Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

Vincent J Cannistraro; George D Glekas; Christopher V Rao; George W Ordal

doi:10.1128/JB.01255-10

. 2011 Jul;193(13):3220–3227. doi: 10.1128/JB.01255-10

Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis^{^▿}

Vincent J Cannistraro ^1,^§, George D Glekas ^1,^§, Christopher V Rao ², George W Ordal ^1,^*

PMCID: PMC3133262 PMID: 21515776

Abstract

The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B. subtilis. Significantly higher levels of chemoreceptors and much lower levels of CheA kinase were measured in B. subtilis than in Escherichia coli. The resulting cellular ratio of chemoreceptor dimers per CheA dimer in B. subtilis is roughly 23.0 ± 4.5 compared to 3.4 ± 0.8 receptor dimers per CheA dimer observed in E. coli, but the ratios of the coupling protein CheW to the CheA dimer are nearly identical in the two organisms. The ratios of CheB to CheR in B. subtilis are also very similar, although the overall levels of modification enzymes are higher. When the potential binding partners of CheD are deleted, the levels of CheD drop significantly. This finding suggests that B. subtilis selectively degrades excess chemotaxis proteins to maintain optimum ratios. Finally, the two cytoplasmic receptors were observed to localize among the other receptors at the cell poles and appear to participate in the chemoreceptor complex. These results suggest that there are many novel features of B. subtilis chemotaxis compared with the mechanism in E. coli, but they are built on a common core.

INTRODUCTION

Motile organisms, such as the soil-based bacterium Bacillus subtilis, have the ability to sense their chemical environment and move to more favorable conditions through a process known as chemotaxis. The chemotactic pathway in B. subtilis involves 10 different chemoreceptors and eight soluble proteins (for a review, see reference 43). The core of the chemotaxis system contains the histidine kinase CheA and the coupling protein CheW (7, 15). These proteins interact with the typically membrane-bound chemoreceptors, or MCPs (methyl-accepting chemotaxis proteins), which can sense various ligands in the extracellular environment (16, 33). Once attractant binds to the receptor, CheA autophosphorylates and then transfers its phosphoryl group to CheY, the response regulator (2, 8). Phosphorylated CheY (CheYp) binds to the flagellar motor and changes the direction of its rotation, which leads to a change in the swimming behavior of the bacterium.

B. subtilis employs three adaptation systems to sense chemical gradients (35). The methylation system consists of CheR, a methyltransferase, and CheB, a methylesterase, enzymes that, respectively, add and remove methyl groups at specific glutamate residues on the MCPs (4, 12, 13). A second adaptation system, the CheC-CheD-CheY system, consists of three proteins. CheC has been shown to weakly dephosphorylate CheYp, and its activity is enhanced in the presence of CheD (32, 38, 42). CheD, apart from interacting with CheC, can also interact with the chemoreceptors and deamidate specific glutamine residues on the MCPs, meaning it has two binding partners in the cell (24, 37). The third adaptation system contains CheV, a hybrid protein with a CheW-like coupling domain and a CheY-like response regulator domain that is phosphorylated by CheA (19).

The concentrations and ratios of the chemotaxis proteins in B. subtilis are not known. Using quantitative immunoblotting, we determined the concentrations and stoichiometries of all 10 chemoreceptors and the eight soluble chemotaxis signaling proteins in this study. We also examined the cellular localization of the two soluble chemoreceptors using fluorescence microscopy.

MATERIALS AND METHODS

Plasmid and strain construction.

All of the B. subtilis strains are derived from the chemotactic strain (che⁺) OI1085 (45). All cloning and plasmid propagation was performed in Escherichia coli strains TG1 (GE Healthcare) and JM101 (New England BioLabs). Recombinant protein overexpression was done in E. coli strain BL21 or BL21(DE3) (GE Healthcare). All of the strains and plasmids used in this study are listed in Tables 1 and 2.

Table 1.

Strains used in this study

Strain	Relevant genotype or description	Reference
OI1085	Che⁺; trpF7 hisH2 metC133	46
OI1840	cheA::cat	7
OI2057	ΔcheY	3
OI2652	cheR::cat	21
OI2836	cheB8::cat	22
OI2934	cheD1::cat	37
OI3627	cheB7 unkU29::spc (cheC cheD)501::cat cheR3::cat (cheRBCD)	39
OI4356	mcpA-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4357	mcpB-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4358	mcpC-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4359	tlpA-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4360	tlpB-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4361	tlpC-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4362	yfmS-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4363	yoaH-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4368	yvaQ-6×His gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4369	yvaQ-6×His gene fusion; Che⁺; trpF7 hisH2 metC133 cheB8::cat	This work
OI4370	yvaQ-6×His gene fusion; Che+; trpF7 hisH2 metC133 cheD1::cat	This work
OI4371	yvaQ-6×His gene fusion; Che⁺; trpF7 hisH2 metC133 cheR::cat	This work
OI4372	yvaQ-6×His gene fusion; Che⁺; trpF7 hisH2 metC133(cheRBCD)	This work
OI4373	yfmS-YFP gene fusion; Che⁺; trpF7 hisH2 metC133	This work
OI4374	hemAT-YFP gene fusion; Che⁺; trpF7 hisH2 metC133	This work
HB4007	cheW::cat cheV::kan	36
BL21	E. coli protease-deficient expression host	Amersham
BL21(DE3)	E. coli protease-deficient expression host	Novagen
TG1	E. coli cloning host	Amersham
JM101	E. coli K-12 dam DNA methyltransferase mutant	New England Biotechnology

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Table 2.

Plasmids used in this study

Plasmid	Description	Reference
pBluescriptSK(−)	E. coli cloning vector; Amp^r	Stratagene
pGEX-6P-2	E. coli GST tag expression vector; Amp^r	Amersham
pET-42b(+)	E. coli His tag expression vector; Amp^r	Novagen
pMUTIN-YFP	E. coli YFP fusion vector; Amp^r Em^r	18
pMUTIN-FLAG	E. coli FLAG fusion vector; Amp^r Em^r	18
pMUTIN-HIS	E. coli His tag fusion vector; Amp^r Em^r	This work
pHS101	pGEX-6P-2::cheA1	41
pHS102	pGEX-6P-2::cheY1	41
pUSH1	B. subtilis-E. coli shuttle vector for His tag fusions	40
pAIN620	pUSH1 expressing His6-McpA cytoplasmic domain	24
pMH1	pMUTIN-HIS::mcpA	This work
pMH2	pMUTIN-HIS::mcpB	This work
pMH3	pMUTIN-HIS::mcpC	This work
pMH4	pMUTIN-HIS::tlpA	This work
pMH4	pMUTIN-HIS::tlpB	This work
pMH5	pMUTIN-HIS::tlpC	This work
pMH6	pMUTIN-HIS::yfmS	This work
pMH7	pMUTIN-HIS::yoaH	This work
pMH8	pMUTIN-HIS::yvaQ	This work
pTM18	pGEX-6P-2::cheC2	42
pTM25	pGEX-6P-2::cheD2	42
pVC13	pGEX-6P-2:: hemAT	This work
pVC15	pGEX-6P-2:: cheV	This work
pVC20	pET-42b(+)::cheR	This work
pVC21	pET-42b(+)::yfmS	This work
pVC43	pMUTIN-YFP::yfmS	This work
pVC51	pMUTIN-YFP::hemAT	This work

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The plasmid pMUTIN-HIS was generated by PCR using pMUTIN-FLAG as a template (18). The primers were designed to mutate the last 6 amino acids of the FLAG peptide Lys-Asp-Asp-Asp-Asp-Lys to His-His-His-His-His-His. The pMUTIN-HIS vector was then transformed into the E. coli strain JM101 to yield unmethylated vector DNA. The ClaI restriction site in the multiple cloning site of pMUTIN-HIS cannot be cleaved in methylated DNA, and subsequent pMUTIN-HIS restriction digests were performed in JM101-propagated plasmids.

All the pMUTIN-HIS and pMUTIN-YFP plasmid constructs were made by amplifying the last 300 bp of the select chemoreceptor genes by PCR using genomic OI1085 DNA as the template. The primers were also designed to include KpnI and ClaI restriction enzyme recognition sites for subsequent cloning into the pMUTIN-HIS and pMUTIN-YFP vectors. The final product contains the last 300 bp of the specific gene, followed by either a 6×His tag or a yellow fluorescent protein (YFP) gene fusion.

The plasmids pVC13 and pVC15 were constructed by amplifying the full-length hemAT and cheV genes from OI1085 genomic DNA using PCR and introducing 5′-BamHI and 3′-EcoRI restriction sites for subsequent cloning into the pGEX-6P2 vector (GE Healthcare). The plasmids pVC20 and pVC21 were constructed by amplifying the full-length cheR and yfmS genes from OI1085 genomic DNA using PCR and introducing 5′-NdeI and 3′-XhoI restriction sites, respectively. The PCR products were then ligated into pET-42b(+) (Novagen) and transformed into strain BL21(DE3) for protein expression.

Protein purification.

All proteins were purified as previously described (42). The glutathione S-transferase (GST) fusion expression plasmids (pGEX-6P-2 variants) were expressed in E. coli strain BL21. GSTrap columns (5 ml; GE Healthcare) were used with an Akta Prime system (GE Healthcare) for purification as prescribed by the manufacturer.

To purify the GST fusion proteins, pGEX-6P-2 with the assorted chemotaxis proteins cloned into the multiple-cloning site were grown in 2 liters of LB (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) with 100 μg/ml ampicillin at 37°C and shaking at 250 rpm until the A₆₀₀ was equal to 0.8. Expression was then induced by the addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), and the culture was grown at 25°C with 250 rpm shaking for 12 h. For CheA and FliY, the cultures were induced at 37°C for 4 h, and for CheY, the cultures were induced at 14°C for 12 h.

The 6×His fusion expression plasmids (pET-42b variants) were expressed in E. coli strain BL21(DE3). HisTrap columns (5 ml; GE Healthcare) were used with an Akta Prime system (GE Healthcare) for purification, generally as the manufacturer described. Growth and induction conditions were identical to the conditions for the GST fusion constructs. Cells were collected by centrifugation at 10,000 × g for 10 min. The cell pellet was then resuspended in 3 ml of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) for every 1 g of cell pellet. The cells were then disrupted by sonication (5 10-s pulses), the cell debris was removed by centrifugation at 15,000 × g for 15 min, and the lysate was further centrifuged for 30 min at 75,000 × g. Finally, for further clarification, the lysate was passed through a 0.2-μm filter to remove any other aggregates or insoluble particles. The cell lysate was then passed through a 5-ml HisTrap column and washed with at least 5 bed volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0). The fusion proteins were eluted from the column with a 0 to 100% gradient of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). Fractions were collected and run on an SDS-PAGE gel to check for the elution of the desired 6×His fusion protein. The pure protein flowthrough was collected and dialyzed against four changes of 1 liter of TKMD (50 mM Tris, pH 8.0, 50 mM KCl, 5 mM MgCl₂, 0.1 mM dithiothreitol [DTT], 10% [vol/vol] glycerol) and then stored at −80°C.

Protein quantification.

All proteins were quantified with the BCA Protein Assay Kit (Thermo Scientific) with a bovine serum albumin (BSA) standard curve. To confirm the values obtained by the bicinchoninic acid (BCA) method, the protein concentrations were confirmed using two other methods. In the first, the proteins were run over a C₄ reverse-phase high-performance liquid chromatography (RP-HPLC) column. Their concentrations were quantified by UV absorption measurements at 214 nm using the molar extinction coefficient calculated from the ratio of all 20 amino acids and the peptide bonds present in each protein (25). The second method involved measuring the absorption at both 280 and 288 nm under denaturing conditions, based on the molar extinction coefficient calculated by the ratio of tryptophan, tyrosine, and cysteine residues present in each protein (14). These two independent methods generated protein concentrations that correlated very well with the BCA results.

Western blot preparation.

The bacterial strains that were used for quantitative Western blot analysis were grown on tryptose blood agar base (TBAB) plates and incubated overnight at 30°C. Colonies from these plates were then used to start 50-ml day cultures in capillary assay minimal medium plus tryptone broth (CAMM+) [50 mM K₃PO₄, pH 7.0, 1.2 mM MgCl₂, 0.14 mM CaCl₂, 1 mM (NH₄)₂SO₄, 0.01 mM MnCl₂, 0.2% (vol/vol) tryptone broth (TBr), 20 mM sorbitol, 50 μg/ml (each) histidine, methionine, and tryptophan] with an initial A₆₀₀ of 0.02. The cells were incubated at 37°C with aeration until they reached mid-exponential phase (approximately 6 h.). The cells were then diluted 1:10 (vol/vol) into 50 ml of capillary assay minimal medium (CAMM) [50 mM K₃PO₄, pH 7.0, 1.2 mM MgCl₂, 0.14 mM CaCl₂, 1 mM (NH₄)₂SO₄, 0.01 mM MnCl₂, 20 mM sorbitol, 50 μg/ml (each) histidine, methionine, and tryptophan] and further incubated at 37°C until they reached mid-exponential phase (approximately 6 h.). The cells were then diluted an additional time to an A₆₀₀ of 0.01 and were incubated until they reached mid-exponential phase (approximately 10 h) in CAMM. The culture was then diluted 1:10 (vol/vol) into multiple flasks (to a total volume of 50 ml) and returned to the incubator until an A₆₀₀ of 0.6 was reached in CAMM. The cells were then pelleted, washed once with 5 ml of protoplast buffer, diluted to an A₆₀₀ of 1.0 in 10 ml of protoplast buffer (25 mM K₃PO₄, pH 7.0, 20% [wt/vol] sucrose, 10 mM MgCl₂, 1 mM EDTA, 30 mM sodium lactate) with 250 μg/ml chloramphenicol and 4 mg/ml lysozyme, and incubated at 37°C for 30 min. Protoplasts were collected by centrifugation and resuspended in 1,000 μl of SDS solubilizer buffer (1×). The number of cells in each lysozyme digest was calculated by serial dilution before the addition of lysozyme or chloramphenicol. There were 4 × 10⁷ cells loaded per lane of an SDS-PAGE gel, except for the CheW (1.04 × 10⁷), McpA (3.94 × 10⁶), and YoaH (4.26 × 10⁷) gels.

Purified protein standards were also made for each quantitative Western blot to give a range of concentrations that was previously determined to be the approximate range for the protein of interest. The amounts loaded on the gel for each protein standard curve were as follows: 20 to 200 fmol CheA, 10 to 200 fmol CheB, 20 to 250 fmol CheC, 20 to 200 fmol CheD, 10 to 100 fmol CheR, 50 to 500 fmol CheV, 1 to 20 fmol CheW, 10 to 1,000 fmol CheY, 200 to 2,000 fmol HemAT, 50 to 500 fmol YfmS (for YfmS-only quantification), 10 to 50 fmol YfmS-His (for 6×His tag receptor quantification), 1 to 25 fmol YfmS-His (for McpA-His quantification), and 1 to 20 fmol YfmS-His (for YoaH-His quantification). Cell lysates from the corresponding deletion strain were also added to the purified protein standard lanes at concentrations identical to that of the deletion-strain-only lanes to compensate for any variation in antibody affinity in lanes either with or without whole-cell lysates.

Western blot analysis.

Ten microliters of the samples was separated by 12% or 4 to 20% gradient SDS-PAGE and subsequently transferred to a polyvinylidene difluoride (PDVF) membrane (Millipore) by semidry transfer for 1 h at 25 V. The membrane was blocked for 1 to 2 h with blocking buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 5% [wt/vol] milk powder, 0.05% Tween 20); primary antibody was added and incubated for 15 h. Antibody concentrations varied in the degree of specificity, and thus the following dilutions were used: 1:500,000 anti-CheA, 1:200,000 anti-CheB, 1:2,000 anti-CheC, 1:2,000 anti-CheD, 1:500,000 anti-CheR, 1:50,000 anti-CheV, 1:500,000 anti-CheW, 1:200,00 anti-CheY, 1:500,000 anti-HemAT, and 1:1,000,000 anti-YfmS. The antibody concentrations were optimized to give a linear standard protein curve for each protein. To minimize nonspecific interactions, the antibodies were preadsorbed using acetone powder derived from mutants of the respective proteins against which antibody had been generated. After incubation with the primary antibody, the membranes were washed in 50 ml of blocking buffer for 30 min. They were then incubated with a 1:20,000 dilution of horseradish peroxidase (HRP)-conjugated goat-anti-rabbit-IgG (Pierce) in blocking buffer for 3 h, washed in 50 ml of blocking buffer for 30 min, and treated with ECL Plus signal solution according to the manufacturer's specifications (GE Healthcare). The blots were visualized on a phosphorimager (Molecular Dynamics Storm PhosphorImager), and the image analysis was performed using the LabWorks Imaging Software (UVP BioImaging Systems). The integrated optical density (IOD) was measured and plotted on Microsoft Excel. The purified protein standard was used to generate a standard curve. The Western blot samples from the B. subtilis strains expressing the chemotaxis proteins were then compared to the standard curve.

For Western blots that were probing for 6×His gene fusions, 1:1,000 His probe (HIS.H8 mouse monoclonal IgG2b; 100 μg/ml; Santa Cruz Biotechnology) was used. The rest of the blotting and probing procedure was performed as previously described, except for the use of HRP-conjugated goat anti-mouse IgG (Pierce) at a dilution of 1:10,000.

Fluorescence microcopy.

HemAT-YFP and YfmS-YFP fusion strains were grown in 5 ml of CAMM+ and harvested at an A₆₀₀ of 0.4. A 20-μl aliquot of the cell cultures was then attached to the slides by treatment with poly-l-lysine solution (Sigma Aldrich). The localizations of HemAT-YFP and YfmS-YFP were visualized on an upright Leica DMR microscope with an HCL FL Fluotar 100× oil objective (numerical aperture [NA] = 1.30). Images were captured on a monochrome Retiga EXI charge-coupled device (CCD) camera (Qimaging) with In vivo software version 3.2.0 and processed with Adobe Photoshop CS version 8.0.

RESULTS

Quantitative immunoblots of the soluble signaling proteins.

The alternative sigma factor SigD substantially controls the expression of the genes responsible for flagellar synthesis, motility, and chemotaxis in B. subtilis (29). Previous work has shown that nutritional signals modulate the expression of SigD. Growth in rich medium results in changes in the expression levels of SigD-dependent genes, with expression increasing exponentially and peaking at the transition phase between exponential and stationary phases. However, growth in minimal medium results in constant and high expression of SigD-dependent genes (30). Similarly, in E. coli, chemotaxis gene expression is higher in minimal medium than in rich medium (27).

Quantitative Western blot analysis was used to determine the in vivo protein expression levels of the chemotaxis proteins. Growth conditions were optimized to maintain relatively stable expression of the chemotaxis genes through many generations of growth in minimal medium (CAMM). Using CAMM in the absence of any rich medium for a number of cell generations insured against the effect of any growth transitions in changing from rich medium to CAMM.

Examples of quantitative immunoblots for the soluble chemotaxis proteins are shown in Fig. 1. The concentration of each protein was determined from the derived standard curve with known amounts of purified proteins. The total numbers of protein molecules per cell are shown in Table 3, with the standard deviations expressing the variations in the measurements.

Table 3.

Cellular chemotaxis protein contents in B. subtilis

Component	Content (no. of molecules/cell) in OI1085^a
CheA	2,600 ± 560
CheB	2,100 ± 400
CheC	770 ± 330
CheD	1,200 ± 250
CheR	1,100 ± 280
CheY	7,100 ± 1,000
Coupling protein (total)^b	9,600 ± 2,000
CheV	7,500 ± 2,000
CheW	2,100 ± 430
Receptor (total)^c	59,960 ± 5,960
HemAT	19,000 ± 3,900
McpA	15,900 ± 3,000
McpB	6,200 ± 1,900
McpC	2,800 ± 640
TlpA	2,100 ± 890
TlpB	2,500 ± 740
TlpC	5,400 ± 1700
YfmS	4,200 ± 800
YoaH	460 ± 150
YvaQ	1,400 ± 380

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Mean ± the standard deviation (n ≥ 9).

Sum of CheV and CheW.

Sum of all 10 receptors.

As shown in Table 3, the absolute numbers of many of the soluble signaling proteins in B. subtilis appear to be somewhat lower than what was found previously in E. coli, but the ratios between the various proteins appear to be similar (27). The ratios between CheW and CheA dimers are 1.6 to 1 in both B. subtilis and E. coli. Furthermore, the ratios of CheB to CheR (1.9 in B. subtilis and 1.7 in E. coli) and the overall levels of CheY are similar.

Quantitative immunoblots for the chemoreceptors.

There are two soluble chemoreceptors present in B. subtilis, HemAT and YfmS. HemAT is a soluble chemoreceptor that is capable of sensing oxygen (17), whereas the ligand for YfmS is not known. HemAT and YfmS were both quantified with specific antibodies raised against purified recombinant protein (Fig. 1C and D, respectively). YfmS appeared as two distinct bands on its Western blot. The two bands were quantified separately and summed to give the total protein expressed in the wild-type cell lysate (Fig. 1D). YfmS has been shown to undergo posttranslational modification similar to that of the other chemoreceptors in B. subtilis (unpublished results). The migration of YfmS as multiple bands is most likely due to the presence or absence of multiple posttranslational modifications.

The eight transmembrane chemotaxis receptors (McpA, McpB, McpC, TlpA, TlpB, TlpC, YoaH, and YvaQ) were expressed as 6×His fusions under the gene's native promoter. The high sequence identity (in some cases over 60%) of the transmembrane receptors, especially McpA and McpB, leads to cross-reactive antibodies, making quantification of each receptor in a wild-type background very difficult. Thus, each receptor was separately 6×His tagged, and a commercially available His probe antibody (Santa Cruz Biotechnology) was used in these immunoblots. To test the effect on the expression of a chemoreceptor upon the addition of a 6×His tag, YfmS-His was tested with an anti-YfmS antibody (Fig. 2). Under these conditions, the levels of YfmS-His appeared to be similar to the levels of the native YfmS protein observed in the wild-type strain (82% ± 14% of wild-type levels). When the copy number of YfmS-His was determined using the anti-His antibody (Fig. 3A) and that of YfmS was determined using an anti-YfmS antibody (Fig. 2), the ratio was found to be 80% ± 20%, a similar ratio. This result implies that the amount of chemoreceptors in the cell, based on the anti-His antibody, provides a reasonable estimate of the cellular concentration.

Fig. 2. — Immunoblots of YfmS versus YfmS-His expression. Shown are the expression levels of the native YfmS protein expressed in the wild-type (WT) strain OI1085 and a YfmS-His fusion construct expressed under the native promoter on the chromosome. The multiple bands are due to the presence of covalent modification of the chemoreceptor. The higher migration of YfmS-His is due to the six carboxy-terminal histidine residues.

Fig. 3. — Quantitative immunoblots of His tag receptors. Shown are the immunoblots with His tag receptors. In each experiment, two (panels A, B, and C) or three (panels D and E) cultures of the His tag receptor strain were grown in CAMM as previously described. All samples were adjusted to contain the same amount of total cellular material by the addition of cellular material to the Δ10 chemoreceptor null strain. The YfmS-His standard curve consisted of 10, 20, 30, and 50 fmol. (A) McpA-His, McpB-His, McpC-His, the YfmS positive control to show transfer to the membrane, the Δ10 chemoreceptors, and purified YfmS protein standard. (B) TlpA-His, TlpB-His, TlpC-His, the YfmS positive control, Δ10 chemoreceptors, and the purified YfmS protein standard. (C) YoaH-His, YvaQ-His, and the YfmS positive control, Δ10 chemoreceptors, and the purified YfmS protein standard. (D) An additional blot for McpA quantification shows McpA-His, Δ10 chemoreceptors, and 1 to 25 fmol of the purified YfmS protein standard. (E) An additional blot for YoaH quantification shows McpA-His, Δ10 chemoreceptors, and 1 to 20 fmol of the purified YfmS protein standard.

It should be noted that YfmS-His appears to migrate at a slightly higher molecular weight than the native YfmS. The slightly higher molecular weight is likely due to the addition of the 6×His tag. Furthermore, the multiply modified bands are still present in the YfmS-His blot, indicating a receptor that is likely fully functional. Finally, motility swarm plates were performed on the McpB-His strain, with asparagine (a known ligand for McpB) used as the attractant (data not shown), and the 6×His tag showed no effect on swarm size, again indicating a fully functional receptor.

The method of quantifying each chemoreceptor was based on choosing a suitable protein standard that could behave similarly to the 6×His fusion receptors. Given the sequence and probable structural homology between YfmS and the other nine chemoreceptors in B. subtilis, especially in the cytoplasmic region (1), YfmS was chosen as both an internal control and the purified protein standard for all 6×His receptor fusion Western blots (Fig. 3). Each blot had YfmS-His expressed as a gene fusion from the chromosome under its native promoter to verify that the transfer of the proteins to the membrane occurred normally. Additionally, the immunoblot transfer time was optimized for maximal transfer of all chemoreceptors based on the transfer of a molecular weight standard similar to the weight of the chemoreceptors. Purified YfmS-His protein was also added to each blot as the purified protein standard curve. For each receptor, duplicate cultures expressing the 6×His fusion were used. The in vivo expression levels for all the proteins were determined by this method to yield the expression levels outlined in Table 3. Additional gels were run for McpA and YoaH because they fell out of the linear range in the initial blots.

For the 10 chemoreceptors, the concentrations ranged from a few hundred to several thousand per cell. Overall, the most highly expressed class of chemotaxis proteins under these conditions was the chemoreceptors (59,960 ± 5,960). Out of all 10 chemoreceptors, HemAT (19,000 ± 3,900) and McpA (15,900 ± 3,000) were expressed at the highest levels and constituted a majority of the total chemoreceptors expressed in B. subtilis (Table 3). The two best-studied chemoreceptors, which mediate nearly all taxis to amino acids and sugars (16, 33), showed medium expression, 6,200 ± 1,900 for McpB and 2,800 ± 640 for McpC.

When combined with the previous results, the cellular ratio between the chemoreceptors and CheA is much higher than previously reported in E. coli. For every CheA homodimer, there are approximately 23 receptor dimers. This ratio is approximately 6 times higher than in E. coli (3.4 receptor dimers per CheA dimer).

CheD expression in a cheC null mutant and in a mutant lacking all chemoreceptors.

CheD is expressed at levels of 1,200 ± 250 molecules per cell compared to 770 ± 330 CheC molecules per cell (Table 3). These copy numbers correspond to roughly 1.5 CheD molecules per CheC molecule in vivo. CheD binds to both the receptor and CheC in order to play a role in adaptation (23, 24, 32). Other work has suggested that CheD protein levels in the cell may be linked to the presence of CheC and perhaps other proteins (31).

Quantitative immunoblots were performed to measure the relative CheD expression levels in mutants lacking potential CheD binding partners: CheC and all 10 chemoreceptors (Fig. 4). The expression levels in the cheC null mutant are approximately 32% of wild-type levels. In the Δ10 receptor background, CheD expression levels are reduced to approximately 19% of wild-type levels. These results imply that excess or unbound CheD is degraded.

Fig. 4. — Quantitative CheD immunoblots in Δ10 receptor and *cheC* mutants. Shown are CheD expression levels in various genetic backgrounds. The protein standard is 20, 50, 75, 100, and 150 fmol from left to right. CheD protein levels in the Δ10 background are approximately 19% of wild type. CheD protein levels in the *cheC* null strain are approximately 32% of wild-type levels. The integrated optical densities of the bands were quantified by LabWorks.

YfmS and HemAT cellular localization.

Previous work has shown that the transmembrane chemoreceptor McpB localizes at the cell poles, much like its counterparts in E. coli (20, 26, 28). This polar clustering of the chemoreceptors allows all the receptors to function collectively in an array (49). However, not all chemotaxis receptors cluster at the poles. In the case of the soluble chemoreceptors TlpC and TlpT in Rhodobacter sphaeroides, they reside in the cytoplasm rather than at the cell poles with the transmembrane chemoreceptors and are associated with separate species of CheW, CheA, and CheR (46, 47). To determine if the two soluble chemoreceptors in B. subtilis, YfmS and HemAT, localize to the cell poles or to clusters near the center of the cytoplasm, YFP fusions of the two were constructed.

A wild-type strain expressing YfmS-YFP (OI4373) was grown in CAMM and imaged by fluorescence microscopy (Fig. 5A). A snapshot of a field of cells illustrates that YfmS-YFP tends to cluster at the poles, unlike the soluble chemoreceptors in R. sphaeroides. HemAT-YFP fusion proteins were also shown to cluster at the cell poles (Fig. 5B). Even though they lack a transmembrane domain, the two soluble receptors cluster at the poles with the transmembrane chemoreceptors. The fact that these two receptors cluster at the cell poles suggests that they might be working in association with other chemoreceptors.

DISCUSSION

Chemotaxis is a process that involves numerous proteins working in conjunction with one another to bring about a physiological change. In order for this process to work efficiently, these proteins must be expressed in absolute amounts and ratios that are highly tuned for optimal performance. In the work presented in this study, the copy numbers of all the proteins in the B. subtilis chemotactic pathway were determined using quantitative immunoblots.

The total number of chemoreceptors per cell in B. subtilis is 59,960 ± 5,960, compared to 26,000 ± 1,800 for E. coli strain RP437 and 41,000 ± 1,400 for E. coli strain OW1 grown in minimal medium (Table 3) (27). The environmentally diverse habitat of B. subtilis might necessitate a wider variety of receptors in order for the bacterium to most efficiently navigate its surroundings. E. coli, in comparison, seems to be able to make do with fewer types of receptors, again most likely due to its simpler environment. The most abundant receptor found in B. subtilis is HemAT, which senses oxygen. Since B. subtilis is an obligate aerobe, it seems quite reasonable that HemAT concentrations are high. In fact, if HemAT were not present, then B. subtilis would have only 41,000 receptors, which is comparable to the number found in E. coli. As a comparison, in the facultative anaerobe E. coli, the oxygen sensor Aer is expressed in very low numbers (44). The other highly abundant receptor is McpA, which is known to play a minor role in chemotaxis toward glucose (16). However, its high expression levels mean that it likely functions in the cell's ability to move toward some other, unknown molecules. McpB and MpcC, which coordinate migration toward all amino acids and many sugars, are found at medium levels. Exactly how these receptors might function together in a larger receptor array is not known and is a topic for further investigation.

The addition of the 6×His tag to the 3′ ends of the respective chemoreceptor genes yielded a straightforward method of quantifying each receptor with a specific, commercially available antibody without any cross-reactivity with any other chemoreceptor. However, the effect on the expression level of each chemoreceptor after the addition of the 6×His tag is unknown, although in the case of McpB, receptor function seems unaffected. The expression of the YfmS-His fusion construct in Fig. 2 appears to be slightly lower than the expression level of the native YfmS but within the error of the experimental conditions. The high sequence and predicted structural homologies of the cytoplasmic domain of the chemoreceptors suggest that using YfmS-His as a protein standard for all 10 chemoreceptors should yield reasonable quantification results. While obtaining chemoreceptor antibodies that are able to detect the native chemoreceptors with little or no cross-reactivity would serve to confirm the chemoreceptor expression levels, the high sequence similarity in the cytoplasmic domains of the receptors means such antibodies would be difficult to find.

In spite of having more chemoreceptors, CheA levels are much lower in B. subtilis (2,600 ± 190) than in E. coli (7,700 ± 440). The resulting cellular ratio of chemoreceptor dimers to CheA homodimers in B. subtilis is roughly 23.0 ± 4.5 compared to the ratio found in E. coli, 3.4 ± 0.8 receptor dimers per CheA dimer, as shown in Fig. 6. Unlike B. subtilis, E. coli has two CheA species, which might be partly responsible for the different levels shown here. A truncated version of the kinase, CheA_short, has no phosphorylation domain but has been shown to bind to CheZ (5), a CheYp phosphatase not found in B. subtilis. In spite of differences in the cellular ratio of the chemoreceptors to CheA, the cellular ratios of the coupling protein CheW to CheA homodimer are nearly identical in the two organisms (Fig. 6). These similar ratios may suggest that the functional ratio in both organisms, and possibly in many other chemotactic organisms, as well, is one CheW protein to one CheA molecule in the ternary complex (9, 10). It seems likely that CheA has considerably higher affinity for receptors associated with CheW, which agrees with the observation that the polar receptors in B. subtilis are typically associated with both CheA and CheW, whereas the lateral receptors are typically associated with neither (48).

Fig. 6. — Comparison of the cellular stoichiometries of *B. subtilis* and *E. coli*. Shown is the cellular stoichiometry of the chemotaxis signaling proteins in this study compared to the ratios observed in *E. coli* (27). These models are normalized to show the cellular ratios to CheA dimers present in the cell.

B. subtilis has an additional protein that contains a CheW-like coupling domain in CheV, which is present at a ratio of 5.8 ± 2.0 for every CheA dimer. Previous experiments have shown that cell membranes isolated from cheW null strains exhibit severely decreased CheA activity in vitro, despite the presumed presence of CheV (8). The fact that the ratios of the levels of CheA and CheW in E. coli and B. subtilis are very similar supports the hypothesis that CheV normally plays a quite different role. In fact, it is known to function in adaptation (19).

The overall number of receptor-modifying enzymes, CheB and CheR, is much higher in B. subtilis than in E. coli. This difference may simply be due to the larger overall number of chemoreceptors found in B. subtilis, but the significance of the greater abundance of CheB and CheR in B. subtilis is not clear at this point. We note that the ratios of CheB to CheR are similar in the two organisms (1.7 in E. coli and 1.9 in B. subtilis), and the presence of a third modifying enzyme (CheD) may also have some effect on these differing levels.

CheC and CheD are two proteins that are not found in E. coli but are present in many other chemotactic species. There are 1,200 ± 250 copies of CheD per cell compared to 770 ± 330 for CheC. The quantitative Western blots performed on CheD may suggest that in the absence of binding partners in the cell, CheD is subject to proteolysis. CheD protein levels are reduced to 32% of wild-type levels in the cheC null strain. It appears that in the absence of CheC, CheD expression levels drop approximately 810 copies per cell, from approximately 1,200 to 390. The loss of roughly 810 CheD protein molecules per cell roughly equals the amount of CheC protein present in the wild-type cell (770 ± 330). The CheD expression levels in the Δ10 receptor mutant showed even more severe decrease than in the cheC null mutant. In the Δ10 receptor genetic background, CheD levels were reduced to approximately 19% of wild-type levels. While the affinity of CheD to both CheC and the chemoreceptors has not been measured experimentally, the binding is strong enough to exhibit a result in a GST pulldown assay (6, 38).

Previous work has shown that CheW is subject to ClpP- and ClpX-mediated proteolysis during glucose starvation (11). Perhaps CheD is subject to similar proteolysis when unbound from CheC or not associated with the receptors. The degradation of unbound CheD suggests that there may be a mechanism in B. subtilis for optimally tuning the expression levels of the chemotaxis proteins posttranslationally. In this way, fluctuations in expression amounts can be rectified so that chemotaxis works most efficiently. This principle also appears to operate in the cases of CheR and CheB, as the level of CheB expression in a cheR mutant is only about 19% (22), probably due to its poor affinity for the unmethylated receptors (unpublished data).

When fused to fluorescent proteins, the two soluble receptors in B. subtilis, YfmS and HemAT, were both found to localize to the poles of the cells. In contrast, the soluble receptors TlpC and TlpT in R. sphaeroides are localized in the middle of the cell and interact with a dedicated CheA kinase different from the one associated with the poles. One implication might be that YfmS and HemAT are integral parts of the polar receptor complex and undergo conformational changes upon the binding of attractant to other receptors. For instance, in an mcpB mutant where McpB cannot be methylated or demethylated, methanol can still be released upon the addition and removal of asparagine (49). This finding suggests that other receptors in the cell undergo methylation and demethylation upon the addition of an attractant specific to another receptor (49), thus fostering adaptation. Similarly, methanol evolves upon addition and removal of oxygen despite HemAT not being itself subject to methylation, a phenomenon that also may help adaptation, since aerotaxis is more efficient when the other receptors are present than with HemAT as the sole receptor (17).

In our opinion, the greatest contrast between the values of chemotaxis proteins in B. subtilis versus E. coli is the ratio of receptors to CheA, which is much larger (23 versus 3.4) in B. subtilis. This result raises the possibility that the structure of the receptor-CheA-CheW complex might be in some ways significantly different than the structure in E. coli, a result suggested by others (34). It should be of considerable interest to determine the stoichiometry of a complex of CheA and CheW with an individual receptor, such as YfmS or HemAT, to see whether this lopsided ratio can be verified in a particular case. How this different ratio, as well as the presence of novel chemotaxis proteins, plays a role in chemotaxis across different species is certainly a very interesting avenue for further research that would lead to greater understanding of bacterial chemotaxis.

ACKNOWLEDGMENTS

We thank Snehal Patel for fluorescence microcopy technical assistance and Michel Bellini for the use of the microscope.

This work was supported by National Institutes of Health Grant GM054365 to G.W.O and C.V.R. and Cellular and Molecular Biology Training Grant T32 GM007283 to V.J.C.

Footnotes

^▿

Published ahead of print on 22 April 2011.

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[B1] 1. Alexander R. P., Zhulin I. B. 2007. Evolutionary genomics reveals conserved structural determinants of signaling and adaptation in microbial chemoreceptors. Proc. Natl. Acad. Sci. U. S. A. 104:2885–2890 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Bischoff D. S., Bourret R. B., Kirsch M. L., Ordal G. W. 1993. Purification and characterization of Bacillus subtilis CheY. Biochemistry 32:9256–9261 [DOI] [PubMed] [Google Scholar]

[B3] 3. Bischoff D. S., Ordal G. W. 1991. Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis. J. Biol. Chem. 266:12301–12305 [PubMed] [Google Scholar]

[B4] 4. Burgess-Cassler A., Ullah A. H., Ordal G. W. 1982. Purification and characterization of Bacillus subtilis methyl-accepting chemotaxis protein methyltransferase II. J. Biol. Chem. 257:8412–8417 [PubMed] [Google Scholar]

[B5] 5. Cantwell B. J., et al. 2003. CheZ phosphatase localizes to chemoreceptor patches via CheA-short. J. Bacteriol. 185:2354–2361 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Chao X., et al. 2006. A receptor-modifying deamidase in complex with a signaling phosphatase reveals reciprocal regulation. Cell 124:561–571 [DOI] [PubMed] [Google Scholar]

[B7] 7. Fuhrer D. K., Ordal G. W. 1991. Bacillus subtilis CheN, a homolog of CheA, the central regulator of chemotaxis in Escherichia coli. J. Bacteriol. 173:7443–7448 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Garrity L. F., Ordal G. W. 1997. Activation of the CheA kinase by asparagine in Bacillus subtilis chemotaxis. Microbiology 143:2945–2951 [DOI] [PubMed] [Google Scholar]

[B9] 9. Gegner J. A., Dahlquist F. W. 1991. Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA. Proc. Natl. Acad. Sci. U. S. A. 88:750–754 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Gegner J. A., Graham D. R., Roth A. F., Dahlquist F. W. 1992. Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway. Cell 70:975–982 [DOI] [PubMed] [Google Scholar]

[B11] 11. Gerth U., et al. 2008. Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis. J. Bacteriol. 190:321–331 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Goldman D. J., Nettleton D. O., Ordal G. W. 1984. Purification and characterization of chemotactic methylesterase from Bacillus subtilis. Biochemistry 23:675–680 [DOI] [PubMed] [Google Scholar]

[B13] 13. Goldman D. J., Ordal G. W. 1984. In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis. Biochemistry 23:2600–2606 [DOI] [PubMed] [Google Scholar]

[B14] 14. Greenfield N. J. 2006. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1:2876–2890 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Hanlon D. W., Marquez-Magana L. M., Carpenter P. B., Chamberlin M. J., Ordal G. W. 1992. Sequence and characterization of Bacillus subtilis CheW. J. Biol. Chem. 267:12055–12060 [PubMed] [Google Scholar]

[B16] 16. Hanlon D. W., Ordal G. W. 1994. Cloning and characterization of genes encoding methyl-accepting chemotaxis proteins in Bacillus subtilis. J. Biol. Chem. 269:14038–14046 [PubMed] [Google Scholar]

[B17] 17. Hou S., et al. 2000. Myoglobin-like aerotaxis transducers in Archaea and Bacteria. Nature 403:540–544 [DOI] [PubMed] [Google Scholar]

[B18] 18. Kaltwasser M., Wiegert T., Schumann W. 2002. Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis. Appl. Environ. Microbiol. 68:2624–2628 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Karatan E., Saulmon M. M., Bunn M. W., Ordal G. W. 2001. Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis. J. Biol. Chem. 276:43618–43626 [DOI] [PubMed] [Google Scholar]

[B20] 20. Kirby J. R., Niewold T. B., Maloy S., Ordal G. W. 2000. CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis. Mol. Microbiol. 35:44–57 [DOI] [PubMed] [Google Scholar]

[B21] 21. Kirsch M. L., Peters P. D., Hanlon D. W., Kirby J. R., Ordal G. W. 1993. Chemotactic methylesterase promotes adaptation to high concentrations of attractant in Bacillus subtilis. J. Biol. Chem. 268:18610–18616 [PubMed] [Google Scholar]

[B22] 22. Kirsch M. L., et al. 1993. Chemotactic methyltransferase promotes adaptation to repellents in Bacillus subtilis. J. Biol. Chem. 268:25350–25356 [PubMed] [Google Scholar]

[B23] 23. Kristich C. J., Ordal G. W. 2004. Analysis of chimeric chemoreceptors in Bacillus subtilis reveals a role for CheD in the function of the McpC HAMP domain. J. Bacteriol. 186:5950–5955 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Kristich C. J., Ordal G. W. 2002. Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis. J. Biol. Chem. 277:25356–25362 [DOI] [PubMed] [Google Scholar]

[B25] 25. Kuipers B. J., Gruppen H. 2007. Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J. Agric. Food Chem. 55:5445–5451 [DOI] [PubMed] [Google Scholar]

[B26] 26. Lamanna A. C., Ordal G. W., Kiessling L. L. 2005. Large increases in attractant concentration disrupt the polar localization of bacterial chemoreceptors. Mol. Microbiol. 57:774–785 [DOI] [PubMed] [Google Scholar]

[B27] 27. Li M., Hazelbauer G. L. 2004. Cellular stoichiometry of the components of the chemotaxis signaling complex. J. Bacteriol. 186:3687–3694 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Maddock J. R., Shapiro L. 1993. Polar location of the chemoreceptor complex in the Escherichia coli cell. Science 259:1717–1723 [DOI] [PubMed] [Google Scholar]

[B29] 29. Márquez L. M., et al. 1990. Studies of sigma D-dependent functions in Bacillus subtilis. J. Bacteriol. 172:3435–3443 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Mirel D. B., et al. 2000. Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression. J. Bacteriol. 182:3055–3062 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Muff T. J., Foster R. M., Liu P. J., Ordal G. W. 2007. CheX in the three-phosphatase system of bacterial chemotaxis. J. Bacteriol. 189:7007–7013 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis^{^▿}

Vincent J Cannistraro

George D Glekas

Christopher V Rao

George W Ordal

Abstract

INTRODUCTION