INTRODUCTION

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To survive, bacteria must adapt to their rapidly changing environment, and two-component regulatory systems are key to this process. More than 250 two-component pairs have been discovered, and these systems sense and respond to a variety of environmental parameters and insults (5). A two-component system is generally composed of a sensor histidine kinase and a response regulator. The sensor kinase is often localized to the cytoplasmic membrane, where it senses external stimuli and transduces this information to the response regulator. The response regulator, which is normally a transcriptional regulatory protein that controls expression of a set of related genes (9, 18), mediates the proper cellular response to the stimuli.

A key feature of the information flow from the sensor to the regulator is protein phosphorylation and dephosphorylation (references 9 and 18 and references therein). The sensor can be autophosphorylated by ATP at a conserved histidine residue and subsequently transfer the phosphoryl group onto a conserved aspartic acid residue in the regulator. In many cases, the sensor kinase can dephosphorylate the phosphorylated response regulator. Thus, unlike their counterparts in eukaryotic signal transduction systems, three enzymatic activities are often associated with bacterial two-component sensor kinases: the autokinase, response regulator kinase, and response regulator phosphate phosphatase. How the catalytic domain of one protein catalyzes these three different reactions is a topic of considerable interest.

In this study, we approached this question by mutational analysis of EnvZ. EnvZ is a well-characterized sensor kinase that is involved in osmoregulation in Escherichia coli (6, 7). It shares basic architectural features with many other sensor kinases, having two transmembrane segments that divide the protein into an N-terminal sensory domain and a C-terminal catalytic domain. The C-terminal domain contains all of the conserved motifs common to the sensor family: H, N, D/F, G1, and G2 boxes (Fig. 1) (18, 25). Thus, mechanisms underlying the functions of EnvZ have implications for many other transmembrane sensors as well.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Schematic presentation of the structure of EnvZ. Conserved regions in the catalytic cytoplasmic domain are indicated with lettered boxes.

EnvZ forms a two-component pair with its cognate response regulator, OmpR. Together, EnvZ and OmpR enable cells to sense the external osmolarity and respond to it by regulating the transcription of two porin genes: ompF and ompC (4, 16, 19). Genetic evidence suggests that EnvZ can exist in two active but opposed signaling states: the OmpR kinase-dominant (K⁺ P) state and the OmpR-P phosphatase-dominant (K P⁺) state (19, 22, 24). High levels of OmpR-P activate ompC but repress ompF transcription; low levels of OmpR-P activate ompF transcription only. Neither ompC nor ompF is transcribed in the absence of OmpR-P. Thus, the level of ompF and ompC transcription reflects the level of OmpR-P, which is set by the sum of EnvZ kinase and phosphatase activities in vivo. By using lacZ fusions to the porin gene promoters, this can be easily monitored.

To investigate the structural components involved in the OmpR kinase or OmpR-P phosphatase activity of EnvZ, mutations that shift the balance of these enzymatic activities toward either the K P⁺ or K⁺ P state were identified and characterized both in vivo and in vitro. We found that most of the mutations altered a previously defined structural motif, demonstrating their critical importance. In addition, our studies revealed a novel motif that we have termed the X region, which is shared by other two-component sensors.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and phage. The E. coli strains and plasmids used in this study are described in Table 1. Phage P1_vir was used for transduction. Standard microbiological techniques were used for strain construction and bacterial growth (23). Cells were grown at 37 or 30°C with shaking in appropriate media.

Media and reagents. Most media were prepared as described previously (23). ortho-Nitrophenyl--D-galactoside (ONPG) for -galactosidase assay was purchased from Sigma. Restriction enzymes, -agarase, and T4 DNA ligase were from New England BioLabs, Inc. Taq polymerase and reagents used for PCR amplification, T4 DNA polymerase, and Sequenase were from United States Biochemical Corp. [-³³P]ATP (1,000 to 3,000 Ci/mmol; 10 mCi/ml) and [-³³P]ATP (2,000 Ci/mmol; 10 mCi/ml) were from NEN Life Science Products. ATP and ADP were from Boehringer Mannheim. The oligonucleotide primers used for PCR and DNA sequencing were provided by the Princeton University Department of Molecular Biology Synthesis/Sequencing Facility. NuSieve low-melting-point agarose was purchased from FMC BioProducts. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from National Diagnostics. The protein assay reagent was from Bio-Rad.

-Galactosidase assay. -Galactosidase activities were determined by using a microtiter plate assay that has been previously described (24). Cells were grown overnight in appropriate medium at 37°C, subcultured (1:40) into 2 ml of the same medium, and then grown to mid-log phase at 37°C. Activities are expressed as (units/A₆₀₀) × 10³, where 1 U = 1 µmol of ortho-nitrophenol formed per min. A minimum of four independent assays was performed for each strain, and the results were averaged for display as bar graphs.

PCR amplification and DNA sequence analysis. PCR amplification and DNA sequence analyses were performed essentially as previously described (22). DNA was amplified from either the bacterial cells or plasmids. For sequencing analysis, the template DNA was either from PCR amplification or from restriction digestion. The template was purified by gel electrophoresis in 1% NuSieve low-melting-point agarose, and the agarose was digested with -agarase. Sequence analysis was then performed as indicated in the supplier's instructions.

Screening for K P⁺ mutations. pEnvZ was first subjected to UV irradiation and then transformed into WH67. Transformants were plated onto lactose-MacConkey agar. Plasmids were isolated from the Lac white transformants and retransformed into WH67. The plasmids that maintain the Lac phenotype were sequenced by using primers throughout the entire length of envZ.

Isolation of intragenic suppressors for the K P⁺ Mutants. Several independent cultures of each strain (FR247 and FR250) were grown overnight in Luria-Bertani medium. They were washed in minimal salts, and an aliquot of each culture was plated on lactose minimal agar. Multiple isolated colonies appeared after 2 to 3 days of growth, of which two were picked from each plate and purified by restreaking on lactose minimal agar. Each isolate was characterized for its expression of the ompC'-lacZ⁺ fusion by streaking on lactose-MacConkey and lactose-tetrazolium agars to examine different ranges of -galactosidase expression. If both isolates from any given plate were the same color on lactose-tetrazolium, they were considered siblings and only one was kept for further analysis.

Mapping of the suppressor mutations. P1 transduction was performed to map the suppressor mutations. Each isolate was used as the donor. FR1050 and FR1070, which are identical to FR247 and FR250, respectively, except that they do not carry a Tn10 insertion, were used as recipients. In such a transduction, both the donor and recipient carry the same kinase deficiency mutation. The only difference among transductants is the additional presence or absence of a given suppressor mutation. If the suppressor is not linked to the Tn10, which is about 85% linked to envZ, then all transductants should be Lac, but if the suppressor lies in envZ, Lac⁺ progeny will also result. After confirmation of their linkage to Tn10, the suppressors were moved into a clean ompC'-lacZ⁺ and ompF'-lacZ⁺ background by P1 transduction with WH30 and WH40 as recipients. The presence of suppressors in the ompC'-lacZ⁺ transductants was confirmed by the Lac⁺ phenotype. Marker rescue was used for the ompF'-lacZ⁺ transductants.

Separation of the suppressor mutation from the original kinase deficiency mutation. The fragment of DNA that contains only the suppressor mutation was first subcloned onto pFR32, which has a temperature-sensitive origin of replication, and then recombined onto the chromosome as described by Hamilton et al. (8). WH56 was used as the recipient strain, and lactose-tetrozolium agar was used to facilitate the screening of chromosomal recombinants. The presence of each mutation on the chromosomal location was confirmed by PCR amplification and sequence analysis. Each envZ allele was then moved into clean strain backgrounds by P1 transduction with WH10 and WH20 as recipients.

In vitro phosphorylation and dephosphorylation analysis. Membranes that were enriched for EnvZ through the multicopy plasmid pEnvZ were used for these assays. Procedures for protein phosphorylation and dephosphorylation were the same as previously described (10).

UV-cross-linking experiments. Mutant envZ alleles were first subcloned onto pSG115 through in vitro DNA manipulation. Mutant EnvZ115 proteins were then purified from transformants carrying pSG115 with the mutant alleles and solubilized by using a previously described procedure (13, 14). Concentrations of purified EnvZ115 were quantified by use of the Bio-Rad protein assay reagent. The UV-cross-linking procedure was similar to that described by Ninfa et al. (17). The reaction mix contains 6 µl of EnvZ115 (0.3 mg/ml) in phosphate-buffered saline and 0.2% deoxycholate, 0.5 µl of [-³³P]ATP, and 4 µl of assay buffer (0.1 M Tris-HCl buffer [pH 8.0], 50 mM KCl, 5 mM CaCl₂, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol). The cross-linking of ATP onto EnvZ115 was achieved by exposing the reaction mix to UV light for 10 min on ice. SDS sample buffer was then added to the reaction mix. After being boiled for 5 min, the sample was subjected to SDS-PAGE, and the amount of [-³³P]ATP cross-linked onto EnvZ115 was visualized by autoradiography.

	RESULTS

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In order to identify regions in EnvZ that are important for both the kinase and phosphatase activities, we have sought mutations that specifically alter only one of these activities. In particular, we have searched for K P⁺ and K⁺ P mutations.

Identification of K P⁺ mutations. To obtain information on the structural requirements for kinase activity, we performed a genetic screen to isolate mutations that render EnvZ kinase deficient but still maintain the phosphatase activity.

Characterization of the K P⁺ mutations. The two K P⁺ mutations isolated as described above were subcloned onto a plasmid with a temperature-sensitive origin of replication, and subsequently recombined onto the chromosome at the normal location (see Materials and Methods). Neither of the mutations can activate ompC or ompF transcription as indicated by the Lac phenotype in ompC'-lacZ⁺ or ompF'-lacZ⁺ fusion strains. The level of ompF transcription in either mutant envZ strain was similar to that in an envZ-ompR deletion mutant, and it was much lower than that in an envZ null (envZ::Kan) mutant (data not shown). We conclude that these two mutant EnvZ proteins are K P⁺, since each prevents the accumulation of OmpR-P derived from acetyl phosphate.

Identification of K⁺ P mutations. As described above, the phosphatase activity of the K P⁺ mutant EnvZ proteins diminishes OmpR-P in vivo, causing a porin-negative phenotype. The Lac phenotype exhibited by ompC'-lacZ⁺ fusion strains carrying these mutations provided a means to isolate the other type of mutation that we are searching for, K⁺ P mutations, through the analysis of intragenic suppressors that confer a Lac⁺ phenotype.

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Sequence alignment of the X regions from some E. coli sensor kinases. Sequences of the sensor kinases were obtained from Swissbank. A multiple sequence alignment program, PIMA 1.4 (Pattern-Induced [local] Multiple Alignment) was used. The previously identified H, N, D/F, and G boxes were identified as conserved motifs through this program. Highly conserved residues are shaded. Mutational changes within the X region are shown in boldface.

Characterization of the K⁺ P mutations. All of the suppressors restore ompC expression in the original K P⁺ mutant background, suggesting that they all restore kinase activity. To quantitate the strength of suppression, we measured the levels of porin transcription under both high- and low-osmolarity conditions by using ompF'-lacZ⁺ and ompC'-lacZ⁺ fusion strains. Our results showed that despite the presence of the original K P⁺ mutation, most of the suppressors activated ompC and repressed ompF at both low and high osmolarity (data not shown).

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Osmoregulation of porin gene expression in strains carrying the isolated envZ intragenic suppressor mutations. Transcription of ompF and ompC was monitored by using ompF'-lacZ+ and ompC'-lacZ+ fusions. -Galactosidase assays were performed with log-phase cells grown under either high (A medium plus 15% sucrose)- or low (A medium)-osmolarity conditions. Cells of wild-type (w.t.) and suppressor strains were tested. Note the difference in scale for the -galactosidase activities from the ompF and ompC fusion strains. Error bars indicate standard deviations.

Membrane localization and stability of the mutant EnvZ proteins. The K⁺ P mutations in or near TM1 introduce either an arginine or a proline. Accordingly, they could affect the localization of mutant EnvZ proteins to the membrane. To test this, fractionation experiments were performed with cells carrying the mutant genes on the plasmid pEnvZ. We found that although the TM1 mutant EnvZ proteins fractionated with the cell membranes, their levels were generally about one-fourth that of the wild type (data not shown). We suspect that most of the TM1 mutations reduce the efficiency of membrane targeting and that mislocalized molecules are degraded. All of the K⁺ P mutations in the H region caused similar reductions in the yield of mutant EnvZ protein. We suspect that these mutant proteins are unstable. Low yields and mutant protein instability complicate meaningful interpretation of the in vitro activity assays we employ. Accordingly, we limited biochemical analysis to those mutant proteins that are membrane localized at levels equivalent to the wild type (see below). This subset includes those with the X-region mutations (envZ964 and envZ966), the two K P⁺ mutations in the N and D/F boxes (envZ343 and envZ390), and the K⁺ P mutations in TM1 (envZ976) and the G2 box (envZ962).

Enzymatic activities of the mutant EnvZ proteins. To confirm and extend the predictions based on genetic analysis with the lacZ fusion strains, autokinase, OmpR kinase, and OmpR-P phosphatase assays were performed with cell membranes that were enriched for the mutant EnvZ proteins. This was done by using strains carrying the mutant envZ genes on the multicopy plasmid pEnvZ (see Materials and Methods).

(i) The N-box (envZ343) and D/F-box (envZ390) mutations. The envZ343 and envZ390 mutations confer the K P⁺ phenotype when assayed in vivo (see previous section). Under our assay conditions, the EnvZ390 mutant protein exhibited reduced autokinase activity and barely detectable kinase activity (Fig. 4A). The EnvZ343 mutant protein could not be autophosphorylated by ATP (data not shown). Thus, neither protein could phosphorylate OmpR to any significant level. However, both proteins exhibited wild-type levels of OmpR-P phosphatase activity (Fig. 4B). Indeed, EnvZ390 may have elevated phosphatase activity. These results are consistent with the observed K P⁺ phenotype conferred by these mutant proteins in vivo.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   Enzymatic assays of the K P+ mutants in vitro. (A) The autokinase and OmpR kinase activities were measured by incubating cell membranes that were enriched for EnvZ with [-33P]ATP in the absence (autokinase) or presence (kinase) of purified MBP-OmpR. Aliquots were removed at the indicated time points. Proteins were separated by SDS-PAGE and subjected to autoradiography. The EnvZ proteins enriched in cell membranes were either wild type (w.t.) or EnvZ390 (F390L). (B) The OmpR-P phosphatase activities were measured by using [33P]MBP-OmpR as the substrate (see Materials and Methods). Membranes isolated from strains that were enriched for wild-type, EnvZ390, or EnvZ343 were used for the assay. Proteins were displayed as described above. As a control, the phosphatase activity of membranes from the vector control strain (envZ null) was also measured.

(ii) The TM1 (envZ976), X-region (envZ966), and G2-box (envZ962) mutations. As shown in Fig. 3, the envZ976, envZ966, and envZ962 mutations confer a K⁺ P phenotype. These mutant proteins were also subjected to biochemical analysis, and the results are presented in Fig. 5. Each of the mutant proteins retained the ability to autophosphorylate, although they had lower autokinase activity than the wild type (Fig. 5A). The observed OmpR kinase activities of these mutant proteins were significantly lower than that of the wild type, which may be due to their lower autokinase activities. Nonetheless, each of them could phosphorylate OmpR (Fig. 5B). In contrast, none of these mutant proteins exhibited OmpR-P phosphatase activity under conditions in which wild-type EnvZ could dephosphorylate OmpR-P completely in about 10 min. (Fig. 5C). Results similar to those for EnvZ966 were also obtained with proteins altered by two other X-region mutations, envZ964 and envZ965 (data not shown). These results are consistent with the observed K⁺ P phenotype conferred by these mutant proteins in vivo. Although these mutations affected the autokinase and OmpR kinase activities to various degrees, they all severely decreased the phosphatase activity. This will shift the balance between the kinase and phosphatase reactions such that the formation of OmpR-P is strongly favored.

View larger version (69K): [in this window] [in a new window]   FIG. 5.   Enzymatic assays of the K+ P mutations in vitro. Cell membranes that were enriched for wild-type or mutant EnvZ proteins were used for the in vitro phosphorylation and dephosphorylation assays. Mutant EnvZ proteins tested include suppressors near TM1 (envZ976), in the X region (envZ966), and in the G2 box (envZ962). (A) Autokinase assay. Cell membranes were incubated with [-33P]ATP in assay buffer, and the reaction was stopped at different time points. Proteins were separated by SDS-PAGE and subjected to autoradiography. (B) OmpR kinase assay. The procedure was the same as for panel A except that purified MBP-OmpR was added to the reaction mixture. (C) OmpR-P phosphatase assay. MBP-OmpR-33P was used as the substrate for the assay. Reactions were stopped at different time points after incubation of the substrate, cell membranes, and 1 mM cold ATP in the assay buffer. The remaining MBP-OmpR-P was visualized through autoradiography after separation by SDS-PAGE. Membranes from an envZ null strain were also tested as a control.

ATP binding by the mutant EnvZ proteins. The conserved N, D/F, and G boxes of two-component sensor kinases have been implicated in nucleotide binding because of sequence homologies to eukaryotic kinases (25). ATP cross-linking was performed to determine if the mutations that alter these boxes affect ATP binding. The mutant proteins to be tested were purified as a truncated form, EnvZ115, in which the amino terminus of the molecule, including TM1, has been removed. Following purification, the aggregated EnvZ115 proteins are solubilized and renatured (12-14) prior to the cross-linking experiment.

View larger version (24K): [in this window] [in a new window]   FIG. 6.   UV-cross-linking assay. Various mutant alleles were first subcloned onto pSG115, a plasmid that overproduces a truncated version of EnvZ, EnvZ115. After solubilization with deoxycholate, mutant proteins were incubated with [-33P]ATP on ice in the presence (+) or absence () of UV light for 10 min. The amount of ATP cross-linked onto the protein was visualized through autoradiography after SDS-PAGE. The autoradiograph is shown on the right. A similar amount of each mutant EnvZ115 protein was used in the assay, as shown by the Coomassie blue-stained SDS-polyacrylamide gel on the left. envZ alleles tested included the wild type (w.t.), envZ962 (G2 box), envZ966 (X region), envZ390 (D/F box), and envZ343 (N box).

The X-region mutations alter the conformation of EnvZ. Figure 6 shows that envZ966 slows the migration of EnvZ in SDS-polyacrylamide gels. Similar gel migration patterns were also observed with other X-region mutant proteins. We have also observed that the X-region mutant EnvZ proteins have proteolytic patterns different from those of the wild type after limited trypsin proteolysis (data not shown). Thus, we conclude that the X-region mutations alter the conformation of EnvZ.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We sought to identify the structural components of EnvZ that are involved in either the OmpR kinase or the OmpR-P phosphatase activity by mutational analysis. Two striking generalities emerge from this analysis. First, although the mutations identified decreased one of these activities more than the other, most affected both simultaneously. This supports and strengthens our previous proposal that these active sites overlap substantially (10). Second, all of the mutations lie in the conserved structural motifs previously identified by sequence homologies. Even the newly discovered X region is weakly conserved among all the sensors (5). Clearly these conserved structural motifs are critically important for function.

The structural motifs highlighted by these envZ mutations will be discussed in turn.

The newly identified X region. The majority of the K⁺ P mutations that alter the cytoplasmic domain of EnvZ fall within a region approximately 40 residues downstream of histidine-243. We have termed this the X region, and as noted above, this region is weakly conserved among all sensors. A representation of the alignment in this region is illustrated in Fig. 2. One codon in particular, that for tyrosine-287, is the site of mutations causing three different amino acid substitutions, one of which (Y287D) was independently isolated twice. Interestingly, neither this position nor other positions altered by X mutations in this study are highly conserved. Tyrosine-287 is located at the end of a fairly conserved sequence that could form an amphipathic -helix, and it is followed by a conserved positive charge (R289). A mutation at this conserved site (R289C) confers a weak K⁺ P phenotype that decreases but does not completely abolish the phosphatase activity when assayed in vivo and in vitro (data not shown).

The vicinity of the phosphorylated histidine. A number of suppressors of the K P⁺ mutations envZ247 and envZ250 fall into the conserved H region, which contains the autophosphorylation site, histidine-243. After they were separated from the original K P⁺ mutations, these H-region mutations conferred an OmpC⁺ OmpF phenotype, suggesting that they have shifted the balance of the kinase and phosphatase reactions toward the kinase reaction (i.e., K⁺ P), resulting in increased OmpR-P levels in vivo.

The other conserved motifs in the cytoplasmic domain. In addition to the H region, previous sequence alignment revealed three more conserved motifs among the bacterial two-component sensor-kinases: the N, D/F, and G boxes. It is thought that these elements form a nucleotide-binding surface within the active site (18, 25). We obtained mutations in all three boxes.

OmpC⁺ OmpF

The first transmembrane segment. Five of the intragenic suppressors of the periplasmic mutant protein EnvZ250 (P159S), but none of the suppressors of the cytoplasmic mutant protein EnvZ247 (A239T), fall into or near the first transmembrane segment (TM1) of EnvZ (Table 2). Based on hydrophobicity, this segment is predicted to include residues from arginine-14 to serine-42 (27). All of these TM1 mutant proteins are localized to the inner membranes as shown by membrane fractionation experiments. Thus, it does not appear that these mutations work by causing the mislocalization of the mutant EnvZ proteins. These suppressors cause constitutive activation of ompC in the presence or absence of envZ250, and when assayed in vitro, these mutant EnvZ proteins were phosphatase defective. Interestingly, they were still capable of sensing and responding to osmolarity as indicated by the stronger repression of ompF at high osmolarity (Fig. 3). Apparently these functions are not critically dependent on TM1 (but see reference 15).

The model. The external stimulus (15) modulates the relative amount of time that the external domain spends in either of two distinct conformations. The two conformations affect the transmembrane signaling differently. A specific face on TM1 is critical for this signaling process. In response to the transmembrane signaling, the region surrounding the critical histidine-243 is positioned into one of two different states by movements in which the X region is critically involved. In one state this histidine is properly aligned with ATP, which is bound at a surface composed of the N, D/F, and G boxes, for autophosphorylation; in the other histidine-243 is inaccessible to this bound ATP. EnvZ-P always functions as an OmpR kinase (K⁺ P), and EnvZ is a phosphatase for OmpR-P (K P⁺). The sum of these opposing enzymatic activities determines the amount of intracellular OmpR-P, and this in turn determines the relative levels of ompF and ompC transcription (22). We believe that this theme, a balance between two extreme states by regulation of the accessibility of the critical histidine residue to ATP, explains the function of EnvZ and probably applies to homologous sensors as well.