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.
We know that in an envZ
null background, ompF
is expressed to a certain degree, because OmpR-P, which is formed by an acetyl phosphate-dependent mechanism, accumulates to significant levels in strains lacking EnvZ phosphatase activity (10
). Thus, an envZ
null strain with an ompF′-lacZ+
fusion is Lac+
and forms red colonies on lactose-MacConkey agar. Subsequent introduction of a plasmid carrying a K−
allele will decrease the accumulated OmpR-P and confer a Lac−
phenotype; therefore, the colony will appear white on this medium. This forms the basis for a screen for K−
mutations of envZ
Plasmid pEnvZ was first mutagenized by UV light and then transformed into an envZ null ompF′-lacZ+ recA strain background. The transformants were plated onto lactose-MacConkey agar, and Lac− (white) colonies were picked and purified. After confirmation of the linkage of the Lac− phenotype to the plasmid by retransforming the plasmid into the original strain background, the entire envZ gene was sequenced. Two novel mutations were identified, envZ343 (N343K) and envZ390 (F390L), which have changed conserved residues in the N and D/F boxes, respectively (Fig. and Table ).
TABLE 2 Mutations isolated in thisstudy 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.
The nature of the original K−
mutation dictates the type of suppressor expected. Information flow within EnvZ occurs by a strictly ordered pathway that involves different domains of the receptor in turn. Environmental cues sensed by the periplasmic domain are transduced by the transmembrane helices to the catalytic cytoplasmic domain (Fig. ). The defects caused by the original lesion should not be suppressed by mutations that affect steps upstream in this signaling pathway. To obtain a broader spectrum of mutations, we used two well-characterized K−
mutations for the suppressor analysis: envZ250
), which causes a P159S change at the junction between the periplasmic domain and the second transmembrane segment (TM2) of EnvZ, and envZ247
), which causes an A239T change in the conserved H box.
The two strains used to isolate intragenic suppressors were FR247 and FR250. Each carries an ompR101
null allele at the normal chromosomal locus, which is complemented in trans
by the ompR+
prophage λpSG10 integrated at λatt
. FR247 carries envZ247
linked to ompR101
, while FR250 carries envZ250
. Both strains harbor a Tn10
that is 85% linked to envZ
. These features were chosen to facilitate suppressor mapping: suppressors in envZ
must be linked to the Tn10
. In addition, both strains carry the ompC-lacZ+
transcriptional fusion. In the presence of either envZ250
is not transcribed, and the strains are phenotypically Lac−
. Any suppressor mutation that restores expression from the ompC
promoter will increase the production of β-galactosidase and allow the strain to grow on lactose. A fusion to the ompC
promoter, as opposed to ompF
, was chosen for two reasons. First, an envZ
null mutation allows residual expression of the ompF-lacZ+
owing to OmpR-P produced from acetyl phosphate (10
), and this residual expression is sufficient to support growth on lactose. The use of an ompC
fusion excludes envZ
null mutations from the selection. The second reason is that the phenotype of a known K+
, is OmpF−
. Such potentially interesting mutations would be excluded if the selection was performed in an ompF-lacZ+
fusion background. Twelve suppressors for envZ250
and 10 suppressors for envZ247
were isolated. Linkage mapping showed that 10 suppressors of envZ250
and 9 suppressors of envZ247
were linked to envZ
To determine the DNA sequence changes in the isolated suppressor strains, the entire envZ gene from each strain was amplified by PCR, and primers throughout the gene were used to sequence the products. All of these mutant envZ genes contain the original mutation plus a suppressor. All of the suppressor mutations are single-base-pair changes, except for one deletion, and most of them are transversions. As predicted, we obtained a different array of suppressors from each initial mutation. Suppressors of the envZ250 (P159S) mutation mapped within two domains (Table ): five suppressors affect the TM1 region, and five affect the catalytic domain. Among the latter, two missense changes and one deletion fall immediately upstream of the conserved autophosphorylation site, histidine-243, and two additional mutations fall near each other in a region slightly downstream of the H box, which we have termed the X region. The suppressors of envZ247 (A239T) affect the catalytic domain only (Table ). Two of these fall in the H box, six affect the X region (the Y287D mutation was isolated twice independently), and one affects a residue in the conserved G2 box. Q283P, which lies in the X region, was the only mutation isolated as a suppressor of both kinase deficiency alleles.
The X region of EnvZ has not generally been recognized as a conserved motif. However, scattered but significant homology within this region can be found in all two-component sensors (5
). The sequence alignment in this region of some representative sensors is shown in Fig. .
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. (more ...) 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).
As noted in the introduction, levels of porin gene transcription are determined by the amount of OmpR-P, which is set by the sum of the kinase and phosphatase activities of EnvZ. At high osmolarity, EnvZ is mainly K+ P−, resulting in high levels of OmpR-P, which in turn will activate ompC and repress ompF. At low osmolarity, however, EnvZ is shifted toward K− P+, resulting in low levels of OmpR-P, which can activate ompF only. The constitutive activation of ompC and repression of ompF in most of the double envZ mutants indicate high levels of OmpR-P even under low-osmolarity conditions. This suggests that these suppressor mutations shifted the balance of enzymatic activities from the original K− P+ state toward K+ P−. The balance was reset such that even at low osmolarity, the level of OmpR-P was still higher than that in the wild type at high osmolarity.
To determine if the increased kinase activity caused by the intragenic suppressors depends on the original K− P+ mutation (envZ250 or envZ247), the suppressors were isolated by in vitro DNA manipulation and recombined in single copy at the chromosomal location (see Materials and Methods). The kinase and phosphatase activities conferred by the resulting single mutant envZ alleles were then assessed in vivo by assaying the β-galactosidase activities in ompF′-lacZ+ and ompC′-lacZ+ fusion strains following growth at different osmolarities. As illustrated in Fig. , all of the isolated suppressor mutations tested activate ompC and repress ompF constitutively, suggesting that these suppressor mutations alone reset the balance of the enzymatic activity toward K+ P−: the restoration of kinase activity does not depend on the original K− P+ mutation. This is consistent with the locations of the suppressor mutations at or downstream from the original mutations in the intramolecular signal transduction pathway (Fig. ). All further analyses were performed with these single mutant (K+ P−) genes or their products.
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. β-Galactosidase (more ...) 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. A). 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. B). Indeed, EnvZ390 may have elevated phosphatase activity. These results are consistent with the observed K− P+ phenotype conferred by these mutant proteins in vivo.
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 [γ-33P]ATP in the absence (autokinase) or (more ...)
Note that the total 33P labeling in the EnvZ390 kinase reaction (Fig. B) is much lower than that in the EnvZ390 autokinase reaction (Fig. A). One possible explanation is that in the kinase reaction, the 33P label on EnvZ390 is efficiently transferred to OmpR but subsequently undergoes rapid hydrolysis due to an elevated phosphatase activity of EnvZ390. Such a hypothesis predicts an increased yield of 33Pi in the EnvZ390 kinase reaction compared to the wild-type EnvZ kinase reaction. To test this, we examined the levels of ATP and Pi in these kinase reactions by thin-layer chromotography. Our results showed much higher concentrations of 33Pi in the wild-type EnvZ kinase reaction than in the EnvZ390 reaction (data not shown). Thus, we favor an alternative explanation: OmpR, the substrate for the kinase reaction, may inhibit the autophosphorylation of EnvZ390 by ATP (see Discussion).
(ii) The TM1 (envZ976), X-region (envZ966), and G2-box (envZ962) mutations.
As shown in Fig. , 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. . Each of the mutant proteins retained the ability to autophosphorylate, although they had lower autokinase activity than the wild type (Fig. A). 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. B). 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. C). 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.
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 (more ...)
Interestingly, similar to the case for EnvZ390, the total 33P labeling in the EnvZ962 kinase reaction (Fig. B) is much lower than that in the EnvZ962 autokinase reaction (Fig. A). Again, no increased ATP cleavage to yield 33Pi by EnvZ962 during the kinase reaction can be detected by using the thin-layer chromatography assay (data not shown). Thus, we suspect that OmpR may inhibit the autophosphorylation of EnvZ962 by ATP as well (see Discussion).
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
) prior to the cross-linking experiment.
The mutant EnvZ115 proteins were incubated with [α-33
P]ATP, and UV light was used to cross-link radioactive label onto the protein (17
). As shown in Fig. , under our assay conditions with ATP concentrations of approximately 0.25 μM, cross-linking was observed with all of the mutant EnvZ115 proteins tested, including those containing lesions in the X region (envZ966
) and the N (envZ343
), D/F (envZ390
), and G2 (envZ962
) boxes. Cross-linking to the N-box mutant (envZ343
) is noticeably less efficient than that to the others, and we suggest that this mutation decreases affinity for ATP. However, the other mutants behave in a manner indistinguishable from that of the wild type.
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 [α-33P]ATP (more ...)
The use of truncated EnvZ115 mutant proteins complicates biochemical analysis because the truncation itself may alter activity. We have performed the UV-cross-linking assay with cell membranes enriched for the full-length mutant EnvZ proteins. The results showed that all of the mutant EnvZ proteins can bind ATP. However, the strength of binding varied from assay to assay and was less reproducible.
The X-region mutations alter the conformation of EnvZ.
Figure 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.