Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.     

Location of the Genes Controlling Alkaline Phosphatase on the F′13 Episome of Escherichia coli

Interrupted mating experiments between F'13 and F(-) cells showed that the location on the F'13 episome of the genes controlling alkaline phosphatase is on the end proximal to the point of entry, in the order phoA proC phoB phoR tsx.     

Overlapping and separate controls on the phosphate regulon in Escherichia coli K12

The physiological and genetic controls operating on phosphate-regulated promoters were studied in greater detail. This was done by defining the control for three phosphate-regulated genes: phoA, psiE, and psiO. Each is highly inducible by phosphate starvation. Individually, these phosphate-starvation-inducible, psi, genes at the same time show common and differing features in their molecular control. The phoA gene, encoding alkaline phosphatase, is specifically induced by phosphate starvation. It is negatively controlled by phoR as well as by the phosphate-specific transport (PST) system in Escherichia coli. phoA induction is positively controlled by the phoB, M, and R products; it is unaffected by the cAMP and CAP system. The psiE and psiO genes were studied by using strains with lacZ fused to their respective promoters. psiE-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth. Genetically, psiE-lacZ induction is partially phoB and phoR-dependent. However, its expression is phoM-independent. This implies that phoB/phoR coupled control differs from phoB/phoM coupled control. Repression of psiE-lacZ is substantially altered in only some PST mutants, such as phoT. In addition, psiE-lacZ is negatively controlled by the cAMP and CAP system. psiO-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth or by anaerobiosis. Its expression is unaffected by any pho mutation that has been previously described. A cell density-dependent induction of psiO-lacZ is observed in lon mutants. Also, psiO-lacZ is negatively controlled by the cAMP-CAP system. In summary, these results demonstrate that co-ordinately regulated promoters can have some common regulatory elements while, at the same time, not sharing other controlling factors.     

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

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.     

Analysis of regulation of phoB expression using a phoB-cat fusion.

The phoB gene, which encodes a positive control factor for a number of phosphate-regulated genes in Escherichia coli, was cloned into multicopy plasmid pBR322. A phoB-cat fusion that expressed chloramphenicol transacetylase from the phoB promoter was constructed. Studies of the expression of the phoB-cat fusion showed that the pattern of regulation of the phoB gene was similar to that of the phoA gene, the structural gene for alkaline phosphatase. The phoB gene was derepressed under conditions of phosphate starvation, was constitutively expressed in a phoR background, and required the phoM gene product for expression in a phoR strain. Finally, a functional phoB product was required for its own synthesis. Our results indicate either that phoA gene expression responds directly to the concentration of the phoB gene product in cells or that the phoA and phoB controlling elements are quite similar.     

The PHOA and PHOB cyclin-dependent kinases perform an essential function in Aspergillus nidulans

Unlike Pho85 of Saccharomyces cerevisiae, the highly related PHOA cyclin-dependent kinase (CDK) of Aspergillus nidulans plays no role in regulation of enzymes involved in phosphorous acquisition but instead modulates differentiation in response to environmental conditions, including limited phosphorous. Like PHO85, Aspergillus phoA is a nonessential gene. However, we find that expression of dominant-negative PHOA inhibits growth, suggesting it may have an essential but redundant function. Supporting this we have identified another cyclin-dependent kinase, PHOB, which is 77% identical to PHOA. Deletion of phoB causes no phenotype, even under phosphorous-limited growth conditions. To investigate the function of phoA/phoB, double mutants were selected from a cross of strains containing null alleles and by generating a temperature-sensitive allele of phoA in a deltaphoB background. Double-deleted ascospores were able to germinate but had a limited capacity for nuclear division, suggesting a cell cycle defect. Longer germination revealed morphological defects. The temperature-sensitive phoA allele caused both nuclear division and polarity defects at restrictive temperature, which could be complemented by expression of mammalian CDK5. Therefore, an essential function exists in A. nidulans for the Pho85-like kinase pair PHOA and PHOB, which may involve cell cycle control and morphogenesis.     

Genetic characterization of the gene hupB encoding the HU-1 protein of Escherichia coli

The gene hupB encoding the HU-1(HU beta) protein of Escherichia coli was mapped between proC at min 9 and minA at min 10 on the K-12 genome by plasmid integration and chromosome transfer studies. Genetic studies using plasmid rescue techniques demonstrated that the lon gene is located very close to the 5' end of hupB and that the two genes are both transcribed clockwise on the E. coli map [Bachmann, Microbiol. Rev. 47 (1983) 180-230].     

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.     

Regulation of pho regulon gene expression by the carbon control protein A, CcpA, in Bacillus subtilis

Bacterial regulons involved in carbon, nitrogen and phosphorus metabolism must interact for purposes of coordination, but the mechanisms involved are not understood. We here report that the carbon control pro-tein-A (CcpA) of Bacillus subtilis, primarily concerned with carbon metabolism, influences expression of various phosphorus (pho) regulon genes including the two alkaline phosphatase structural genes, phoA and phoB. The directions and magnitudes of the effects of glucose and the loss of CcpA on these two genes depend on growth conditions, but they always correlate inversely. Absolute expression levels of phoA and phoB depend on a rich nitrogen source, and gene activation by a fermentable substrate such as glucose depends on the presence of a respiratory substrate such as succinate. We show that these CcpA-dependent glucose effects can be explained by the effects of glucose and CcpA acting on the phoPR operon. Although a good CcpA-binding site (CRE) is found in the control region of the phoPR operon, direct regulation of phoPR gene expression by CcpA via this CRE could not account for the effects of glucose and CcpA on phoA and phoB gene expression. We conclude that CcpA exerts indirect control over the pho regulon by a mechanism that involves CcpA and PhoRP but does not involve the phoPR operon CRE.