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. 2015 May 5;6(3):e00546-15. doi: 10.1128/mBio.00546-15

Adenylate Charge Regulates Sensor Kinase CheS3 To Control Cyst Formation in Rhodospirillum centenum

Kuang He 1,*, Vladimira Dragnea 1, Carl E Bauer 1,✉
Editor: Caroline S Harwood2
PMCID: PMC4436063  PMID: 25944862

ABSTRACT

Rhodospirillum centenum forms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che3 signaling cascade. The Che3 cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3 and CheR3, two CheW3 linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3 cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3 does not serve as a phosphor donor for CheY3; instead, CheA3 inhibits a CheS3→CheY3 two-component system by phosphorylating an inhibitory receiver domain of CheS3. In this study, we show that in addition to phosphorylation by CheA3, the phosphorylation state of CheS3 is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occur in vivo upon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicated in vitro, the phosphorylation level of CheS3 is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3 phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3.

IMPORTANCE

Upon starvation, Rhodospirillum centenum undergoes a developmental process that forms metabolically dormant cysts, which withstand desiccation and nutritional limitation. This study explores the role of the cellular energy state as measured by the ratio of ATP to ADP as an important regulator of cyst formation in Rhodospirillum centenum. We show that R. centenum cells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When this in vivo level of energy starvation is simulated in vitro, CheS3 phosphorylation is reduced by 75%. This profound reduction in CheS3 autophosphorylation is contrasted with a much lower 25% decrease in CheA3 phosphorylation in response to a similar downward shift in ATP/(ATP + ADP). We argue that even though adenylate energy affects all ATP-dependent enzymes to an extent, the enhanced inhibition of CheS3 activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.

INTRODUCTION

Rhodospirillum centenum is a photosynthetic alphaproteobacterium that is also a member of the Azospirillum clade (1–4). Members of this clade exhibit a complex life cycle manifested as individual rod- or vibrio-shaped vegetative cells that can differentiate into metabolically dormant round clusters of cells during times of nutrient deprivation or desiccation (3, 5, 6). Rhodospirillum and Azospirillum constitute part of the plant root rhizosphere, where they are known to provide plants with fixed nitrogen and several plant growth hormones that they synthesize (7–9). Inoculation of seeds with Azospirillum species is known to enhance crop yields in a wide variety of cultivars (8, 10).

Unlike extensive analysis of sporulation by Gram-positive cells, little is known about how Gram-negative cells regulate development of metabolically dormant cysts. Recently, we have been studying R. centenum, which is a genetically amendable model organism for cyst development by members of the Azospirillum clade (5, 11, 12). Genetic studies have indicated that the control of cyst cell development is quite complex, involving numerous histidine kinases, cell cycle regulators, a sigma factor, and the use of cGMP as a signaling molecule (11–15). One of the better-studied regulatory components is a novel Che-like signal transduction cascade (the Che3 cascade) that controls the timing of cyst development (12, 16). This signal transduction cascade, representing a variation of the classic chemotaxis signaling pathway (17), contains homologs to a methyl chemoreceptor protein (MCP3), a methyltransferase CheR (CheR3), a methylesterase CheB (CheB3), two linker CheWs (CheW3a and CheW3b), a CheA-CheY hybrid histidine kinase (CheA3), and a standalone CheY response regulator/receiver (CheY3) (Fig. 1). By analogy to the Escherichia coli chemotaxis signaling paradigm (18), CheA3 is thought to dock with CheW3a-CheW3b, with its kinase activity regulated through signals received by MCP3. The che3 gene cluster also codes for a second hybrid histidine kinase, CheS3, which contains two N-terminal receiver (REC) domains followed by a Per-Arnt-Sim (PAS) domain and an N-terminal HWE class kinase domain. Genetic characterization of the che3 gene cluster showed that cheA3 null mutants exhibited a defect in cyst formation while cheS3 and cheY3 null mutants constitutively formed cysts (19). We recently demonstrated that CheS3 and CheY3 constitute a two-component system (TCS) and that under cyst-inducing conditions the kinase activity of CheS3 is inhibited by phosphorylation of the first receiver (REC) domain in CheS3 by CheA3 (12).

FIG 1 .

FIG 1 

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The Che3 signal transduction cascade that controls cyst development. (A) CheS3 and CheY3 comprise a two-component system that is phosphorylated under favorable growth conditions. (B) During the vegetative-to-dormant transition, CheA3 is activated by an external signal (indicated by a red wedge) via the chemoreceptor MCP3 and phosphorylates the receiver domain of CheS3. When the REC domain is phosphorylated by CheA, it inactivates the ability of the HWE domain to autophosphorylate, thereby deactivating the CheS3-CheY3 two-component system. Abbreviations: REC, receiver domain; PAS, Per-Arnt-Sim domains; HWE, HWE superfamily of histidine kinases; Hpt, histidine phosphotransfer domain.

Even though autophosphorylation activity of CheS3 is known to be regulated by CheA3 phosphorylation, there remains the possibility that CheS3 may also be regulated by additional intracellular signals. In this report, we demonstrate that the level of CheS3 phosphorylation is indeed fine-tuned by the relative molar ratio of ATP and ADP as explained by the adenylate energy charge (AEC) theory put forth in the late 1960s by Atkinson and Walton (20). AEC is a measure of intracellular energy level based on the amount of high-energy chemical bonds present in ATP and ADP [AEC = (ATP + 1/2 ADP)/(ATP + ADP + AMP)] (20). Cells appear to sustain vegetative growth by maintaining a narrow range of high-energy charge (high ATP relative to ADP and AMP). They do so by regulating the activity of many ATP-utilizing enzymes through inhibition by ADP and/or AMP (21). Since AMP is known to have a regulatory effect on only eukaryotic serine/threonine protein kinases (22), ATP and ADP are thought to be the key components that affect energy charge in bacterial cells. Interestingly, ATP depletion is a characteristic of a dormant energy-starving state in bacteria (23, 24). For example, sporulating cells such as Bacillus megaterium contain strikingly low ATP concentrations relative to vegetatively grown cells (3 nmol ATP/g [dry weight] of spores versus 725 nmol ATP/g [dry weight] of cells) (23). Since nutrient deprivation is one of the triggers for inducing cyst formation (2), we have investigated whether there is a reduction in intracellular energy state as represented by changes in the ratios of adenine nucleotides as R. centenum develops cysts. Here, we report that ATP levels are indeed significantly reduced as cells enter dormancy and that CheS3 phosphorylation is also regulated in vitro by the ATP/ADP ratio. These results suggest that a reduction in cellular energy charge may constitute an additional regulatory signal for the promotion of encystment.

RESULTS

ATP decreases relative to ADP during encystment.

Bacteria typically maintain an AEC between 0.8 and 0.9 during vegetative growth, where high levels of ATP production are sustained (20, 25, 26). However, AEC is reduced in cells entering stationary phase or dormancy, due to nutrient deprivation that limits ATP production. For example, it has been reported that E. coli reduces its AEC from 0.92 during logarithmic growth to 0.45 during stationary phase (21). Actively growing Bacillus megaterium cells maintain an AEC above 0.79, while stationary pre-sporulation-phase Bacillus has a reported AEC of 0.36 and spores of several Bacillus species have AEC values below 0.1 (21). We argue that under nutritional starvation, R. centenum cells should experience similar ATP depletion that would result in a substantial decline in AEC. Since to our knowledge AMP does not have any known regulatory effects on enzymes of bacterial origin, we evaluated the AEC of R. centenum by measuring the ATP/(ATP + ADP) ratio using a luciferase-based ATP luminescence assay. For this analysis, we first grew R. centenum in nutrient-rich CENS medium and then shifted the cells into nutrient-limiting CENBA medium to induce encystment (2). Aliquots of the culture were removed at various time points, rapidly disrupted by perchloric acid, and analyzed for adenylate nucleotide pool levels. As shown in Fig. 2A, the intracellular ATP/(ATP + ADP) ratio remained fairly constant at approximately 85% for the first 30 h poststarvation. Within 48 h of growth in CENBA medium, ATP/(ATP + ADP) dropped to approximately 55%, which was sustained for the next 3 days as the cysts matured. Microscopic visual signs of cyst formation (i.e., the formation of highly retractile round cells forming multicell clusters) occurred on day 4.

FIG 2 .

FIG 2 

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Changes in ATP/(ATP + ADP) observed in encysting R. centenum cells affect kinase phosphorylation levels in vitro. (A) In vivo changes in ATP/(ATP + ADP) in cells experiencing nutrient limitation. Within the first 30 h of incubation, cells propagate at ~85% intracellular ATP/(ATP + ADP), which then drops to ~55% in the next 18 h and maintains the low level in the following 3 days. (B) CheS3 33P labeling is highly sensitive to changes in ATP/(ATP + ADP) ratios. (C) CheA3 33P labeling is minimally affected within the 90% to 50% ATP/(ATP + ADP) range. One hundred percent phosphorylation is set to the maximum 33P labeling observed for the duration of the experiments. The error bars represent standard deviations from three replicates.

CheS3 has a lower binding affinity to ATP than does CheA3.

Analysis of the intracellular nucleotide concentrations in E. coli indicates that logarithmically growing cells typically contain ~3 mM ATP and ~110 µM ADP (27). As discussed above, these levels change as cells transition from logarithmic to stationary phase, with ATP dropping to ~1 mM and ADP increasing to ~400 µM. To address whether similar changes in intracellular nucleotide concentration would equally affect the activity of CheS3 and CheA3, we measured the substrate binding constant for nonhydrolyzable ATPγS and of ADP to both CheS3 and CheA3 using microscale thermophoresis. This technique measures the mobility of molecules in a temperature gradient that depends on molecular weight as well as structural or conformational changes in protein as a consequence of ligand binding. The results of this analysis (Fig. 3) indicate that CheS3 has a significant 8-fold-lower affinity toward ATPγS than does CheA3 (112 nM versus 13 nM, respectively). These values indicate that both kinases should be fully saturated with ATP during logarithmic and stationary phases of growth; however, under severe starvation conditions, as would be expected in metabolically dormant cyst cells, the activity of CheS3 may be selectively inhibited due to its low ATP binding affinity. In the case of ADP, we observed that the two kinases were indeed capable of binding ADP with similar binding constants (8 µM and 14 µM, respectively) (Fig. 3).

FIG 3 .

FIG 3 

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Kinase-nucleotide binding assays using microscale thermophoresis. Increasing concentrations of ATPγS and ADP were incubated with fluorescently labeled CheS3 (A) and CheA3 (B). The thermophoresis signal (a fluorescence “jump” between two states—laser off versus laser on—as a result of movement of fluorescently labeled protein away from the heat spot) was plotted against nucleotide concentrations. Dissociation constant (Kd) values were fitted with sigmoidal binding curves using NanoTemper software.

Phosphorylation of CheS3 is highly sensitive to elevated ADP concentrations.

It has been proposed elsewhere that a reduction in AEC that occurs during starvation could itself function as a metabolic regulator (28). The reason for this conclusion is that an alteration in AEC is known to affect the activity of several ATP-utilizing and ATP-generating enzymes (25). For example, the activity of phosphofructokinase in E. coli that controls the flux of metabolites in glycolysis is inhibited by high levels of ATP and stimulated by high levels of ADP (29). Consequently, we investigated whether the in vitro kinase activity of CheS3 and CheA3 could be affected by a 30% decrease in ATP/(ATP + ADP) ratio as is observed during a nutrient downshift of R. centenum cells. To replicate high, medium, and low ATP/ADP ratios, we formulated a series of ATP/ADP mixes containing a fixed amount of ATP with ADP at various concentrations to achieve ATP/(ATP + ADP) ratios that varied between 90% and 10%. CheS3 and CheA3 were allowed to autophosphorylate in the presence of these ATP/ADP mixes, and the amounts of 33P labeling representing the overall phosphorylation of the kinases were then compared. Figure 2B shows that the level of CheS3 phosphorylation at each time point drops with decreasing ATP/(ATP + ADP) ratios. A profound 70.3% reduction in overall CheS3 phosphorylation at 26 min was observed when the ATP/(ATP + ADP) ratio was decreased from 90% to 50%. This level of reduction in the ATP/(ATP + ADP) ratio is similar to that observed when cells are shifted to the cyst-inducing medium. At an ATP/(ATP + ADP) ratio of 30%, CheS3 contains only 10% 33P incorporation compared to the amount of 33P incorporation observed when ATP/(ATP + ADP) was fixed at 90% (Fig. 2B). When the ADP concentration further increased such that the ATP/(ATP + ADP) was at 13.9%, then CheS3 autophosphorylation was essentially deactivated (Fig. 2B).

We next tested whether ADP inhibition of kinase activity was a characteristic feature of CheS3 or whether it was a general feature of histidine kinases. To address this, we also assayed CheA3’s sensitivity to changes in ATP/(ATP + ADP). As shown in Fig. 2C, CheA3 phosphorylation is also ADP sensitive. However, in contrast to CheS3, 33P labeling of CheA3 is only slightly diminished (23.9%) by a similar 90% to 50% decrease in the ATP/(ATP + ADP) ratio at 57 min (Fig. 2C). CheA3 starts to exhibit a greater reduction in overall phosphorylation as ATP/(ATP + ADP) drops below 50%.

ADP affects the stability of phosphorylated CheS3.

Since CheS3 exhibits an unusually sensitive response to changes in the adenylate energy state as measured by ATP/(ATP + ADP), we next investigated the role of ADP in regulating CheS3. One possibility is that an excessive amount of ADP occupies and blocks the active site, preventing the enzyme from autophosphorylating. Another hypothesis is that ADP drives the reverse reaction of autophosphorylation to form ATP. It is also possible that ADP boosts the intrinsic hydrolysis of the phosphohistidine bonds or promotes intramolecular phosphoryl transfer to its REC1 domain, which is known to undergo rapid autodephosphorylation. We tested these possibilities by first assessing the stability of purified CheS3~P in the presence of ADP, ATP, or AMP. For this analysis, we first autophosphorylated CheS3 at room temperature for 30 min with excess [33P]ATP and then removed unincorporated ATP with a desalting column. Isolated CheS3~P was then exposed to 100 µM ATP, ADP, or AMP and then assayed for phosphate stability by SDS-PAGE analysis. The results shown in Fig. 4A indicate that exposure of CheS3~P to 100 µM ADP resulted in greatly accelerated dephosphorylation (half-life of 0.02 min) relative to the stability observed in the presence of ATP and AMP (half-life of 53 min) or in the absence of any nucleotides (55.4 min) (12).

FIG 4 .

FIG 4 

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The stability of phosphorylated CheS3 is greatly reduced in the presence of ADP. (A) Purified CheS3~P dephosphorylates within 30 s in the presence of 0.1 mM ADP, which was not observed with the same concentrations of ATP or AMP. (B) ADP drives dephosphorylation of CheS3~P to form inorganic phosphates (Pi) as assayed by thin-layer chromatography (TLC).

We next analyzed the nature of the products formed as a result of ADP destabilization using thin-layer chromatography (Fig. 4B). CheS3~P was immobilized onto Ni2+-Sepharose resin and rinsed 5 times with buffer before 100 µM ADP was introduced. The purification step was not thorough, as a trace amount of ATP was still present at the 20-s time point (Fig. 4B). However, ATP disappeared at the 40-s time point, indicating that despite excess ADP, ATP was still hydrolyzed presumably by CheS3 in an autophosphorylation event. Additionally, CheS3~P, which should remain at the origin, also disappeared at 40 s, suggesting that the phosphoprotein had released its phosphoryl group. Since no ATP was reformed beyond 40 s, ADP does not drive the reverse reaction of autophosphorylation. Finally, ADP does not promote intramolecular transfer of phosphates from the HK domain to the REC1 domain, as the phosphorylated form of CheS3 and its nonphosphorylatable REC domain mutant CheS3:D54A are both base resistant in the presence of ADP (Fig. 5). These results suggest that ADP interacts with CheS3~P in a manner that leads to destabilization of the phosphor-histidine bond.

FIG 5 .

FIG 5 

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Acid-base stability test of CheS3 and its receiver mutant. Autophosphorylation was carried out in the presence of 100%, 90%, 70%, 50%, and 30% ATP/(ATP + ADP). Under these conditions, both phosphorylated CheS3 and CheS3:D54A are acid labile and alkaline stable, suggesting that both forms of CheS3 are phosphorylated only at the conserved histidine site despite the varied adenylate energy levels.

DISCUSSION

While a nutrient downshift seems to initiate the formation of resting cells in many bacterial species, the actual signals that regulate the development of dormancy remain unclear. For example, the multicomponent Spo0F-Spo0B-Spo0A phosphorelay that controls sporulation in Bacillus subtilis involves three sensor kinases: KinA and KinB, which phosphorylate Spo0F (30), and KinC, which directly phosphorylates the DNA binding response regulator Spo0A (31). The only clue that might reveal the input signal for these sporulation kinases is that the transmembrane kinase KinC responds to small-molecule natural products that cause potassium leakage from the cytosol (32). However, it remains unclear what input signals are actually being sensed by KinA and KinB.

In this report, we propose that CheS3 responds to alterations in ATP/ADP ratios upon starvation. The intracellular ATP-to-ADP ratio has previously been shown to be sufficient to entrain circadian oscillators reconstituted in vitro with isolated cyanobacterial kinases (33). In this case, light illumination drives in vivo oscillations of ATP/ADP ratios, which in turn generate a circadian KaiC oscillator where the phosphorylation of KaiC is regulated by two other proteins, KaiA and KaiB. Furthermore, darkness induces a phase shift of the KaiABC system, which can be simulated in vitro by lowering the ATP/ADP ratios alone. Here, we show that a nutrient downshift causes a decrease in the ATP/ADP ratio in R. centenum cells and that changes in the ATP/ADP ratio similar to those that occur during cyst formation modulate CheS3 phosphorylation levels in vitro. In response to the measured 35% drop between the initial high and final low ATP/(ATP + ADP) ratios that occurs upon a nutrient downshift, the CheS3 phosphorylation level is reduced by approximately 75% relative to its peak level. This 35% drop in the ATP pool relative to the total amount of ATP and ADP could be an underestimate due to samples containing a mixed population of mature cysts and cells that have not proceeded entirely through the encystment process. So, there is a possibility that CheS3 can be deactivated even more than what we have observed. The great sensitivity of CheS3~P to fluctuations in the ATP/ADP ratio during encystment is distinct from moderate resistance exhibited by CheA3~P. As ATP/(ATP + ADP) drops from the vegetative level to the dormant level, CheA3 loses only 25% of its phosphorylation. This difference in inhibition by these two sensor kinases may at one level be due to differences in the ATP binding affinities of CheA3 and CheS3. CheA3 binds ATP 8-fold more tightly and therefore remains more active in utilizing ATP even at ultralow ATP intracellular concentrations that would occur during cyst formation. Furthermore, CheA3 phosphorylation of the Rec1 domain of CheS3 is known to inhibit the formation of a phosphor-histidine bond in CheS3. Thus, a sharp step down in ATP levels would favor this inhibition event, as CheA3 is more resistant to a reduction in ATP levels, allowing it to continue to phosphorylate Rec1 in CheS3. In fact, the ATP binding affinity for CheA3 is about 2 to 200 times higher than that reported for other histidine kinases (34–37). This tight binding ensures that CheA3 remains active even during the energy starvation periods. We further demonstrate that ADP stimulates hydrolysis of CheS3~P to generate Pi by affecting the stability of the phosphohistidine bond. We speculate that when ADP builds up during a nutrient downshift, any phosphohistidine present on CheS3 is rapidly removed by ADP-stimulated dephosphorylation (Fig. 6). To our knowledge, this is a novel mechanism of dephosphorylation not described for other histidine kinases.

FIG 6 .

FIG 6 

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Revised Che3 signal transduction cascade under the control of adenylate energy charge. (A) During vegetative growth, the CheS3-CheY3 two-component system is active at high intracellular ATP/ADP ratios. (B) Upon energy starvation, ATP is quickly depleted and ADP (indicated by the molecular mechanism) is accumulated, leading to dephosphorylation of the HK domain of CheS3; in the meantime, CheA3 is activated by an external signal (indicated by a red wedge) via the chemoreceptor MCP3 and inhibits the CheS3 kinase activity by phosphorylating its receiver domain.

Gram-negative cysts are nonreplicative dormant cells like that of dormant Gram-positive spores. In both cases, there appear to be many input signals and checkpoints harbored by these cells to regulate these developmental processes so as to ensure that the cell enters a nonreplicative survival pathway only when conditions for survival are harsh. The inputs to control Rhodospirillum/Azospirillum Gram-negative encystment are complex, involving production of cGMP (11), the complex Che3 signaling cascade (12, 17, 19), and the cellular energy charge. In the Gram-positive bacterium Bacillus subtilis, there are many histidine sensor kinases (KinA, KinB, KinC, KinD, and KinE) that are involved in controlling the decision to induce encystment (31, 38–41). Despite years of study, it remains unclear what signal most of these kinases receive to control their autophosphorylation activity. Although untested, it is possible that the cellular energy charge may also be an input that affects one or more Kin kinase activities so as to induce sporulation at times when the cells are undergoing a drop in energy production.

MATERIALS AND METHODS

Overexpression and purification of CheS3 and CheA3.

CheS3 and CheA3 were overexpressed in E. coli BL21 Rosetta 2(DE3) cells (Novagen) and isolated as previously described (12).

Kinase assays.

Kinase assays for CheS3 utilized buffer S (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 3 mM CaCl2, 10% glycerol), and those for CheA3 utilized buffer A (25 mM Tris-HCl, pH 7.4, 100 mM KCl, 6 mM CaCl2, 10% glycerol). Protein freezer stocks were diluted in the appropriate buffers to yield 2 to 5 µM working aliquots for CheS3 and CheA3. For ATP/ADP ratio experiments, a series of radioactive ATP/ADP solutions was prepared by mixing fixed amounts of [γ-33P]ATP (>3,000 Ci/mmol; PerkinElmer) and unlabeled ATP with varied amounts of ADP to achieve 0.8 mM total ATP with ATP/(ATP + ADP) ranging from 10% to 90%. Autophosphorylation was initiated at room temperature by adding 1 volume of ATP/ADP mix to 3 volumes of CheS3 or CheA3. For all kinase assays, reaction aliquots were removed and reactions were stopped by mixing with 6× SDS-PAGE sample loading buffer. Protein samples were subjected to SDS-PAGE, and gels were examined by autoradiography on a Typhoon 9100 scanner (GE Healthcare). Phosphorylation of the kinases was quantified using ImageJ by integrating the grayscale density of the radioactive bands.

Nucleotide extraction and ATP and ADP determination.

Cells from overnight cultures were washed two times with 40 mM phosphate buffer and inoculated in CENBA medium at a 1:40 ratio. These subcultures were aliquoted 3 ml/test tube and shaken at 37°C. At each time point, 675 µl of chilled 3 M perchloric acid and 75 µl of chilled 500 mM EDTA (pH 8.0) were simultaneously added into each tube. The acid-treated cultures were quickly shaken and placed on ice. Cell lysis was performed using FastPrep with Lysis Matrix B (MP Biomedicals), and cell debris was cleared by centrifugation at 13,000 rpm for 10 min. The supernatant was then neutralized with a solution of 1 M Tris-HCl, 0.5 M KOH, and 0.5 M KCl to pH 7.5. KClO4 precipitate was removed by centrifugation, and the nucleotide extracts were diluted in 20 mM Tris-HCl, pH 7.5, containing 2 mM EDTA before storage at −80°C. The ATP/(ATP + ADP) ratios were determined using a commercial bioluminescence-based assay kit (EnzyLight; Bioassay Systems).

Measurement of ADP and ATP binding constants with microscale thermophoresis.

CheS3 protein (20 µM) was fluorescently labeled using the Monolith NT protein labeling kit Blue-NHS (NanoTemper Technologies) according to the manufacturer’s instructions. Labeled protein was diluted to 100 nM, mixed with 16 increasing concentrations of ADP or ATPγS (various ranges were tested to optimize the signal), incubated for a short period, and transferred into 16 standard capillaries. Thermophoresis measurements were performed using a Monolith N-115 (NanoTemper Technologies) and automated Monolith data acquisition software. Data were fitted using the standard temperature jump and thermophoresis model using NanoTemper analysis software.

ADP stability assays.

Three aliquots of 10 µM CheS3 were allowed to autophosphorylate at room temperature for 30 min, with excess ATP and [γ-32P]ATP subsequently being removed by filtration using Zeba spin desalting columns (Thermo Scientific). Forty-five microliters of each aliquot was mixed with 30 µl of buffer 5 (12) to which 15 µl of 1 mM ATP, ADP, or AMP was added and then incubated at room temperature for 15 min. Protein samples were subjected to SDS-PAGE and autoradiography.

Thin-layer chromatography (TLC).

CheS3~P was immobilized onto Ni2+-Sepharose resin and rinsed 5 times with buffer, and CheS3~P-loaded resin was suspended in 100 µM ADP at time zero. Aliquots of the resin suspension were treated with equal volumes of 1 M formic acid before spotting on polyethyleneimine cellulose plates, which were then developed in 0.75 M KH2PO4, pH 3.75.

Acid-base stability assays.

The CheS3 phosphorylation state was determined by assaying phosphoprotein stability after autophosphorylation in the presence of acids, Tris buffer, or bases as previously described (12).

ACKNOWLEDGMENT

Funding for this study was provided by a National Institutes of Health grant GM099703 to C.E.B.

Footnotes

Citation He K, Dragnea V, Bauer CE. 2015. Adenylate charge regulates sensor kinase CheS3 to control cyst formation in Rhodospirillum centenum. mBio 6(3):e00546-15. doi:10.1128/mBio.00546-15.

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