Isolation and characterization of a temperature-sensitive conditional mutant of Escherichia coli altered for the control of phosphate-regulated proteins

We have identified a conditional mutation which confers a ple?otropic phenotype to Escherichia coli cells: no growth at temperature higher than 36 degrees C, an altered control of the synthesis of several phosphate-regulated polypeptides (including alkaline phosphatase, sn-glycerol-3-phosphate binding protein, phosphate binding protein and outer membrane porin protein PhoE) after growth at 36 degrees C and a wild-type phenotype at 30 degrees C. This mutation was located at minute 89.5 on the E. coli chromosome in a gene we have called cpr for conditional phosphate-regulated.     

Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60-65 degrees C and pH 11.0 and had K(m) = 0.055 mM as estimated with p-nitrophenyl phosphate (pNPP). The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60 degrees C and being completely inactivated in 2 h at 80 degrees C. In substrate specificity assays, the highest enzymic activity was observed with pNPP and dATP. Vanadate, inorganic phosphate, and SDS inhibited M. ruber alkaline phosphatase, while thiol-reducing agents had virtually no effect. The enzymic activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.     

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.     

A SINGLE-CELL IMMUNOASSAY FOR PHOSPHATE STRESS IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, and determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. This would be improved by the development of single-cell methods for monitoring in situ physiology. Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minimum (Pavillard) Schiller. Antiserum was raised against a cell-surface alkaline phosphatase purified from P. minimum. Western screening indicated that the antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the identification of this protein as an alkaline phosphatase. Based on Western blots, the antiserum appeared to be specific for phosphate-regulated proteins in P. minimum because there is no discernible cross-reaction with closely related P. micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P. minimum from Narragansett Bay, Rhode Island. The percentage of labeled P. minimum cells in this environment during the summer of 1998 decreased through time as the inorganic phosphate concentration increased. The percentage of antibody-labeled cells significantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibody-based method developed for monitoring cell-specific physiology in a dinoflagellate, and the method described here may serve as a model for developing similar tools in other species of phytoplankton.     

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Cortisol modification of HeLa 65 alkaline phosphatase. Decreased phosphate content of the induced enzyme.

Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues.     

Characterization of a phosphotyrosyl protein phosphatase activity associated with a phosphoseryl protein phosphatase of Mr = 95,000 from bovine heart

A cytosolic phosphoprotein phosphatase of Mr = 95,000 purified from bovine cardiac muscle, which contains a catalytic subunit of Mr = 35,000, is known to be associated with a Mg2+-activated p-nitrophenyl phosphatase activity. We have found that the enzyme preparation is also active toward phosphotyrosyl-IgG and -casein phosphorylated by pp60v-src, the transforming gene product of Rous sarcoma virus. The properties of this phosphotyrosyl protein phosphatase activity closely resemble those of the p-nitrophenyl phosphatase activity but sharply differ from those of the phosphorylase phosphatase activity. Comparative studies of the activities of the Mr = 95,000 phosphatase, bovine kidney alkaline phosphatase, and ATP X Mg-dependent phosphatase toward phosphoseryl, phosphothreonyl, and phosphotyrosyl proteins and p-nitrophenyl phosphate under various conditions have been carried out. The results indicate that the Mr = 95,000 enzyme exhibits higher activity toward phosphoseryl and phosphothreonyl proteins than toward phosphotyrosyl proteins, while the kidney alkaline phosphatase preferentially dephosphorylates phosphotyrosyl proteins. ATP X Mg-dependent phosphatase is inactive toward phosphotyrosyl proteins.     

Distribution of the sites of alkaline phosphatase(s) activity in vegetative cells of Bacillus subtilis

Sites of alkaline phosphatase activity have been located by an electron microscopic histochemical (Gomori) technique in vegetative cells of a repressible strain SB15 of Bacillus subtilis, derepressed and repressed by inorganic phosphate, and in a mutant SB1004 which forms alkaline phosphatase in a medium high in phosphate. The sites of enzyme activity were revealed as discrete, dense, and largely spherical bodies of varying sizes (20 to 150 nm). Cells of both repressible and repression-resistant strains acted on a wide variety of phosphate esters (p-nitrophenylphosphate, beta-glycerophosphate, adenosine-5'-phosphate, glucose-6-phosphate, glucose-l-phosphate, adenosine triphosphate, and sodium pyrophosphate) to produce inorganic phosphorus under conditions of alkaline phosphatase assay [0.05 m tris(hydroxymethyl)aminomethane buffer (pH 8.4) containing 2 mm MgCl(2)]. The purified alkaline phosphatase also acted on all these esters, although much less effectively on adenosine triphosphate and sodium pyrophosphate than did the cells. Comparison of the relative utilization of the various substrates by repressed and derepressed cells and purified enzyme suggested the presence of multiple enzymes in the cells. Thus, the cytochemical method of trapping the newly generated inorganic phosphorus determines the location of an alkaline phosphatase of broad substrate profile, and in addition locates the sites of other enzymes generating inorganic phosphorus under identical conditions of assay. It is intriguing that all of these enzymes usually exist in a few clusters attached to the peripheral plasma membrane. In addition to this predominant location, there were a few sites of enzyme activity in the cytoplasm unattached to any discernible structure, and also in the cell wall of the repression-resistant and of the derepressed, repressible strains.     

Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm

The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase.     

Relationship between alkaline phosphatase and neomycin formation in Streptomyces fradiae

Studies on phosphatase activity of Streptomyces fradiae 3535 grown in three different media indicate that neomycin formation varies directly with enzyme activity, sodium nitrate-maltose-mineral salts medium giving the highest yields of alkaline phosphatase and neomycin. S. fradiae contains more than one alkaline phosphatase and the phosphatase responsible for hydrolysis of neomycin phosphate appears to be substrate specific. The same enzyme apparently hydrolyses both the N-P and P-O-P bonds of neomycin pyrophosphate. The enzyme is stimulated by Ca(2+), is inactive at a pH below 7 and is inhibited by EDTA. Enzymic activity increases when mycelia are incubated in mineral salts medium, but decreases when phosphate or glucose is included in the medium, although the latter is more effective. The inhibitory effect of EDTA on neomycin formation by resting mycelia is completely reversed by Ca(2+).     

Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.     

Use of gene fusions to determine a partial signal sequence of alkaline phosphatase.

We have isolated strains of Escherichia coli in which an amino-terminal portion of the cytoplasmic enzyme beta-galactosidase is replaced by an amino-terminal portion of the periplasmic enzyme alkaline phosphatase. The synthesis of these hybrid proteins is regulated by inorganic phosphate and they are located in the cytoplasm. One of these proteins was purified, and 14 amino acids of the amino-terminal sequence were determined. The first five amino acids, Met-Lys-Gln-Ser-Thr, appear to represent a portion of the signal sequence of the precursor of alkaline phosphatase, and the remaining sequence corresponds to that of beta-galactosidase, beginning at amino acid residue 20. The approach described here could be used for the analysis of signal sequences of exported proteins and for partial amino acid sequence determination of certain of certain other proteins.     

Co-Regulation in Escherichia coli of a Novel Transport System for sn-Glycerol-3-Phosphate and Outer Membrane Protein Ic (e, E) with Alkaline Phosphatase and Phosphate-Binding Protein

Mutants constitutive for the novel outer membrane protein Ic (e or E) contained a recently discovered binding protein for sn-glycerol-3-phosphate. The corresponding parental strains missing the outer membrane protein Ic (e, E) were negative or strongly reduced in the synthesis of the binding protein. In addition, strains that were previously isolated as mutants constitutive for the sn-glycerol-3-phosphate transport system (ugp⁺ mutants) and that produced the novel periplasmic proteins GP1 to GP4 also synthesized a new outer membrane protein with the same electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as protein Ic. Screening of different ugp⁺ mutants revealed the existence of three types in respect to the four novel periplasmic proteins GP1, -2, -3, and -4: (i) one containing all four proteins; (ii) one containing only proteins GP1, -2, and -3; (iii) one containing only proteins GP1, -2, and -4. In confirmation of the data presented in the accompanying paper by Tommassen and Lugtenberg (J. Bacteriol. 143:151–157, 1980), we found that purified GP1 is identical to alkaline phosphatase, whereas purified GP3 has binding activity of inorganic phosphate and is identical to the phosphate-binding protein. Moreover, growth conditions that lead in a wild-type strain to the derepression of alkaline phosphatase synthesis also derepressed the synthesis of the sn-glycerol-3-phosphate-binding protein as well as the corresponding transport system. Thus, the new sn-glycerol-3-phosphate transport system is part of the alkaline phosphatase regulatory system.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

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.