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' a residue that is conserved in all homologous response regulator proteins (5). Two additional highly conserved residues, Asp-12 and Asp-13, bind a Mg2+ ion that is essential for phosphorylation (6, 7). CheY, like many other response regulators, has an associated autophosphatase activity; phospho-CheY has a half-life of =10 sec. Phosphatase activity is enhanced by an auxiliary regulatory protein, CheZ (8).The activity of response regulators is controlled by a family of histidine kinases that are autophosphorylated in the presence of ATP (1-3). The phosphotransfer mechanism involves the intermediate formation of a phosphohistidine residue in the kinase. In chemotaxis, the rate of phosphorylation of the histidine residue in the kinase, CheA, is stimulated by membrane chemoreceptor proteins (9, 10). The phosphoryl group is then rapidly transferred to CheY to control motility. Another chemotaxis response regulator, CheB, also accepts phosphoryl groups from CheA. -CheB provides a feedback adaptation mechanism. Phosphorylation of its N-terminal regulatory domain stimulates a C-terminal catalytic activity that modifies the chemoreceptors to attenuate CheA kinase activity (9, 11).Although in many cases a specific kinase has been implicated in the regulation of a given response regulator, considerable cross specificity has also been observed (12-14). The phenotypes of kinase mutants have indicated that response regulators can be phosphorylated in the absence of their cognate kinases (15-20). Here we show that CheY and CheB are enzymes that catalyze their own phosphorylation using low molecular weight phospho-donors. Thus, the enzymology of aspartate phosphorylation is an inherent property of the response regulators that can occur independently of any other protein. Proteins. Wild-type and mutant CheY proteins as well as CheZ and CheB were purified as described (23-25). Protein purity was estimated to be >95% on the basis of SDS/PAGE. MATERIALS AND METHODS Materials[3H]Methyl-labeled Tar receptor in Escherichia coli membranes was prepared as described (25 718The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRI, (NtrB), which both phosphorylates and dephosphorylates NRI. Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo. We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate. With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis. Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro. Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRn, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase ginAp2 promoter.The Ntr regulon of enteric bacteria is a collection of genes and operons that are regulated by the availability of ammonia and whose products facilitate survival under nitrogenlimiting growth conditions. The most important enzyme for the assimilation of ammonia under nitrogen-limiting conditions is glutamine synthetase (GS), the product of the glnA gene. Under nitrogen-excess conditions, a low intracellular concentration of this enzyme results from transcription initiated at a o70-dependent promoter known as gbnApl. Under nitrogen-limiting conditions, a much higher intracellular concentration of this enzyme results from transcription from a nitrogen-regulated promoter known asglnAp2. The activation of transcription from the glnAp2 promoter of enteric bacteria and other nitrogen-regulated promoters in intact cells and by purified components has been studied in some detail (reviewed in references 12, 13, 19, and 32). Transcription from theglnAp2 promoter requires RNA polymerase containing the alternative sigma factor a54 (8,9). This polymerase binds tightly to the glnAp2 promoter sequence, but it lacks the capacity to melt the DNA in the region surrounding the transcription start site (26,29). The formation of an open complex by or" RNA polymerase requires a transcriptional activator; for the glnAp2 promoter, this activator is the phosphorylated form of NR, (NtrC [21]). The efficient action of phosphorylated NR, (NR,-P) in bringing about the activation of transcription is facilitated by high-affinity binding sites on the template; these sites can be located far from the promoter and are functionally analogous to enhancer sequences (23, 28). The enhancers serve to increase the local concentration of NRI-P near the promoter (38). NRI-P, bound to its enhancer, interacts with &rI4 RNA polymerase at the promoter by means of a DNA loop (33) and, by so doing, somehow brings abou...

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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.

Robust growth in many bacteria is dependent upon proper regulation of the adaptive response to phosphate (P i ) limitation. This response enables cells to acquire P i with high affinity and utilize alternate phosphorous sources. The molecular mechanisms of P i signal transduction are not completely understood. PhoU, along with the high-affinity, P i -specific ATP-binding cassette transporter PstSCAB and the two-component proteins PhoR and PhoB, is absolutely required for P i signaling in Escherichia coli. Little is known about the role of PhoU and its function in regulation. We have demonstrated using bacterial two-hybrid analysis and confirmatory coelution experiments that PhoU interacts with PhoR through its PAS (Per-ARNT-Sim) domain and that it also interacts with PstB, the cytoplasmic component of the transporter. We have also shown that the soluble form of PhoU is a dimer that binds manganese and magnesium. Alteration of highly conserved residues in PhoU by site-directed mutagenesis shows that these sites play a role in binding metals. Analysis of these phoU mutants suggests that metal binding may be important for PhoU membrane interactions. Taken together, these results support the hypothesis that PhoU is involved in the formation of a signaling complex at the cytoplasmic membrane that responds to environmental P i levels.

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.

Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P i ). Under these conditions, the high-affinity PstSCAB 2 protein (i.e., with two PstB proteins) is the primary P i transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR twocomponent regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P tac promoter and the lacO ID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P phoB ::P tac and P pstS ::P tac strains to characterize phenotypes resulting from various ⌬phoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB 2 transporter, as well as its abundance within the cell. In addition, we used the P phoB ::P tac ⌬phoU strain as a platform to begin characterizing new phoU mutations in plasmids.

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 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.The ability to sense and respond to changing environmental conditions by genetic regulatory systems is an essential feature that enables bacteria to survive and adapt to numerous stresses. One way bacteria sense and respond to changing environments is through the use of two-component regulatory systems (9). In their simplest forms, two-component systems consist of histidine kinases and response regulators (21, 26). Histidine kinases transduce environmental cues into intracellular signals by interacting with and modifying response regulator proteins. Histidine kinases contain conserved catalytic (CA) and dimerization and histidine phosphorylation (DHp) domains (4). The DHp domain donates phosphate from a histidine residue to a universally conserved aspartate residue located within the response regulator. Phosphorylation of response regulators alters their activity, thereby allowing these proteins to function as phosphorylation-based biochemical switches (21,26). Most response regulators consist of multiple domains: an N-terminal receiver domain that contains the site of phosphorylation and a C-terminal output domain that often binds DNA and activates transcription (28).In Escherichia coli, the adaptive response to limiting phosphate is regulated by a two-component signal transduction system (27,30). PhoB is the response regulator, and PhoR is the histidine kinase. PhoR is a transmembrane protein that modulates the activity of PhoB by promoting specific phosphorylation and dephosphorylation of PhoB in response to the phosphate signal (14-16). PhoB binds to specific DNA sequences and interacts with the 70 subunit of RNA polymerase to control the transcription of more than 30 genes that comprise the Pho regulon (10, 24). This regulon includes operons and g...