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By making operon fusions with lambda placMu53, we identified, cloned, and analyzed the phoH gene belonging to the phosphate (pho) regulon. We mapped the phoH gene at 23.6 min in the Escherichia coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Its nucleotide sequence revealed an open reading frame of 354 amino acids which contains sequences for nucleotide-binding motifs. From comparison of the DNA sequences, phoH was found to be identical to psiH, which had been identified as a phosphate starvation-inducible gene (W.W. Metcalf, P.M. Steed, and B.L. Wanner, J. Bacteriol. 172:3191-3200, 1990). The PhoH protein was overproduced by the T7 promoter system, identified as a protein of about 39 kDa, and purified. The amino-terminal amino acid sequence of the PhoH protein agreed with the one deduced from the DNA sequence. We demonstrated that PhoH has an ATP-binding activity by a photoaffinity labeling experiment. Two transcriptional initiation sites (P1 and P2) were identified by S1 nuclease mapping. The upstream P1 promoter contains a pho box, the conserved sequence shared by the pho regulon genes. The region containing the pho box was bound by PhoB protein, the transcriptional activator of the pho regulon, as revealed by footprinting. Regulation of phoH expression in vivo was studied by constructing plasmids containing transcriptional fusions of the phoH promoters with a promoterless gene for chloramphenicol acetyltransferase. Transcription from the P1 promoter required the phoB function and was induced by phosphate limitation, while transcription from the P2 promoter was independent of phoB and constitutive under tested conditions.

The ugp operon of Escherichia coli includes genes involved in the uptake of sn-glycerol-3-phosphate and glycerophosphoryl diesters and belongs to the pho regulon which is induced by phosphate limitation. This operon has two transcriptional initiation sites, as determined by S1 nuclease mapping of the in vivo transcripts. The downstream promoter has multiple copies of the pho box, the consensus sequence shared by the pho promoters; the upstream promoter has a consensus sequence for the promoters regulated by cyclic AMP and its receptor protein, CRP. PhoB protein, which is the transcriptional activator for the pho regulon, protected the regulatory region with the pho boxes in DNase I footprinting experiments and activated transcription from the downstream promoter in vitro. Studies with transcriptional fusions between ugp and a promoterless gene for chloramphenicol acetyltransferase show that the upstream promoter is induced by carbon starvation in a manner that required the cya and crp genes. PhoB protein may act as a repressor for this upstream promoter, which also overlaps the upstream third pho box. The downstream promoter was induced by phosphate starvation and requires the PhoB protein for its activation as do the other pho regulon promoters. These results suggest that the two promoters function alternately in responding to phosphate or carbon starvation, thus providing the cell with a means to adapt to these physiological stresses.

The phoM gene is one of the positive regulatory genes for the phosphate regulon of Escherichia coli. We analyzed the nucleotide sequence of a 4.7-kilobase chromosomal DNA segment that encompasses the phoM gene and its flanking regions. Four open reading frames (ORFs) were identified in the order ORF1-ORF2-ORF3 (phoM)-ORF4-dye clockwise on the standard E. coli genetic map. Since these ORFs are preceded by a putative promotor sequence upstream of ORF1 and followed by a putative terminator distal to ORF4, they seem to constitute an operon. The 157-amino-acid ORF1 protein contains highly hydrophobic amino acids in the amino-terminal portion, which is a characteristic of a signal peptide. The 229-amino-acid ORF2 protein is highly homologous to the PhoB protein, a positive regulatory protein for the phosphate regulon. The ORF3 (phoM gene) protein contains two stretches of highly hydrophobic residues in the amino-terminal and central regions and, therefore, may be a membrane protein. The 450-amino-acid ORF4 protein contains long hydrophobic regions and is likely to be a membrane protein.

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.

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.

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.

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.

The 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 gel-mobility 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.

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.

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.

phoB encodes a positive regulator for a number of the genes belonging to the phosphate regulon of Escherichia coli, including phoA, phoS, phoE and ugpAB. We have determined the nucleotide sequence of the chromosomal segment containing the promoter and the coding region of the phoB gene. The sequence data combined with the known amino-terminal amino acid sequence of a PhoB-LacZ hybrid protein suggest that the PhoB protein consists of 228 amino acid residues with a Mr of 26,302. In the regulatory region of the gene, a consensus nucleotide sequence shared by the regulatory regions of the phoA, phoS and phoE genes, which we name the "phosphate box", was found. Since these genes are positively regulated by the product of phoB, this suggests that transcription of the phoB gene is also regulated positively by its own product. Extensive homology was found in the amino acid sequences of the products of phoB, ompR and dye, all of which are positive regulatory genes for a number of genes coding for envelope proteins. This implies that these genes were originally duplications of a protogene that evolved to have divergent but related functions.

The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.