Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli.

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growth in phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvation conditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion of the phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is not responsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expression of the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excess growth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphatase constitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of being constitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulon expression was supported by the fact that the two nucleotides appearing under phosphate starvation induced the synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphate excess.     

A constitutive mutation, phoT, of the repressible acid phosphatase synthesis with inability to transport inorganic phosphate in Saccharomyces cerevisiae

Wild-type cells of Saccharomyces cerevisiae cultivated in low-Pi medium actively accumulate inorganic phosphate (Pi), while the same cells cultivated in high-Pi medium do not. A recessive constitutive mutant (phoT), for repressible acid phosphatase (EC 3.1.3.2) synthesis, is described. It shows severely reduced potency of Pi uptake, while the recessive constitutive mutants, phoR and phoU, in the same system show wild-type potency as.     

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.     

Transcriptome analysis of phosphate starvation response in Escherichia coli

Escherichia coli has a PhoR-PhoB two-component regulatory system to detect and respond to the changes of environmental phosphate concentration. For the E. coli W3110 strain growing under phosphate-limiting condition, the changes of global gene expression levels were investigated by using DNA microarray analysis. The expression levels of some genes that are involved in phosphate metabolism were increased as phosphate became limited, whereas those of the genes involved in ribosomal protein or amino acid metabolism were decreased, owing to the stationary phase response. The upregulated genes could be divided into temporarily and permanently inducible genes by phosphate starvation. At the peak point showing the highest expression levels of the phoB and phoR genes under phosphate-limiting condition, the phoB- and/or phoR-dependent regulatory mechanisms were investigated in detail by comparing the gene expression levels among the wild-type and phoB and/or phoR mutant strains. Overall, the phoB mutation was epistatic over the phoR mutation. It was found that PhoBR and PhoB were responsible for the upregulation of the phosphonate or glycerol phosphate metabolism and high-affinity phosphate transport system, respectively. These results show the complex regulation by the PhoR-PhoB two-component regulatory system in E. coli.     

Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli.

The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon. Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism. Here we show that envZ11-procaine act differently on the mal and pho regulons. In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation. In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment. The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action. Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin. The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain.     

Cloning of and complementation tests with alkaline phosphatase regulatory genes (phoS and phoT) of Escherichia coli.

The regulatory genes of alkaline phosphatase, phoS and phoT, of Escherichia coli were cloned on pBR322, initially as an 11.8-kilobase EcoRI fragment. A restriction map of the hybrid plasmid was established. Deletion plasmids of various sizes were constructed in vitro, and the presence of phoS and phoT genes on the cloned DNA fragments was tested by introducing the plasmids into phoS64 and phoT9 strains for complementation tests. One set complemented only phoS64 but not phoT9; the other set complemented only phoT9 but not phoS64. We conclude that phoS64 and phoT9 mutations belong to different complementation groups and probably to different cistrons. The hybrid plasmid with the 11.8-kilobase chromosomal fragment also complemented the phoT35 mutation. A smaller derivative of the hybrid plasmid was constructed in vitro which complemented phoT35 but did not complement phoS64, phoT9, or pst-2. Our results agree with the suggestion that phoT35 lies in a different complementation group from phoS, phoT, or pst-2 (Zuckier and Torriani, J. Bacteriol. 145:1249--1256, 1981). Therefore, we propose to designate phoT35 as phoU. The effect of amplification of phoS or phoT on alkaline phosphatase production was examined. It was found that multiple copies of the phoS gene borne on pBR322 repressed enzyme production even in low-phosphate medium, whether it was introduced into wild-type strains (partially repressed) or phoR (phoR68 or phoR17) strains (fully repressed), whereas the introduction of multicopy plasmids bearing the phoT gene did not affect the inducibility of the enzyme.     

From cell membrane to nucleotides: the phosphate regulon in Escherichia coli

Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated. The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium. Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms. Since the Pi concentration is normally low in E. coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation. The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions. PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein. It phosphorylates the regulator protein PhoB which is also Pi starvation-induced. The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes. The genes controlled by phoB constitute the Pho regulon. The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR. The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic. The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.     

Control of phoR-dependent bacterial alkaline phosphatase clonal variation by the phoM region.

phoR mutants with the wild-type Escherichia coli K-12 Var+ phoM region showed clonal variation of bacterial alkaline phosphatase synthesis, whereas mutants with the pho-510 Var- allele did not. The pho-510 mutation is responsible for the phoR mutant constitutive phenotype and probably arose in E. coli K-12 58F+ after X-ray mutagenesis over 40 years ago. I propose that the phoM region controls a change in state of bacterial alkaline phosphatase synthesis, at least in phoR mutants. Four possible molecular mechanisms for how phoM may act are discussed.     

Evidence for a trans-dominant regulator of purine nucleoside phosphorylase expression in rat hepatoma cells

Purine nucleoside phosphorylase (PNP) levels are modulated during the growth cycle of rat hepatoma cells and increase two- to three-fold as cells go from early exponential growth phase to stationary growth phase. A mutant of these hepatoma cells has been isolated which is deficient in PNP activity. Quantitative immunoprecipitation tests indicate that the decrease in enzyme activity is due to a decrease in the number of PNP molecules. The low level of PNP enzyme produced by the mutant, however, is indistinguishable from the wild-type enzyme, suggesting that the mutant may be defective in the ability to modulate PNP levels. Fusion of the mutant cells to wild-type parental cells results in hybrids that express the mutant phenotype. Segregants that arise from the hybrids show chromosome loss and reexpression of the wild-type parental phenotype, the mutant parental phenotype, and a 2S wild-type phenotype. These indicate the following about the defect in modulation in the mutant PNP-100: (1) it is trans dominant to the wild-type; (2) its effect is negative; (3) some genomic element is required for its continued effect; and (4) it does not act by obliterating its functioning counterpart in hybrid cells.     

Disruption of sucA, which encodes the E1 subunit of alpha-ketoglutarate dehydrogenase, affects the survival of Nitrosomonas europaea in stationary phase

Although Nitrosomonas europaea lacks measurable alpha-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.     

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Phosphate-Accumulator Mutant of Arabidopsis thaliana

We have characterized a novel mutation of Arabidopsis thaliana at a locus designated pho2. pho2 mutants accumulated up to 3-fold more total P in leaves, mostly as inorganic phosphate (Pi), than wild-type seedlings. In addition, we isolated a mutant (locus designated pho1-2, an allelle of pho1-1 described by Y. Poirier, S. Thoma, C. Somerville, J. Schiefelbein [1991] Plant Physiol 97: 1087-1093) with low Pi concentrations in leaves. When grown under high transpiration conditions, leaves of pho2 seedlings became severely P intoxicated, whereas shoots of pho1-2 mutants were P deficient and wild-type seedlings were normal. A pho1/pho2 double mutant resulting from a cross between the single mutants was identified in the F2 generation and shown to have a pho1 phenotype. Prior to the development of P toxicity symptoms, P was the only mineral nutrient whose concentration was greater in pho2 mutants than wild-type seedlings. Compared to wild-type, pho2 mutants had greater Pi concentrations in stems, siliques, and seeds, but roots of pho2 mutants had similar or lower Pi concentrations than either pho1 mutants or wild-type seedlings. We suggest that the pho2 mutation affects a function normally involved in regulating the concentration of Pi in shoots of Arabidopsis.     

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli.

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.