Kinetic Characterization of the WalRK_Spn (VicRK) Two-Component System of Streptococcus pneumoniae: Dependence of WalK_Spn (VicK) Phosphatase Activity on Its PAS Domain

Abstract

The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalR_Spn (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalR_Spn is phosphorylated by the WalK_Spn (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalK_Spn signal transduction, we performed a kinetic characterization of the WalRK_Spn autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalK_Spn. Consequently, these analyses were performed using two truncated versions of WalK_Spn lacking its single transmembrane domain. The longer version (Δ35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Δ195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Δ35 and Δ195 WalK_Spn were similar [K_m(ATP) ≈ 37 μM; k_cat ≈ 0.10 min⁻¹] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK_Spn∼P were also similar in the phosphoryltransfer reaction to full-length WalR_Spn. In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalK_Spn for WalR_Spn∼P. Deletion and point mutations confirmed that optimal WalK_Spn phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalK_Spn DHp domain and ΔPAS mutations led to attenuation of virulence in a murine pneumonia model.

The WalRK two-component regulatory system (TCS) plays pivotal roles in maintaining cell wall and surface homeostasis in low GC Gram-positive bacteria (14, 41, 93). Recent global transcription analyses suggest that it may also respond to cell wall stresses, such as antibiotic addition (22, 38, 68). In Bacillus and Staphylococcus species, both the WalR (YycF) response regulator and the WalK (YycG) histidine kinase are essential in that they cannot be depleted (20, 23, 53). In contrast, the WalR (VicR) response regulator of Streptococcus species is essential, whereas the WalK (VicK) histidine kinase is not essential under standard growth conditions, and the corresponding gene can be knocked out (18, 58, 72, 89). The WalRK TCS was initially discovered in Bacillus subtilis, where it was designated as YycFG (20), but it is widespread in other species, where it has other names. A recent proposal was made to unify this nomenclature (14, 15), and we refer to this TCS from S. pneumoniae as WalRK_Spn, instead of VicRK as used previously (58, 72, 89).

WalRK regulons include genes that mediate peptidoglycan biosynthesis, cell division, and cell surface proteins (4, 9, 15, 54, 58, 59), but the specific genes regulated are dissimilar in different species (reviewed in references 14 and 93). In B. subtilis, WalRK_Bsu positively regulates several cell wall hydrolase genes and the ftsZ cell division gene and negatively regulates genes that modulate hydrolase activity (9, 23, 34). Likewise, the WalRK_Sau regulon of Staphylococcus aureus contains several murein hydrolases (15, 16). In these species, the essentiality of the WalRK TCS has been ascribed to misregulation of a combination of genes, since none of the hydrolase genes is individually essential (15), and ftsZ is transcribed from other promoters not regulated by WalRK (23). In S. pneumoniae, the essentiality of WalR_Spn is due to its positive regulation of pcsB, which encodes a putative hydrolase that plays a critical role in cell wall biosynthesis and division (4, 27, 57, 58). Besides cell wall hydrolases and division proteins, the WalRK regulons of different Streptococcus species includes genes encoding surface virulence factors and enzymes of exopolysaccharide biosynthesis (1, 42, 51, 59, 72).

The WalK histidine kinases of Streptococcus species have sensing domains that are structurally different from those of Bacillus, Staphylococcus, and most other species (60, 84). WalK_Bsu, which is typical of one class, contains two transmembrane domains flanking an extracytoplasmic domain. The transmembrane domains of WalK_Bsu interact with the membrane domains of the ancillary WalHI (YycHI) proteins to negatively regulate phosphorylation levels of the WalR_Bsu response regulator (84-86). In addition, WalK_Bsu colocalizes with FtsZ at the septa of dividing B. subtilis cells (24). In contrast, WalK_Spn, which exemplifies the other class, contains only a single transmembrane domain connected to an extracellular peptide of only 12 amino acids (Fig. 1, line 1) (47, 60, 89). Streptococcus species also lack homologues of WalHI. On the other hand, the cytoplasmic domains of both classes of WalK histidine kinases are highly similar and include HAMP (linker), PAS (potential signal binding made up of PAS and PAC motifs), DHp (dimerization and histidine phosphorylation), and CA (catalytic ATPase) domains typical of other histidine kinases (Fig. 1, line 1) (14, 25, 60, 93). The signals that are sensed by WalK histidine kinases are not yet known in any species, although it has been speculated that Lipid II derivatives may act as signals of the class that includes WalK_Bsu and WalK_Sau (14).

FIG. 1.

Domain organization of the protein constructs used in the present study. Full-length WalK_Spn (VicK) (line 1) contains 449 amino acids organized into five architectural and functional domains based on the SMART database (smart.embl-heidelberg.de): TM (anchoring transmembrane domain; amino acids 13 to 32), HAMP linker domain (amino acids 16 to 84), PAS domain consisting of PAS and PAC motifs (amino acids 94 to 202), DHp (dimerization histidine phosphoryltransfer [HisKA]; amino acids 208 to 274), and CA (kinase catalytic domain [HATPase]; amino acids 323 to 435). Histidine residue 218 (H218) is phosphorylated in the autokinase reaction. Numbering of full-length WalK_Spn was extended to the soluble, truncated WalK_Spn derivatives purified and characterized in the present study (lines 2 to 10; Materials and Methods; see Fig. S1 in the supplemental material). The affinity tags on the constructs are indicated. Full-length WalR_Spn (VicR) contains 234 amino acids organized into two domains: a receiver domain (amino acids 2 to 112) and an effector domain (amino acids 154 to 230). Aspartate residue 52 in the receiver domain is phosphorylated in the transferase reaction with WalK_Spn∼P constructs, and the effector domain contains the helix-turn-helix DNA binding motif. See the text for further details.

Given their regulatory importance, relatively little enzymological characterization of WalK histidine kinases and WalR response regulators has been reported. Autophosphorylation of WalK and phosphoryl group transfer between WalK∼P and WalR was demonstrated for homologues from B. subtilis (34, 35, 91, 96) and Enterococcus faecalis (52). In addition, phosphoryl transfer that reflects physiologically relevant cross talk was detected between PhoR_Bsu∼P and WalR_Bsu (34, 35). A refolded, soluble construct of WalK_Spn lacking the transmembrane domain was shown to have autokinase activity and to carry out phosphoryltransfer to purified WalR_Spn (18). However, the only quantitative analysis of WalK autophosphorylation was reported for highly truncated versions of WalK_Sau and WalK_Spn containing only the DHp and CA domains (12). We report here a comparison of the kinetics of the autokinase activity of a nearly full-length construct of WalK_Spn with a highly truncated version and their activities in phosphoryltransfer to full-length WalR_Spn and dephosphorylation of WalR_Spn∼P. We also analyzed the effects of an internal PAS domain deletion and changes of key amino acids in the DHp domain of WalK_Spn on these activities. We show that the autokinase and phosphoryltransfer reactions were largely unaffected by the absence of the HAMP and PAS domains but unexpectedly, optimal WalK_Spn phosphatase activity for WalR_Spn∼P depended on the PAS domain. We also show that the WalK_Spn internal PAS deletion and the point mutations in the DHp domain characterized here biochemically are important for pneumococcal virulence in a mouse model of infection.

MATERIALS AND METHODS

Bacterial strains and plasmids.

The bacterial strains and plasmids used in the present study are listed in Tables S1 and S2 in the supplemental material. Genomic DNA used to construct protein expression plasmids was obtained from S. pneumoniae serotype 2 strains R6 and D39 (see reference 48). In most cases, deletion and point mutations in cloned walK_Spn were constructed by fusion PCR (58, 59) using mutagenic primers and primers containing appropriate restriction sites (see Table S3 in the supplemental material). In three cases where mutations already existed, appropriate regions were simply amplified from the S. pneumoniae genome. PCR amplicons were cloned into the BamHI and BsaI or BsmFI sites of plasmid pSumo (LifeSensors, Inc.) and into NotI and XhoI sites of plasmid pET28a (Novagen, Inc.) to generate protein expression vectors (see Table S2 in the supplemental material). Recombinant expression plasmids were transformed into competent Escherichia coli strain DH5α and then into strain BL21(DE3)Rosetta/pLysS (see Table S1 in the supplemental material). All expression plasmids were verified by sequencing.

WalK_Spn mutants in S. pneumoniae were constructed by the Janus method of allele replacement used previously (67, 82, 88). A ΔwalK_Spn::[kanR-rpsL⁺] amplicon was transformed into strain IU1781 (D39 rpsL1 [resistant to 150 μg of streptomycin per ml]), resulting in strain IU1885 that is resistant to 250 μg of kanamycin per ml and sensitive to streptomycin. Markerless amplicons containing mutations in walK_Spn (ΔwalK_Spn), walK_Spn (H218A), walK_Spn (T222R), and walK_Spn (ΔPAS [absence of amino acids 104 to 198])) were constructed by fusion PCR (58, 59) using the primers listed in Table S2 in the supplemental material that introduce the desired amino acid substitutions or deletions. The ΔwalK_Spn deletion retained 60 bp at the 5′ and 3′ ends of walK_Spn to maintain any transcription or translation signals that might affect the expression of the closely spaced adjacent walR_Spn and walJ_Spn genes. Transformation of IU1885 with the markerless amplicons crossed out the walK_Spn::[kanR-rpsL⁺] region, resulting in colonies resistant again to 150 μg of streptomycin per ml and sensitive to kanamycin. Mutants were checked for gene duplications by PCR, and mutations were confirmed by DNA sequencing of genomic DNA (48). The presence of capsule was confirmed in each transformant by the Quellung reaction (48).

Overexpression and purification of proteins.

E. coli strains were grown with shaking at 30°C in standard LB media (MP Biomedicals) supplemented with antibiotics required to maintain expression vectors (see Table S1 in the supplemental material) and other additives as indicated (see Table S4 in the supplemental material). After reaching an optical density at 620 nm (OD₆₂₀) of ca. 0.2 to 0.6, cultures were induced by addition of IPTG at concentrations listed in Table S4.

Protein expression and solubility were estimated by SDS-PAGE (70). Cells from 1 ml of uninduced and induced cultures, adjusted to equal OD₆₂₀ levels, were collected by centrifugation for 3.5 min at 16,100 × g, resuspended in 100 μl of Laemmli sample buffer (Bio-Rad) containing 5% (vol/vol) β-mercaptoethanol, and boiled for 5 min. Equal volumes (≈15 μl) of samples were resolved by 10% Tris-glycine SDS-PAGE. Gels were stained with Coomassie brilliant blue dye (70), and the levels of protein induction were estimated visually by comparing uninduced and induced samples relative to molecular weight markers (Invitrogen). To estimate the solubility of recombinant proteins, 5 to 10 ml of induced cultures were collected by centrifugation as described above and resuspended in 3 ml of cold buffer A (20 mM NaPO₄, 0.5 M NaCl, 40 mM imidazole [pH 7.4]). Cells were lysed by passage through a chilled French pressure cell (20,000 lb/in²), and insoluble material was collected by centrifugation at 8,000 × g for 10 min at 4°C. Insoluble inclusion bodies in pellets were resuspended in 2 ml of buffer A. An equal volume of 2× Laemmli sample buffer was added to the supernatants and resuspended insoluble material. After boiling for 5 min, supernatant and pellet samples were loaded and analyzed by SDS-PAGE. If a band of the correct size was detected in the soluble fraction of the induced culture, then larger cultures were grown for protein purification.

Proteins were purified as described previously (59) with the following modifications. Induced cultures (0.3 to 1 liter) were chilled on ice and centrifuged at 8,000 × g for 10 min, and cell pellets were resuspended in 20 to 40 ml of buffer A supplemented with protease cocktail inhibitor III (Calbiochem). All remaining steps were performed at 4°C. Cells were lysed by two passes through a French press cell (20,000 lb/in²). Lysates were centrifuged twice at 8,000 × g for 20 min and filtered in a 50-ml disposable manifold containing a 0.22-μm-pore-size membrane (Millipore) to remove debris. The filtrate was applied to a HisTrap HP column (GE Healthcare) preequilibrated with buffer A using a peristaltic pump at a flow rate of 0.5 ml per min. Loaded columns were attached to a Shimadzu 10A Biocompatible high-pressure liquid chromatography (HPLC) system, and proteins were eluted by using a linear 60-min gradient of 40 to 500 mM imidazole in buffer A at a flow rate of 0.5 ml per min. Proteins were detected by monitoring A₂₂₀ and A₂₈₀. Fractions containing recombinant proteins were checked for contaminants by SDS-PAGE and pooled. Purified protein samples were concentrated and exchanged into final optimized storage buffers (see Table S4 in the supplemental material) by using Amicon ultracentrifugal filters (Millipore) according to the manufacturer's instructions. Alternatively, overnight dialysis in Slide-A-Lyzer cassettes (Thermo Scientific) was used when fast exchange to storage buffers in Amicon filters caused protein aggregation (see Table S4 in the supplemental material). To improve protein solubility, the composition of storage buffers was optimized by testing for aggregation by centrifugation at 100,000 × g for 15 min and reassaying protein concentrations in supernatants. Protein purities were estimated visually on stained SDS gels to be >95% (see Fig. S1 in the supplemental material). Similar results were obtained in the assays described below for several different preparations of the purified WalK_Spn constructs and WalR_Spn.

Determination of protein concentration.

The concentrations of purified proteins were determined by using the DC protein assay kit (Bio-Rad) as instructed by the manufacturer using bovine serum albumin (Sigma Fraction V) dissolved in storage buffer as the standard (see Table S4 in the supplemental material). For CD measurements, protein concentrations were determined by using a MicroBCA protein assay kit (Pierce) as instructed by the manufacturer.

Determination of WalK_Spn autophosphorylation kinetic parameters.

Autophosphorylation kinetic parameters were determined by using an SDS-PAGE method described previously (21) with the following modifications. Various WalK_Spn constructs (1.1 to 1.7 μM; see Table S4 in the supplemental material) were preequilibrated in reaction buffer B (50 mM Tris-HCl [pH 7.8], 200 mM KCl, 5 mM MgCl₂) for 10 min at 25°C. Reducing agents were omitted, because addition of 2 mM dithiothreitol diminished WalK_Spn autophosphorylation activity. The autophosphorylation reactions were started by adding various concentrations (6, 12.5, 50, 100, and 225 μM) of [γ-³²P]ATP (specific activity, 1.1 to 2.5 Ci/mmol; Perkin-Elmer, catalog no. BLU502Z). At designated times (15, 30, 45, and 60 s), 15-μl samples were removed, and reactions were stopped by adding the samples to 15 μl of 2× Laemmli sample buffer containing 5% (vol/vol) β-mercaptoethanol. Final samples (20 μl) were analyzed without heating by 10% Tris-glycine SDS-PAGE (21). After electrophoresis, gels were soaked for 20 min in 2% (vol/vol) glycerol and dried for 1 h at 80°C on a vacuum gel dryer (Bio-Rad). Dried gels were exposed to a storage phosphor screen (GE Healthcare) and analyzed by using a Typhoon Variable Mode Imager 9200 (Amersham) and ImageQuant 5.2 software (Molecular Dynamics). The amount of WalK_Spn∼P in each lane was quantified by using a standard curve generated by spotting known concentrations of [γ-³²P]ATP. Initial rates were calculated from linear regression plots of WalK_Spn∼P formed versus time (Fig. 2), and Michaelis-Menten kinetic parameters (K_m and k_cat) (see references 12, 21, 28, 50, 61, and 78 for precedents) were obtained by fitting velocities to ATP concentrations using a nonlinear regression program (GraphPad Prism). The autophosphorylation of the H218A or T222R mutant WalK_Spn (N)-Sumo construct was not detectable or very low, respectively. These constructs (2.4 to 3.3 μM; see Table S4 in the supplemental material) were preequilibrated in reaction buffer B for 5 min at 25°C, and reactions were initiated by adding 12.5 μM [γ-³²P]ATP (specific activity, 5 to 10 Ci/mmol). Samples were removed at different times (1, 2.5, 5, 10, 15, and 20 min) and processed as described above.

FIG. 2.

Progress curves of autophosphorylation reactions. Representative curves used to determine the kinetic parameters in Table 1 are shown. (A) Time course used to calculate the initial rates of WalK_SpnΔN35 (C)-His autophosphorylation (Fig. 1, line 2) at different ATP concentrations. (B) Velocity versus [ATP] curve based on A used to calculate K_m(ATP) and k_cat for WalK_SpnΔN35 (C)-His autophosphorylation. See Materials and Methods for details. These reactions contained 1.1 μM WalK_SpnΔN35 (C)-His in a volume of 100 μl at 25°C.

Combined assay of WalK_Spn autophosphorylation and phosphoryltransfer to WalR_Spn.

Combined reactions were carried out based as described previously (12, 65) with the following modifications. WalK_Spn constructs (2.2 to 3.4 μM; see Table S4 in the supplemental material) were autophosphorylated for 3 min in 100-μl reactions containing 50 mM Tris-HCl (pH 7.8), 200 mM KCl, 12.5 μM [γ-³²P]ATP (5 Ci/mmol), 15 to 20% (vol/vol) glycerol (to maintain WalR_Spn solubility later in the reaction), and either 5 mM MgCl₂ or 3.8 mM CaCl₂. The progression of WalK_Spn autophosphorylation was monitored at 0.5, 1, and 3 min by removing 15-μl samples and stopping reactions as described above for the autophosphorylation assay. At 3 min, 9.6 μM WalR_Spn (N)-His was added to the reaction mixtures containing WalK_Spn∼P without removal of excess ATP. Samples (15 μl) were removed 1.5, 4.5, and 19.5 min after WalR_Spn addition and processed and analyzed as described above. Amounts of WalK_Spn∼P and WalR_Spn∼P were calculated relative to the amount of WalK_Spn∼P at the time of WalR_Spn addition, which was set at 100%. To evaluate the effects of the purified PAS domain, we incubated WalK_SpnΔPAS constructs [WalK_SpnΔN195 (N)-Sumo (2.6 μM) or WalK_SpnΔN195 (C)-His (2.9 μM)] with purified PAS domain [PAS (N)-Sumo (7.1 μM) or PAS (C)-His (5.6 μM)] for 10 min at 25°C prior to initiation of the autophosphorylation reaction.

Quantification of phosphoryltransfer efficiency between WalK_Spn∼P constructs and WalR_Spn.

WalK_SpnΔN35 (C)-His (2.5 μM), WalK_SpnΔN35 (N)-Sumo (2.5 μM), WalK_SpnΔN195 (C)-His (3 μM), or WalK_SpnΔN195 (N)-Sumo (2.5 μM) constructs were autophosphorylated for 20 min at 25°C in 100 μl of 50 mM Tris-HCl (pH 7.9), 200 mM KCl, either 5 mM MgCl₂ or 5 mM CaCl₂, 15 to 20% (vol/vol) glycerol (to maintain WalR_Spn solubility later in the reaction), and 500 μM [γ-³²P]ATP (0.5 Ci/mmol). Excess ATP was removed from reactions by using spin desalting columns (Pierce). The recovery of the proteins after desalting was tested by using the DC protein assay and 8% Tris-glycine SDS-PAGE. WalK_Spn∼P concentrations after desalting ranged from 1.3 to 2.0 μM for different preparations. Then, 15-μl samples of desalted WalK_Spn∼P were added to 2× Laemmli buffer to determine the amounts of WalK_Spn∼P. At t = 0, 0.25 μM WalR_Spn (N)-His was added to the remainder of the WalK_Spn∼P sample to start the phosphoryltransfer reaction, and 15-μl samples were taken after 30, 60, 120, and 240 s and processed and analyzed as described above. The phosphoryltransfer efficiency between WalK_Spn∼P and WalR_Spn was evaluated by exponential decay plots of remaining WalK_Spn∼P versus time after addition of WalR_Spn (Fig. 3) (7, 78, 81) rather than measuring the rates of WalR_Spn∼P formation, which is subject to WalK_Spn phosphatase activity. Half-lives of WalK_Spn∼P were corrected for the intrinsic stability of WalK_Spn∼P in the absence of WalR_Spn (average t_1/2 ≈ 660 s).

FIG. 3.

WalK_Spn∼P disappearance due to phosphoryltransfer to added WalR_Spn. A representative reaction progression curve used to determine half-lives in Table 2 is shown. Open symbols, WalK_SpnΔN35 (C)-His∼P decay in the absence of WalR_Spn in reaction mixtures containing Mg²⁺ or Ca²⁺; closed symbols, decay of WalK_SpnΔN35 (C)-His∼P in parallel reaction mixtures containing Mg²⁺ or Ca²⁺ and WalR_Spn added at t = 0. See Materials and Methods for details. These reactions contained 1.5 μM WalK_SpnΔN35 (C)-His∼P and 0.25 μM WalR_Spn in a reaction volume of 100 μl at 25°C.

Phosphorylation of WalR_Spn by acetyl phosphate and quantification of WalR_Spn∼P autodephosphorylation and WalK_Spn-catalyzed dephosphorylation.

Phosphorylation of WalR_Spn with acetyl phosphate was carried out as described earlier (59) with the following modifications. WalR_Spn (N)-His (11.8 μM) was incubated with 40 mM acetyl phosphate (Fluka) in reaction buffer (50 mM Tris-HCl [pH 7.4], 200 mM KCl, 4 mM MgCl₂, and 40% [vol/vol] glycerol) for 75 min at 37°C. Excess acetyl phosphate was removed by using spin desalting columns (Pierce). The recovery of WalR_Spn∼P was ≈50% after desalting as determined by the DC protein assay. Desalted WalR_Spn∼P was incubated at 25°C in the presence or absence of ADP (13.2 μM) (t = 0). At times ranging from 10 min to 22.5 h, samples were removed, and the amounts of WalR_Spn∼P and WalR_Spn were determined by reversed-phase HPLC using a Phenomenex Jupiter 300A C₄ column and a Shimadzu 10A HPLC system (33, 59). Eluent A was composed of 20% (vol/vol) acetonitrile and 0.1% (vol/vol) trifluoroacetic acid in water, and eluent B was composed of 60% (vol/vol) acetonitrile and 0.1% (vol/vol) trifluoroacetic acid in water. A linear gradient from 50% eluent A plus 50% eluent B to 100% eluent B (no eluent A) was formed during a period of 18 min at a flow rate of 1 ml/min. Proteins were detected by monitoring the A₂₂₀. The relative amounts of WalR_Spn∼P and WalR_Spn were calculated from peak areas and normalized to starting samples, which contained ≈85% WalR_Spn∼P. The half-lives of WalR_Spn∼P were calculated from exponential decay plots with time (GraphPad Prism), where the rate constant of autodephosphorylation, k_auto, was ln2/t_1/2 (98).

WalR_Spn∼P dephosphorylation catalyzed by WalK_Spn PAS⁺ constructs in the presence of nucleotide cofactors was determined as follows. Desalted WalR_Spn∼P (5.9 μM) was incubated in the presence of WalK_SpnΔN35 (C)-His (2.0 μM) at 25°C in 50 mM Tris-HCl (pH 7.4 or 7.8), 200 mM KCl, 2 mM MgCl₂, and 40% glycerol. The following nucleotide cofactors were added to some of the reaction mixtures: ATP (13.2 μM), ATP-γS (13.2 μM), or ADP (13.2 or 120 μM). Samples were removed at time points ranging from 10 min to 5 h, and the extent of WalR_Spn∼P dephosphorylation was determined by C₄-HPLC as described above. The rate constant for dephosphorylation of WalR_Spn∼P in the presence of WalK_Spn was determined by the following formula: k = ln2/t_1/2 − ln2/t_{1/2 auto}, where t_1/2auto is the half-life of WalR_Spn∼P in the absence of histidine kinase (98).

WalR_Spn∼P dephosphorylation catalyzed by WalK_Spn DHp mutants was determined at 25°C as described above in reactions containing desalted WalR_Spn∼P (4.3 to 5.8 μM), WalK_SpnΔN35 (N)-Sumo (3.0 μM), WalK_SpnΔN35 H218A (N)-Sumo (2.1 μM), or WalK_SpnΔN35 T222R (N)-Sumo (1.7 μM), and ADP (9.5 to 12.8 μM). WalR_Spn∼P dephosphorylation catalyzed by WalK_Spn PAS domain mutants was determined at 25°C as described above in reactions containing desalted WalR_Spn∼P (4.7 to 5.3 μM), WalK_SpnΔN195 (N)-Sumo (2.1 μM), WalK_SpnΔN195 (C)-His (2.2 μM), WalK_SpnΔN35 D133N,N136Y,L140R (N)-Sumo (2.6 μM), or WalK_SpnΔN35 ΔPAS[104-198] (N)-Sumo (1.8 μM), and ADP (11.0 to 11.9 μM).

CD spectroscopy of wild-type and mutant WalK_Spn constructs.

Circular dichroism (CD) spectra were obtained for purified WalK_SpnΔN35 (N)-Sumo, WalK_SpnΔN35 H218A (N)-Sumo, WalK_SpnΔN35 T222R (N)-Sumo, WalK_SpnΔN35 D133N,N136Y,L140R (N)-Sumo, and WalK_SpnΔN195 (N)-Sumo using a Jasco J-715 CD spectropolarimeter using a previously published protocol (30). Protein concentrations varied from 0.097 to 0.164 mg/ml in 10 mM potassium phosphate buffer (pH 7.4) in a 0.1-cm quartz cell (Starna). Proteins were exchanged into this buffer by using spin desalting columns (Pierce) to remove interfering components. Three independent spectra of each protein were recorded at 25°C by using a scanning speed of 100 nm per min with 0.5-nm intervals. The wavelength range was set from 190 to 240 nm with a bandwidth of 2 nm. Spectra were averaged, smoothed using a Savitsky-Golay filter with a smoothing window of 15 points (30), and corrected for buffer absorbance in the absence of proteins. The raw data was converted to the mean residue ellipticity: [θ] in degrees cm² dmol⁻¹ = (millidegrees × mean residue weight)/(path length in mm × concentration in mg ml⁻¹), where the mean residue weight is the molecular weight of the protein divided by the number of amino acids minus 1. The [θ] values were used to perform secondary structure analyses with Selcon3 software from Dichroweb (see Table S5 in the supplemental material) (79, 80, 92).

Growth of S. pneumoniae strains.

Parent and walK_Spn mutant strains were grown statically in 16-by-100-mm glass tubes at 37°C in an atmosphere of 5% CO₂ as described previously (67, 88). Briefly, bacteria were inoculated from frozen stocks into 5.0 ml of brain heart infusion (BHI) broth (BD), serially diluted in BHI broth, and propagated overnight. Overnight cultures that were still in exponential growth phase (OD₆₂₀ ≈ 0.2 to 0.4) were diluted to a OD₆₂₀ of ≈0.1, and 50 μl of these diluted cultures was inoculated into 5.0 ml of BHI broth lacking antibiotic to give a starting OD₆₂₀ of ≈0.001. Tubes were gently inverted before OD₆₂₀ readings were obtained at approximately 1-h intervals using a Spectronic 20 spectrophotometer.

Murine pneumonia model of infection.

All procedures were approved in advance by the Institutional Animal Care and Use Committee and were performed according to recommendations of the National Research Council. Procedures were carried out as described previously (43) with the following changes. Male ICR (21 to 24 g; Harlan) mice were anesthetized by inhaling 4% isoflurane (Butler Animal Health Supply) delivered by an EZAnesthesia system (Euthanex Corp.) for 5 min. Nine or six mice in replicate experiments were inoculated intranasally with each bacterial strain to be tested. Aliquots (1 ml) of each strain growing exponentially in BHI broth (OD₆₂₀ ≈ 0.240) were microcentrifuged for 10 min at 13,500 × g, and cell pellets were resuspended in 1 ml of phosphate-buffered saline (pH 7.4) solution. A 50-μl sample of this suspension (≈7.0 × 10⁶ CFU) was used as the inoculum. Anesthetized mice were placed on their backs, and their mouths were gently closed to allow inhalation of the 50-μl inoculum, which was delivered in aliquots to the center of the noses. To ensure inhalation, mice were suspended vertically from their teeth after inoculation for ≈1 min until they started to awaken from the anesthesia. CFU in inocula were confirmed by serial dilution and plating. Mice were monitored at ≈6-h intervals. Death was not used as an endpoint. Moribund mice were euthanized by CO₂ asphyxiation, and that time point was used as “time of death” in survival curves. Kaplan-Meier survival curves and log-rank tests were generated by using GraphPad Prism software.

RESULTS

Overexpression and purification of proteins.

We initially attempted to purify active full-length WalK_Spn based on methods published for histidine kinases and other signal transducers (8, 83). Although we could overexpress sufficient amounts of protein, full-length WalK_Spn was insoluble, even using these conditions. Based on extensive precedents from other histidine kinases (12, 21, 32, 61, 76), we turned to truncated versions of WalK_Spn for these initial kinetic analyses. The longest active form of pneumococcal WalK_Spn that retained autokinase activity was truncated for the first 35 amino acids specifying the transmembrane domain and a short section of the HAMP domain (Δ35 constructs, Fig. 1, lines 2 to 6). Several truncations that extended further into the HAMP domain were insoluble or inactive (data not shown). For comparison with the only published kinetic study of WalK_Spn (12), we also characterized WalK_Spn truncated for the transmembrane, HAMP, and PAS domains (Δ195 constructs, Fig. 1, lines 7 to 9). For each WalK_Spn truncation, we added a Sumo or His₆ tag to the amino or carboxyl terminus, respectively, and purified the proteins by affinity chromatography as described in Materials and Methods and Table S4 in the supplemental material. We also purified the PAS domain alone fused to the Sumo or His₆ tag (Fig. 1, line 10). Full-length WalR_Spn response regulator fused to an amino-terminal His₁₀ tag (Fig. 1, line 11) was purified as before (59).

Attempts to remove the affinity tag from the WalK_SpnΔN35 (N)-Sumo construct with Sumo protease were not successful, because the protein lost autokinase activity (data not shown). Therefore, to control for tag-specific effects, we characterized both the N-Sumo and C-His versions of each purified WalK_Spn protein. The tag effects that we observed were generally small. We characterized amino acid substitutions in (N)-Sumo WalK_Spn constructs, because they were generally more soluble than the corresponding (C)-His WalK_Spn proteins (see Table S4 in the supplemental material). CD spectra confirmed that mutant WalK_Spn proteins were not grossly misfolded compared to the wild-type protein (see Table S5 in the supplemental material). Many substitutions and small internal deletions in the PAS domain resulted in insoluble WalK_Spn that could not be purified (see Fig. S2 and Table S4 in the supplemental material). We were able to improve the solubility of full-length WalR_Spn (N)-His by increasing the glycerol and salt concentration in its storage and reaction buffers (see Materials and Methods and Table S4 in the supplemental material) (59).

Kinetic parameters of WalK_Spn autophosphorylation do not depend on the presence of the HAMP and PAS domains.

The autokinase kinetic parameters of the WalK_Spn Δ35 and Δ195 proteins fused to the (C)-His tag were nearly the same within experimental error (Fig. 2 and Table 1, lines 1 and 5). The (N)-Sumo constructs had comparable K_m(ATP) values to their (C)-His counterparts (Table 1, lines, 1, 2, 5, and 6). There was some variation in the k_cat values of the (N)-Sumo compared to the (C)-His constructs, where the k_cat of WalK_SpnΔ35 (N)-Sumo was ≈2.6-fold greater than that of the (C)-His version (Table 1, lines 1 and 2). However, taken together, the absence of the HAMP and PAS domains in the WalK_Spn Δ195 constructs did not appreciably affect the autophosphorylation kinetic parameters, and tag-specific effects, although present for the (N)-Sumo constructs, tended to be marginal. The K_m(ATP) of the WalK_SpnΔ195 (N)-Sumo protein used here (28 μM; Table 1, line 6) was somewhat higher than that reported previously for a comparable WalK_Spn Δ195 construct fused to an N-terminal avidin-His tag (3 μM), although the k_cat rates were comparable for the two constructs (12). The autophosphorylation kinetic parameters of WalK_Spn reported here are similar to those reported for other truncated histidine kinases, such as WalK_Sau, KinA, NarQ, and HpKA (12, 21, 31, 61).

TABLE 1.

Kinetic parameters of WalK_Spn histidine kinase autophosphorylation^a

Enzyme constructb	Mean ± SEM (n)		kcat/Km (M−1 min−1)
	Km for ATP (μM)	kcat (min−1)
WalKSpn PAS+ constructs
1. WalKSpnΔN35 (C)-His	42.0 ± 2.2 (3)	0.084 ± 0.008 (3)	2,000
2. WalKSpnΔN35 (N)-Sumo	43.8 ± 8.3 (4)	0.216 ± 0.02 (4)	4,930
3. WalKSpnΔN35 H218A (N)-Sumo	NA
4. WalKSpnΔN35 T222R (N)-Sumo	LA
WalKSpn PAS mutant constructs
5. WalKSpnΔN195 (C)-His	36.7 ± 1.9 (3)	0.072 ± 0.004 (3)	1,960
6. WalKSpnΔN195 (N)-Sumo	28.4 ± 6.0 (4)	0.030 ± 0.003 (4)	1,060
7. WalKSpnΔN195 H218A (N)-Sumo	NA
8. WalKSpnΔN195 T222R (N)-Sumo	LA
9. WalKSpnΔN35 D133N, N136Y, L140R (N)-Sumo	72.4 ± 29.1 (2)	0.204 ± 0.04 (2)	2,820
10. WalKSpnΔN35 ΔPAS[104-198] (N)-Sumo	257 ± 17 (2)	0.258 ± 0.03 (2)	1,000

^aKinetic parameters were determined at 25°C as described in Materials and Methods. Reaction mixtures contained 1.1 to 1.7 μM concentrations of the indicated WalK_Spn constructs. The means are shown for the indicated number of independent experiments in parentheses (n). “NA” indicates no activity was detected in 20 min. “LA” indicates that autophosphorylation activity was not detected in 1 min and could be detected only in 5-min reactions, in which the relative amount of WalK_Spn T222R∼P was <10% compared to the wild-type protein (data not shown).

^bEach line of data is preceded by a “line number” (“1.”, “2.”, etc.) in the first column. These lines are referenced by number in the text at the corresponding in-text table callouts.

Autophosphorylation of the WalK_Spn ΔN35 and ΔN195 decreased in the presence of 2 mM DTT (data not shown). This result contrasts with a recent report that addition of reducing reagent increased the autokinase activity of full-length WalK_Efa from E. faecalis (52). Different numbers and locations of cysteine residues in WalK_Spn and WalK_Efa may underlie this difference. WalK_Efa contains three cysteine residues, one in the PAS domain and two in the CA domain. In contrast, WalK_Spn contains a single cysteine (C240) in the DHp domain near the phosphorylated histidine (H218). Whether disulfide bond formation and covalent dimerization modulate WalK_Spn function remains to be investigated, especially in the context of the unusual production of high levels of hydrogen peroxide by S. pneumoniae (see reference 67).

H218A, T222R, and ΔPAS mutant WalK_Spn have abolished or reduced autokinase activity.

We determined the autophosphorylation parameters for several mutant WalK_Spn proteins that were soluble. As expected, WalK_Spn H218A mutants (Fig. 1, lines 4 and 9) lacked autokinase activity (Table 1, lines 3 and 7), because they are missing the histidine residue that is phosphorylated. The T222R substitution in the DHp domain (Fig. 1, lines 3 and 8) was tested, because a comparable change in some histidine kinases, such as EnvZ, results in increased autokinase activity, while abolishing phosphatase activity for the cognate phosphorylated response regulator (2, 17). However, the WalK_Spn T222R substitution greatly reduced autokinase activity (Table 1, lines 4 and 8). A refolded WalK_Spn containing a L100R substitution in the PAS domain was also reported to lack autokinase activity (18), although proper folding of this mutant protein was not confirmed. In this first study, we did not introduce other changes at this or other positions in the WalK_Spn DHp domain.

Several amino acid substitutions and small internal deletions in the predicted β strands of the WalK_Spn PAS domain resulted in insoluble protein (see Fig. S2 and Table S4 in the supplemental material). Inability to obtain soluble histidine kinases containing amino acid substitutions in their PAS domains has been reported before (e.g., E. coli NtrB [63]). Mutant WalK_Spn containing three substitutions (D133N, N136Y, and L140R) in a predicted α-helical region of PAS (Fig. 1, line 5) had the same autokinase K_m(ATP) and k_cat as the wild-type protein within experimental error (Table 1, lines 2 and 9). In contrast, an internal deletion of PAS (ΔPAS[104-198]; Fig. 1, line 6) in the one construct that was soluble increased the WalK_Spn autokinase K_m(ATP) by ≈6-fold without affecting the k_cat (Table 1, lines 2 and 10). Therefore, internal deletion of the WalK_Spn PAS domain reduced the relative catalytic efficiency of the autokinase reaction.

Absence of the HAMP and PAS domains of WalK_Spn has a minimal effect on the kinetic preference of the phosphoryltransfer reaction.

We determined half-lives of WalK_Spn∼P during the phosphoryltransfer reaction to WalR_Spn as described in Materials and Methods (see Fig. 3 and Table 2). Phosphorylation of WalK_Spn was carried out in reaction mixtures containing either Mg²⁺ or Ca²⁺, and the same divalent cation was present in the subsequent phosphoryltransfer reactions. As observed for other TCS pairs (32, 81), the initial rates of this reaction were too rapid to measure by steady-state methods in reactions containing excess WalR_Spn substrate. Therefore, we determined the half-lives of WalK_Spn∼P in reactions containing an excess of WalK_Spn over WalR_Spn, which was present at a concentration lower than the typical K_m for TCS pairs (see references 10 and 77). The resulting half-lives of WalK_Spn∼P should reflect the kinetic preference (k_cat/K_m) of the phosphoryltransfer reaction (see references 13, 76, and 77).

TABLE 2.

Half-lives of WalK_Spn∼P in phosphoryltransfer reactions to WalR_Spn^a

Enzyme constructb	Mean ± SEM
	WalKSpn∼P half-life(s) + Mg2+	WalKSpn∼P half-life(s) + Ca2+
WalKSpn PAS+ constructs
1. WalKSpnΔN35 (C)-His	12.5 ± 0.7	60.6 ± 7.1
2. WalKSpnΔN35 (N)-Sumo	26.3 ± 3.1	501 ± 122
WalKSpn ΔPAS mutant constructs
3. WalKSpnΔN195 (C)-His	23.0 ± 3.6	114 ± 31
4. WalKSpnΔN195 (N)-Sumo	15.9 ± 2.2	64.1 ± 13.2

^aReactions were performed at 25°C in buffers containing Mg²⁺ or Ca²⁺ as described in Materials and Methods. Reaction mixtures contained 1.3 to 2.0 μM concentrations of the indicated WalK_Spn constructs and 0.25 μM WalR_Spn (N)-His. The experiment was performed independently twice. The average intrinsic half-life of WalK_Spn∼P was ≈660 s in the presence of either cation.

^bSee Table 1, footnote b.

The kinetic preference of phosphoryltransfer was similar for the WalK_Spn Δ35 and Δ195 constructs in reaction mixtures containing Mg²⁺ ion (Table 2, lines 1 to 4). Although there may be some minor variation due to tag effects, these data indicate that the absence of the HAMP and PAS domains had minimal effect on the kinetic preference of the phosphoryltransfer reaction. The rate of decrease of WalK_Spn∼P amount during phosphoryltransfer depended strongly on Mg²⁺ ion and was reduced severalfold when Ca²⁺ replaced Mg²⁺ in reaction mixtures (Fig. 3 and Table 2). We do not know why substitution of Mg²⁺ with Ca²⁺ had a much more pronounced effect on WalK_Spn Δ35 (N)-Sumo compared to the other constructs (Table 2, line 2).

WalK_Spn phosphatase activity depends on the PAS domain.

Many histidine kinases possess a phosphatase activity that plays a role in preventing unwanted cross talk (3, 49, 74). However, there are several notable exceptions of histidine kinases that lack phosphatase activity, such as KinA and PhoR of B. subtilis (19, 73, 90). Gel-based combined phosphoryltransferase assays revealed a significant phosphatase activity of the WalK_Spn Δ35 constructs (Fig. 4A and B and see Fig. S3 in the supplemental material). In the presence of Mg²⁺ ion, low amounts of WalR_Spn∼P were detected, and there was a clear loss of labeled phosphate from the amount present in the starting WalK_Spn∼P. Previously, it was shown that the phosphatase activity of the EnvZ histidine kinase was strongly reduced in reaction mixtures containing Ca²⁺ instead of Mg²⁺ ion (17, 99). Similar to results with EnvZ, considerably more WalR_Spn∼P was detected in phosphoryltransfer reactions containing Ca²⁺ instead of Mg²⁺ (Fig. 4A and B and see Fig. S3 in the supplemental material), a finding consistent with a WalK_Spn phosphatase activity.

FIG. 4.

Autoradiographs showing autophosphorylation of WalK_Spn Δ35 and Δ195 constructs, phosphoryltransfer to WalR_Spn, and WalK_Spn phosphatase of WalR_Spn∼P. Representative time courses are shown and quantitated in Fig. S3 and S4 in the supplemental material. Combined reactions of WalK_Spn autophosphorylation and WalR_Spn phosphoryltransfer were performed at 25°C in reaction mixtures containing Mg²⁺ or Ca²⁺ as described in Materials and Methods. WalK_Spn autophosphorylation reactions proceeded for 3 min before WalR_Spn was added without removal of ATP (t = 0). Reactions contained the following concentrations of proteins: 2.2 μM WalK_SpnΔN35 (C)-His (A), 2.9 μM WalK_SpnΔN195 (C)-His (B), 3.4 μM WalK_SpnΔN35 (N)-Sumo (C), and 2.6 μM WalK_SpnΔN195 (N)-Sumo (D). Each reaction contained 9.6 μM WalR_Spn.

Unexpectedly, similar amounts of WalR_Spn∼P were detected in combined phosphoryltransfer reactions containing WalK_Spn Δ195 and either Mg²⁺ or Ca²⁺ ion (Fig. 4C and D and see Fig. S4 in the supplemental material). Since the kinetic parameters for the autokinase and phosphoryltransfer reactions were similar for the WalK_Spn Δ35 and Δ195 constructs (Tables 1 and 2), these data imply that the WalK_Spn phosphatase activity was significantly reduced in the absence of the HAMP, PAS, or both domains. Consistent with this interpretation, WalR_Spn∼P continued to accumulate in reactions containing WalK_Spn Δ195 (19.5 min, Fig. 4C and D and Fig. S4), but not WalK_Spn Δ35 (19.5 min, Fig. 4A and B and Fig. S3). In the former case, autophosphorylation of WalK_Spn Δ195 and phosphoryltransfer to WalR_Spn continued to occur, because the WalK_Spn phosphatase activity was significantly reduced. In the latter case, the WalK_Spn phosphatase acted on WalR_Spn∼P, even in reaction mixtures containing Ca²⁺.

The conclusion that the phosphatase activity depends on the WalK_Spn PAS domain was supported by the finding that the WalK_Spn internal ΔPAS[104-198] mutant protein had reduced WalR_Spn∼P phosphatase activity in combined phosphoryltransferase assays. Similar to the results for the WalK_Spn Δ195 construct, WalR_Spn∼P continued to accumulate in reactions containing WalK_Spn(ΔPAS[104-198]) and either Mg²⁺ or Ca²⁺ (Fig. 5B and see Fig. S5 in the supplemental material). In contrast, the mutant WalK_Spn with the triple (D133N, N136Y, and L140R) substitutions in the PAS domain served as a control and did not show diminished WalR_Spn∼P phosphatase activity (Fig. 5A and Fig. S5). Finally, since the phosphatase activity depended on the WalK_Spn PAS domain, we attempted to restore the phosphatase activity of WalK_Spn Δ195 by adding back purified PAS domain in trans (Fig. 1, line 10). Added purified PAS domain did not change the autokinase and phosphoryltransferase activities of the WalK_Spn Δ195 constructs, nor was WalK_Spn phosphatase activity restored (data not shown). However, we do not know whether the purified PAS domain folded correctly.

FIG. 5.

Autoradiographs showing autophosphorylation of WalK_Spn PAS domain mutant constructs, phosphoryltransfer to WalR_Spn, and WalK_Spn phosphatase of WalR_Spn∼P. Representative time courses are shown and quantitated in Fig. S5. Combined reactions of WalK_Spn autophosphorylation and WalR_Spn phosphoryltransfer were performed at 25°C in reaction mixtures containing Mg²⁺ or Ca²⁺ as described in Materials and Methods. WalK_Spn autophosphorylation reactions proceeded for 3 min before WalR_Spn was added without removal of ATP (t = 0). Reactions contained the following concentrations of proteins: 3.4 μM WalK_SpnΔN35 D133N,N136Y,L140R (N)-Sumo (A) and 2.5 μM WalK_SpnΔN35 ΔPAS[104-198] (N)-Sumo (B). Each reaction contained 9.6 μM WalR_Spn.

WalR_Spn∼P autophosphatase activity is extremely low.

We previously showed that WalR_Spn could be phosphorylated with ≈85% efficiency by incubation with acetyl phosphate (59). We used WalR_Spn∼P from this reaction in HPLC-based assays for WalK_Spn phosphatase activity as described in Materials and Methods (Fig. 6). Amino acid alignment predicted that WalR_Spn∼P was likely to have a low rate of autophosphatase activity (see Table S6 in the supplemental material) (87). This prediction was confirmed by phosphatase assays showing that the half-life of WalR_Spn∼P was ≈23 h at 25°C (Table 3, line 1). The WalR_Spn∼P autophosphatase activity was not affected by addition of ADP, similar to other response regulators, including VanR, PhoQ, and DrrA (29, 71, 94).

FIG. 6.

WalK_Spn phosphatase activity of WalR_Spn∼P. Representative reaction progression curves used to determine the rate constants and half-lives in Tables 4 and 5 are shown. Reactions containing 13.2 μM ADP were carried out at 25°C as described in Materials and Methods. (A) Reversed-phase HPLC chromatograms showing dephosphorylation of WalR_Spn∼P by WalK_SpnΔN35 (C)-His with time, where t = 0 was the addition of the WalK_SpnΔ35. Reactions contained 5.9 μM WalR_Spn∼P and 2.0 μM WalK_SpnΔN35 (C)-His. (B) Percent of WalR_Spn∼P remaining with time was calculated from the areas under the WalR_Spn∼P and WalR_Spn, where 100% at t = 0 corresponded to 85% WalR_Spn∼P in the starting sample. The rates of WalR_Spn∼P disappearance and the half-lives were calculated as described in Materials and Methods.

TABLE 3.

Rates of WalR_Spn∼P autodephosphorylation and dephosphorylation in the presence of WalK_Spn constructs^a

Enzyme constructb	Mean ± SEM
	k (min−1)	WalRSpn∼P half-life (min)
WalRSpn∼P autophosphatase activity
1. WalRSpn (N)-His	0.000554 ± 0.000113	1,370 ± 320
WalKSpn PAS+ phosphatase activity
2. WalKSpnΔN35 (C)-His	0.0195 ± 0.0002	34.5 ± 0.3
3. WalKSpnΔN35 (N)-Sumo	0.036 ± 0.0007	18.9 ± 0.4
4. WalKSpnΔN35 T222R (N)-Sumo	0.0025 ± 0.00065	228 ± 47.2
5. WalKSpnΔN35 H218A (N)-Sumo	0.0028 ± 0.0004	210 ± 22
WalKSpn PAS domain mutant phosphatase activity
6. WalKSpnΔN195 (C)-His	0.0042 ± 0.0001	147 ± 3
7. WalKSpnΔN195 (N)-Sumo	0.0023 ± 0.0007	249 ± 58.7
8. WalKSpnΔN35 D133N,N136Y,L140R (N)-Sumo	0.0184 ± 0.0015	36.6 ± 2.9
9. WalKSpnΔN35 ΔPAS[104-198] (N)-Sumo	0.0024 ± 0.0009	234 ± 71.5

^aDephosphorylation rates and half-lives of WalR_Spn∼P were determined at 25°C in reaction mixtures containing Mg²⁺, ADP, and the indicated WalK_Spn constructs as described in Materials and Methods. Reaction mixtures contained 4.3 to 5.9 μM WalR_Spn(N)-His∼P and 1.7 to 3.0 μM concentrations of the indicated WalK_Spn constructs. Experiments were performed at least two times.

^bSee Table 1, footnote b.

WalK_Spn catalyzed dephosphorylation of WalR_Spn∼P is significantly reduced by deletion of the PAS domain.

Addition of WalK_Spn decreased the half-life of WalR_Spn∼P by ≈40- or ≈70-fold for the Δ35 (C)-His or (N)-Sumo constructs, respectively (Fig. 6 and Table 3, lines 2 and 3). This result directly demonstrates a strong WalK_Spn phosphatase activity and indicates a relatively small (<2-fold) tag-specific effect. This decrease in half-life was comparable to that of truncated EnvZ for OmpR∼P in similar reaction mixtures (98, 99). The H218A and T222R substitutions reduced the WalK_Spn phosphatase activity by ≈12-fold (Table 3, lines 3, 4, and 5). Thus, these mutations abolished or greatly reduced both the autokinase and phosphatase activities of these WalK_Spn Δ35 constructs (Tables 1 and 3). However, the H218A mutant WalK_Spn still retained measurable phosphatase activity (Table 3, line 5), whereas it totally lacked autokinase activity (Table 1, lines 3 and 7). Therefore, the WalK_Spn phosphatase activity does not occur by a reversal of the phosphoryltransfer reaction and likely proceeds by release of inorganic phosphate (see references 25, 36, 39, and 75). Similar to other histidine kinases (37, 39, 44, 98, 99), WalK_Spn required ADP or ATP for optimal phosphatase activity (Table 4). In addition, nonhydrolyzable ATPγS stimulated the WalK_Spn phosphatase activity to the same extent as ADP and ATP, a finding consistent with the conclusion that WalK_Spn phosphatase activity is not a simple reversal of the phosphoryltransfer reaction.

TABLE 4.

Rates of WalR_Spn∼P dephosphorylation catalyzed by WalK_Spn in the presence of nucleoside phosphate cofactors^a

Cofactor (concn [μM])b	Mean ± SEM
	k (min−1)	WalRSpn∼P half-life (min)
1. None	0.0044 ± 0.00036	140 ± 10
2. ADP (13.2)	0.0195 ± 0.0002	34.5 ± 0.3
3. ADP, pH 7.8 (13.2)	0.029 ± 0.002	23.3 ± 1.9
4. ADP (120)	0.018 ± 0.0004	37.1 ± 0.9
5. ATP (13.2)	0.032 ± 0.006	21.3 ± 3.8
6. ATPγS (13.2)	0.019 ± 0.0001	35.4 ± 0.2

^aDephosphorylation rates and half-lives of WalR_Spn∼P were determined at 25°C in reaction mixtures containing Mg²⁺ at pH 7.4 (or pH 7.8 where indicated) in the presence or absence of cofactors as described in Materials and Methods. Reaction mixtures contained 5.9 μM WalR_Spn (N)-His∼P and 2.0 μM WalK_SpnΔN35 (C)-His. Experiments were performed independently twice.

^bSee Table 1, footnote b.

Finally, deletion of the PAS domain decreased the phosphatase activity by ≈13-fold for the WalK_Spn (N)-Sumo constructs (Table 3, lines 3, 7, and 9). A smaller decrease (≈5-fold) was detected for the WalK_Spn (C)-His constructs (Table 3, lines 2 and 6). As a control, amino acid substitutions in the WalK_Spn PAS domain minimally affected the phosphatase activity (Table 3, lines 3 and 8). Together, these results support the conclusion from the gel-based combined assays (Fig. 4 and 5 and see Fig. S3 to S5 in the supplemental material) that the PAS domain is required for optimal WalK_Spn phosphatase activity. The relative rates of the phosphatase reaction appeared to be more rapid in the combined gel-based than the HPLC-based assays (Fig. 4, 5, and 6 and Table 3). This difference may reflect some inactivation or conformational changes that occur when WalR_Spn is phosphorylated by acetyl phosphate, which requires an extended incubation and removal of unincorporated acetyl phosphate (Materials and Methods).

ΔPAS and DHp mutations reduce pneumococcal virulence.

To relate the biochemical properties described above to pneumococcus physiology, we tested whether the mutants affected virulence (Fig. 7). Markerless walK_Spn(H218A), walK_Spn(T222R), and walK_Spn(ΔPAS[104-198]) mutations in full-length walK_Spn and a ΔwalK_Spn deletion were crossed into the chromosome of virulent parent strain D39 rpsL1 (Materials and Methods) (see Table S1 in the supplemental material). The rpsL1 mutation was used in the allele exchange procedure and does not affect virulence in this pneumonia model of infection (67). Western blot analyses (see reference 4) confirmed that the walK_Spn(H218A), walK_Spn(T222R), and walK_Spn(ΔPAS[104-198]) mutants produced similar amounts of WalK_Spn protein as the D39 parent strain (data not shown). Attempts to detect mutant WalK_Spn deleted for its transmembrane domain (Fig. 1, line 2) were not successful, possibly due to degradation.

FIG. 7.

Growth and virulence properties of walK_Spn⁺ and ΔwalK_Spn mutant strains. Strain constructions, growths, and survival curve analyses were performed as described in Materials and Methods on the following strains: D39 rpsL1 parent (IU1781), D39 rpsL1 ΔwalK_Spn (IU1896), D39 rpsL1 walK_Spn (H218A) (IU3102), D39 rpsL1 walK_Spn (T222R) (IU3104), D39 rpsL1 walK_Spn ΔPAS[104-198] (IU2306), and D39 rpsL1 walK_Spn⁺ repair (IU2193). (A) Representative growth curve of static BHI broth cultures at 37°C in an atmosphere of 5% CO₂. The experiment was repeated numerous times for each strain. (B) Survival curve analysis of a murine pneumonia model using intranasal inoculation of nine mice for each bacterial strain. Median survival times are in parentheses, where “***” denotes P < 0.005 in log-rank (Mantel-Cox) tests. Similar results were obtained from an independent experiment using six mice per strain.

All strains grew at approximately the same rate (Fig. 7A), although the ΔwalK_Spn and walK_Spn(H218A) mutants, which lacked autokinase activity (Table 1, lines 3 and 7), consistently had lower growth yields (OD₆₂₀ = 0.59 ± 0.03 and 0.60 ± 0.02, respectively) than the walK_Spn⁺ parent and the walK_Spn(T222R) and walK_Spn(ΔPAS[104-198]) mutants (OD₆₂₀ = 0.82 ± 0.02, 0.71 ± 0.07, and 0.79 ± 0.02, respectively). Repair of the ΔwalK_Spn deletion back to wild-type restored the growth yield (OD₆₂₀ = 0.80 ± 0.03). Finally, the walK_Spn(H218A), walK_Spn(T222R), walK_Spn(ΔPAS[104-198]), and ΔwalK_Spn mutants were all significantly attenuated for virulence to approximately the same extent (median survival time ≈ 73 h) in a murine pneumonia model compared to the walK_Spn⁺ parent and repaired strains (median survival time ≈ 52 h) (Fig. 7B). Thus, WalK_Spn containing an intact PAS domain is required for full virulence of serotype strain D39.

DISCUSSION

The data presented here show that the autokinase and phosphoryltransfer reactions of the WalRK_Spn TCS do not depend strongly on the presence of the PAS domain under standard in vitro reaction conditions (Tables 1 and 2; Fig. 2 and 3). In contrast, the PAS domain is required for optimal WalK_Spn phosphatase activity (Fig. 4, 5, and 6; Table 3). Prior to the present study, a WalK_Spn phosphatase activity had been inferred by an inability to detect WalR_Spn∼P in the presence of WalK_Spn bound to membrane vesicles (89); however, a phosphatase activity was not demonstrated directly, although bioinformatic analysis had predicted this activity for WalK_Bsu and WalK_Sau (3). The data from WalRK TCSs in several bacterial species suggest that phosphorylated WalR∼P is required for positive activation of their regulons (18, 23, 58, 59). Since the WalR_Spn∼P autophosphatase activity is extremely low (Table 3), the WalK_Spn phosphatase system may play an important role in resetting the system back to the unphosphorylated WalR “off” state.

Signaling through the PAS domain may predominate in modulating WalR phosphorylation state in Streptococcus species, because WalK_Spn lacks an extracytoplasmic domain and one of the transmembrane domains present in many other WalK homologues (47, 60, 66, 89, 93). In addition, the WalHI (YycHI) extracytoplasmic proteins that modulate WalK activity in B. subtilis and other species (84-86) are absent in Streptococcus species. The signals that impinge on the PAS domains of WalK_Spn and its homologues in other bacteria are unknown (14, 93), and it remains to be determined whether binding of small molecules or proteins to the PAS domain modulates WalK_Spn phosphatase activity in vivo. The mutant WalK_Spn proteins containing the internal deletion of the PAS domain or amino acid replacements in the DHp domain characterized here (Tables 1 and 3) are stable in pneumococcal cells (see Results), and these mutational changes in WalK_Spn attenuated pneumococcal virulence (Fig. 7 and below).

A substantial body of evidence supports a model in which the autokinase and phosphatase activities of EnvZ are balanced to regulate OmpR∼P amount (6, 36, 40, 69, 97). High osmolarity is thought to favor the EnvZ histidine autokinase activity that leads to phosphorylation of OmpR, whereas low osmolarity favors the EnvZ phosphatase activity that dephosphorylates OmpR∼P. In addition, a recently discovered modulator of EnvZ, called MzrA, leads to increased amounts of OmpR∼P, possibly by modulating the autokinase/phosphatase balance (26). However, EnvZ does not contain a cytoplasmic PAS domain, and the exact mechanisms of EnvZ signaling are still largely unknown. There are also some discrepancies between binding and kinetic data that remain to be resolved (45, 97). Modulation of the balance between autokinase and phosphatase activity has also been studied in detail for other histidine kinases that lack cytoplasmic PAS domains. For example, Mg²⁺ binding to an external sensing domain stimulates PhoQ phosphatase activity of serovar Typhimurium (11, 55).

There are a relatively limited number of precedents of cytoplasmic PAS domains regulating the phosphatase activity of histidine kinases. The phosphatase of the ResE histidine kinase is negatively regulated by anaerobiosis, and the ResE PAS domain may contribute to this process, although this has not yet been established experimentally (5, 56). A strong precedent is provided by the NtrB-NtrA TCS of E. coli. The CA catalytic domain of the NtrB histidine kinase inhibits the NtrA∼P phosphatase that resides in the DHp domain (39, 46). The DHp domain alone of NtrB (and EnvZ) is sufficient for phosphatase activity (39, 46, 99). Attempts to purify and test the DHp domain from WalK_Spn were not successful, because the domain was insoluble (see Table S4 in the supplemental material). Inhibition of NtrB phosphatase activity in the DHp domain is relieved by the binding of the PII regulatory protein to the CA catalytic domain in the presence of cofactors, such as AMP-PNP (39, 62-64). The resulting conformational change allows activation of the NtrB phosphatase activity. The PAS domain has been proposed to act as an “anvil” that stabilizes the DHp phosphatase activity (39, 63). walRK operons do not encode a homologue to the gene that encodes PII. However, walRK operons do encode a β-lactamase-fold protein, called WalJ (YycJ; VicX), that can influence WalRK function under some conditions (58). The functions of WalJ_Spn remain to be determined.

Another important precedent appeared in a recent report of the structure of the complex formed between a PAS-containing ThkA histidine kinase and its TrrA cognate response regulator from Thermotoga maritima (95). This structural model has several features relevant to the work reported here. The PAS domain of the ThkA does not dimerize in the complete structure, but rather forms contacts with the CA catalytic domain. This interaction bends the DHp domain toward the CA catalytic domain. In addition, there were two distinct interactions between the DHp domain of ThkA and the TrrA response regulator and an unanticipated third interaction between the PAS domain and TrrA (95). These multiple interactions likely underlie the complex effects that analogous amino acid replacements in the DHp and CA domains have on the autokinase and phosphatase activities of different histidine kinases, such as EnvZ (17, 36, 98), NtrB (39, 63), and WalK_Spn (see above). Similar to the findings presented here, an initial study of the autophosphatase, phosphoryltransferase, and phosphatase activities of the ThkA-TrrA TCS revealed that deletion of the PAS domain strongly reduced the ThkA phosphatase activity without significantly changing the autokinase or phosphoryltransferase activities (95). The interaction between the ThkA PAS domain and TrrA was invoked as a possible explanation for this large decrease in phosphatase activity. The detailed kinetic results in this report suggest a similar type of interaction for the WalK_Spn-WalR_Spn TCS and extend the dependence of the phosphatase activity on the PAS domain from a thermophilic to a mesophilic TCS.

The walK_Spn(H218A) mutation that eliminated autokinase activity in truncated purified WalK_Spn constructs (Table 1) caused reduced growth yields similar to the ΔwalK_Spn deletion mutant (see Results). In contrast, the walK_Spn(T222R) and walK_Spn(ΔPAS[104-198]) mutants did not display reduced growth yields (Results). Microarray analysis of the ΔwalK_Spn mutant compared to the walK_Spn⁺ parent grown under these conditions revealed that significant changes in relative transcript amounts were confined to genes in the WalRK_Spn regulon (unpublished result). Relative transcript amounts decreased in the ΔwalK_Spn mutant by 2.0- to 4.4-fold for different genes in the regulon. Therefore, regulation by the WalK_Spn histidine kinase was specific to its regulon under these condition.

All four walK_Spn mutants were significantly attenuated to about the same extent in a pneumonia model of infection compared to the walK_Spn⁺ parent and repaired strains (Fig. 7B). This result contrasts with a previous paper claiming that a walK_Spn::kan insertion mutant was avirulent in a similar D39 strain (42). A major difference between the present study and the previous one is that we did not passage mutants through mice before characterization because of the possibility of selecting for additional mutations. In addition, the walK_Spn(H218A) and walK_Spn(T222R) mutants contain missense mutations that did not affect WalK_Spn amounts (see Results). This result strongly argues against polarity effects on expression of downstream walJ_Spn as a cause for the reduced virulence. However, we cannot ascribe the reduced virulence of the walK_Spn(H218A), walK_Spn(T222R), and walK_Spn(ΔPAS[104-198]) mutants solely to reduced WalR_Spn∼P phosphatase activity (Table 3; Fig. 5). Purified WalK_Spn(H218A) or WalK_Spn(T222R) also lacked or had very low autokinase activity, respectively (Table 1), and even WalK_Spn(ΔPAS[104-198]) had a moderately increased K_m for ATP in the autokinase reaction, although its relative k_cat was unchanged (Table 1). Therefore, if the pneumococcal ATP pool decreases during infection, then autophosphorylation of WalK_Spn(ΔPAS[104-198]) could become kinetically limited. Nevertheless, taken together, these results indicate that the WalK_Spn histidine kinase through its regulation of the WalK_Spn regulon is required for normal growth in culture and that an intact WalK_Spn PAS domain is required for full virulence in this pneumonia model of infection.