Genetic evidence that the alpha5 helix of the receiver domain of PhoB is involved in interdomain interactions.

Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein. Generally, response regulators are made up of a receiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain. The mechanisms by which receiver domains control the activities of their respective output domains are unknown. To address this question for the PhoB protein from Escherichia coli, we have employed two separate genetic approaches, deletion analysis and domain swapping. In-frame deletions were generated within the phoB gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the phoA gene. However, another deletion mutant that contained the C-terminal alpha-helix of the receiver domain (alpha5) in addition to the entire output domain was unable to activate transcription of phoA. This result suggests that the alpha5 helix of the receiver domain interacts with and inhibits the output domain. We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N-terminal approximately 85% of CheY's receiver domain to the beta5-alpha5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the alpha5 helix of its unphosphorylated receiver domain and its output domain.

The unphosphorylated receiver domain of PhoB silences the activity of its output domain.

PhoB is the response regulator of the Pho regulon. It is composed of two distinct domains, an N-terminal receiver domain and a C-terminal output domain that binds DNA and interacts with sigma(70) to activate transcription of the Pho regulon. Phosphorylation of the receiver domain is required for activation of the protein. The mechanism of activation by phosphorylation has not yet been determined. To better understand the function of the receiver domain in controlling the activity of the output domain, a direct comparison was made between unphosphorylated PhoB and its solitary DNA-binding domain (PhoB(DBD)) for DNA binding and transcriptional activation. Using fluorescence anisotropy, it was found that PhoB(DBD) bound to the pho box with an affinity seven times greater than that of unphosphorylated PhoB. It was also found that PhoB(DBD) was better able to activate transcription than the full-length, unmodified protein. We conclude that the unphosphorylated receiver domain of PhoB silences the activity of its output domain. These results suggest that upon phosphorylation of the receiver domain of PhoB, the inhibition placed upon the output domain is relieved by a conformational change that alters interactions between the unphosphorylated receiver domain and the output domain.

Analysis of the conserved acidic residues in the regulatory domain of PhoB.

The PhoB protein from Escherichia coli is a member of the two-component signal transduction pathway that controls an adaptive response to limiting phosphate. Activation involves its phosphorylation on a conserved aspartate. Site-directed mutations were introduced at conserved acidic residues. The E9D, D10E, D10N, E11A, E11D and E11Q mutants were each able to induce alkaline phosphatase under low phosphate growth conditions whereas the E9A, D10A, D53A, D53E and D53N could not. The E9Q mutant was constitutively active. Phosphorylation assays showed that only the E9D, E11A, E11Q and E11D mutants were phosphorylated by acetyl phosphate. Most mutants also displayed defects in magnesium binding.

The activation of PhoB by acetylphosphate.

PhoB is a response-regulator protein from Escherichia coli that controls an adaptive response to limiting phosphate. It is activated by autophosphorylation of a conserved aspartate residue within its regulatory domain. Its primary phospho-donor is its cognate histidine kinase PhoR; however, it also becomes phosphorylated when incubated with acetylphosphate. To further characterize its activation, PhoB was considered to be an acetylphosphatase whose enzymatic mechanism involves a phospho-enzyme intermediate. The kinetic constants for autophosphorylation were determined using 32P-and fluorescence-based assays and indicated that PhoB has a K(m) for acetylphosphate of between 7 and 8 mM. These constants are not consistent with an in vivo role for acetylphosphate in the normal control of the Pho regulon. In addition, when PhoB was phosphorylated by acetylphosphate it eluted from a high-performance liquid chromatography (HPLC) size-exclusion column in two peaks. The larger form of PhoB eluted from the column in a similar manner to a chemically cross-linked dimer of PhoB. The smaller form of PhoB is a monomer. Phosphorylated PhoB bound pho-box DNA approximately 10 times tighter than PhoB. These observations show that PhoB forms a dimer when phosphorylated and suggest that the characteristics of activated PhoB result from its dimerization.

Acetyl phosphate and the activation of two-component response regulators.

Several bacterial response regulator proteins (CheY, NRI, PhoB, and OmpR) become phosphorylated in vitro when incubated with acetyl phosphate. In the presence of high levels of acetyl phosphate and Mg2+, CheY reached steady state phosphorylation in less than 30 s; NRI and PhoB reached steady state more slowly (t1/2 to steady state of 1.5 and > 15 min, respectively). A simple method was developed to measure acetyl phosphate levels in Escherichia coli grown in defined media. Levels of acetyl phosphate were elevated in cells grown in pyruvate, glucose, and glucuronic acid and were low in cells grown in fructose, glycerol, and fumarate. The effects of varying the intracellular amounts of acetyl phosphate on chemotaxis and the osmo-response were also investigated. Acetyl phosphate was not required but did influence each of these responses. These results suggest that acetyl phosphate may influence either the sensitivity or the magnitude of an adaptive response.

Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors.

Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that receive phosphoryl groups from the histidine kinase, CheA. Here we show that the response regulators catalyze their own phosphorylation in that both CheY and CheB can be phosphorylated in the complete absence of any auxiliary protein. Both CheY and CheB use the N-phosphoryl group in phosphoramidate (NH2PO3(2-)) as a phospho-donor. This enzymatic activity probably reflects the general ability of response regulators to accept phosphoryl groups from phosphohistidines in their associated kinases. It provides a general method for the study of activated response regulators in the absence of kinase proteins. CheY can also use intermediary metabolites such as acetyl phosphate and carbamoyl phosphate as phospho-donors. These reactions may provide a mechanism to modulate cell behavior in response to altered metabolic states.

Effects of farnesylcysteine analogs on protein carboxyl methylation and signal transduction.

Several proteins associated with signal transduction in eukaryotes are carboxyl methylated at COOH-terminal S-farnesylcysteine residues. These include members of the Ras superfamily and gamma-subunits of heterotrimeric G-proteins. The enzymes that catalyze the carboxyl methylation reaction also methylate small molecules such as N-acetyl-S-trans, trans-farnesyl-L-cysteine (AFC). AFC inhibits carboxyl methylation of p21ras and related proteins both in vitro and in vivo. Saturating concentrations of AFC cause a greater than 80% inhibition of chemotactic responses of mouse peritoneal macrophages. Our results suggest that carboxyl methylation may play a role in the regulation of receptor-mediated signal transduction processes in eukaryotic cells.

Myxococcus xanthus protein C is a major spore surface protein.

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.

Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity.

Myxococcus xanthus exhibits multicellular interactions during vegetative growth and fruiting body formation. Gliding motility is needed for these interactions. The frizzy (frz) genes are required to control directed motility. FrzE is homologous to both CheA and CheY from Salmonella typhimurium. We used polyclonal antiserum raised against a fusion protein to detect FrzE in M. xanthus extracts by Western immunoblot analysis. FrzE was clearly present during vegetative growth and at much lower levels during development. A recombinant FrzE protein was overproduced in Escherichia coli, purified from inclusion bodies, and renatured. FrzE was autophosphorylated when it was incubated in the presence of [gamma-32P]ATP and MnCl2. Chemical analyses of the phosphorylated FrzE protein indicated that it contained an acylphosphate; probably phosphoaspartate. FrzE was phosphorylated in an intramolecular reaction. Based on these observations, we propose a model of the mechanism of FrzE phosphorylation in which autophosphorylation initially occurs at a conserved histidine residue within the "CheA" domain and then, via an intramolecular transphosphorylation, is transferred to a conserved aspartate residue within the "CheY" domain.

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.

FrzE of Myxococcus xanthus is homologous to both CheA and CheY of Salmonella typhimurium.

Myxococcus xanthus exhibits multicellular development. The "frizzy" (frz) mutants are unable to complete the developmental pathway. Instead of forming fruiting bodies, these mutants form tangled filaments of cells. We have previously shown that four of the frz gene products are homologous to enteric chemotaxis proteins and have proposed that the frz genes constitute a signal-transduction pathway that controls the frequency at which cells reverse their gliding direction. We show here that frzE encodes a protein with a calculated molecular mass of 83 kDa. FrzE is homologous to both CheA and CheY of Salmonella typhimurium, which are members of a family of "two-component response regulators." It is thought that the modulator components autophosphorylate and transfer a phosphate group to their cognate effector components. FrzE contains an unusual (alanine plus proline)-rich region that might constitute a flexible hinge facilitating phosphate transfer between functional domains. We suggest that FrzE is a second messenger that relays information between the signaling protein FrzCD and the gliding motor.