Mitsuko Amemura

Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins

PhoB protein is the transcriptional activator for genes in the phosphate regulon of Escherichia coli, such as phoA and pstS, that are induced by phosphate deprivation. PhoR protein activates PhoB when phosphate is limiting and inactivates it when phosphate is in excess. We constructed a plasmid with a mutant phoR gene (phoR1084), which encoded a PhoR protein (PhoR1084) lacking the amino-terminal hydrophobic region of the intact protein. The cells carrying the plasmid overproduced PhoR1084, which was recovered in the soluble fraction of the cell lysate. We purified the Phor1084 protein and showed that it was autophosphorylated in the presence of ATP, and the phosphate group on the protein was rapidly transferred to PhoB. The phosphorylation of PhoB protein occurred concurrently with the acquisition of the ability to activate transcription from the pstS promoter. On the basis of these findings, we propose that phosphorylated PhoB protein activates transcription from the promoters of the phosphate regulon, and that the role of PhoR is to catalyze the formation and breakdown of phosphorylated PhoB in response to phosphate concentrations in the medium.

Regulation of the phosphate regulon of Escherichia coli: Activation of pstS transcription by PhoB protein in vitro☆

Expression of the genes in the phosphate regulon, including the pstS (phoS) and phoB genes, is positively regulated by PhoB protein when phosphate is limited. We purified PhoB protein from overproducing cells and studied its interaction with the pstS gene. It binds specifically to the DNA fragment containing the promoter region of pstS. The transcription initiation site of the gene in vivo was identified by S1 nuclease mapping and primer-extension experiments. In-vitro transcription of pstS was activated by the PhoB protein, and the initiation site of transcription agreed with the in-vivo initiation site. Activation of in-vitro transcription by PhoB protein required both the normal sigma factor (sigma 70) and core RNA polymerase. PhoB protein binding sites on the promoter regions of pstS and phoB were determined by footprinting experiments with DNase I and a methylating agent. In both cases the protein binds to the pho box, the concensus sequence shared by regulatory regions of genes in the phosphate regulon. Our findings indicate that PhoB protein recognizes and binds to the pho box and activates transcription of the genes in the phosphate regulon.

Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli.

Genes in the phosphate regulon of Escherichia coli are positively regulated by the products of the phoB and phoR genes with limited phosphate, and negatively regulated by the product of the phoR gene with excess phosphate. We present here the complete nucleotide sequence of the phoR gene. Together with the DNA sequence of the upstream phoB gene that we determined previously, this region shows the following features. The flanking regions of the operon are abundant in A-T base-pairs. A possible stem-and-loop structure of the transcript followed by several U residues characteristic of rho-independent transcriptional terminators was distal to the phoR coding region. The operon is probably composed of only two cistrons. The nucleotide sequence of phoR indicates that its protein consists of 431 amino acid residues and has a molecular weight of 49,666. The amino acid sequence of the PhoR protein has significant homology with that of the EnvZ protein, which is a regulator for the omp regulon. Therefore, the sequences of the PhoB and PhoR proteins have considerable homologies with those of the OmpR and EnvZ proteins, respectively, indicating that the two operons share a common ancestor. The PhoR protein contains an extensive hydrophobic region in the amino-terminal portion. Thus the protein may be a membrane protein and function as a component of a signal transducer.

Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli

The pstA(=phoT), pstB and phoU genes are situated at 84 minutes on the Escherichia coli genetic map. All of them are involved in the negative regulation of the phosphate regulon, and all of them except for phoU are required for the binding-protein-mediated, highly specific phosphate transport. We have determined the DNA sequence of about 4 X 10(3) bases of chromosomal segment containing these genes. Four translational reading frames (TRFs) were detected in the region. We attempted to assign the TRFs to the mutant alleles. Plasmids were constructed so that each contained only one of the TRFs, downstream from the lac promoter, to be used for the complementation tests. By this test, TRF-2, TRF-3 and TRF-4 were identified with the pstA(=phoT), pstB and phoU genes, respectively. Alkaline phosphatase-constitutive mutations of the two strains in our collection were complemented by the plasmid with the TRF-1 region. Therefore, we propose to designate the allele phoW. The order of the genes in this region has been established to be phoS-phoW-pstA(=phoT)-pstB-phoU counterclockwise on the E. coli genetic map.

Identification of the human beta-actin enhancer and its binding factor.

An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

SummaryThe pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.

Mutational analysis of the role of the first helix of region 4.2 of the σ70 subunit ofEscherichia coli RNA polymerase in transcriptional activation by activator protein PhoB

Transcription of the genes belonging to the phosphate (pho) regulon inEscherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, σ70, which is encoded byrpoD. We previously isolated two mutant σ70s(D570G and E575K) that were specifically defective in transcribing thepho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif of region 4.2 of σ70. To study further the role of the first helix of the HTH motif of σ70 in transcriptional activation by PhoB, we made a series ofrpoD mutations that alter the motif and purified the mutant σ70 proteins. RNA polymerases containing the mutant σ70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from thepstS promoter, a representativepho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant σ70 (E574 K) showed enhanced transcription from the promoter. Transcription from the activator-independenttac promoter and thepBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these σ70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to thepho promoters in transcriptional activation.

Signal Transduction of the Phosphate Regulon in Escherichia Coli Mediated by Phosphorylation

In Escherichia coli, expression of genes involved in the phosphate(pho) regulon is regulated by phosphate concentration of the medium. Proteins PhoR and PhoB, which are regulatory factors for the transcriptional regulation of the pho genes, are known to belong to a family of two-component regulatory factors that respond to a variety of environmental stimuli in bacteria. PhoB is the actual activator protein, which binds to the pho gene promoters. PhoR is most likely a transmembrane protein and signal transducer, which exhibits both PhoB-specific phosphorylation and dephosphorylation activities. The phosphorylation of PhoB protein occurs concurrently with the acquisition of the ability to activate transcription from the pho promoters. Cross talk by protein phosphorylation appears to occur from PhoM, a PhoR like protein, to PhoB. Mutational analyses of PhoB protein indicated that the two domains, which are consisted of phosphate-acception and transcriptional activation domains, are separable and the non-phosphorylated domain blocks the transcriptional activation domain.