trp in this position, whereas CheY2 and CheY6 contain Phe and Val residues, respectively. In spite of having Trp106, CheY1 did not show fluorescence quenching upon addition of AcP. It is possible that the conformational change provoked by phosphorylation does not affect the environment of Trp106; thus, the fluorescence remains unchanged. Further study should be carried out to determine whether Trp106 is critical for signaling in these response regulators.

The existence of at least one CheY acting as a phosphate sink is a central idea in the present model proposed to explain the chemotactic response in R. sphaeroides (18, 20). Our data do not contradict this idea, provided that the CheY proteins that act as phosphate sinks bind to the flagellar motor without promoting switching.

It can be hypothesized that in R. sphaeroides the chemotactic control is carried out by the concerted action of the different CheY proteins. By the same token, it is possible that some CheY proteins bind FliM without promoting switching thus competing for motor occupancy. This kind of control would require a complex balance among all of the reactions in which these response regulators are involved (i.e., binding to FliM and to CheA, phosphorylation and dephosphorylation rates, etc.). Therefore, the biochemical characterization of these parameters seems compulsory for understanding this complex response. In this regard, determination of the affinity of each CheY protein for FliM is currently one of our main interests.

It has been previously shown that R. sphaeroides strains expressing mutant FliM proteins (Δ13 or Δ20) showed a stopped phenotype. From these results it was suggested that the binding of CheY to FliM promotes CW rotation hence swimming, whereas the lack of interaction between FliM and CheY induces a stop event (16). In the light of the results reported here, it is conceivable that the stop events could also occur by the binding of a CheY protein that does not promote clockwise rotation. We are currently investigating this possibility.