Purification of the Escherichia coli purine regulon repressor and identification of corepressors.

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.     

Purification and characterization of adenylate cyclase from Escherichia coli K12

Adenylate cyclase of Escherichia coli K12 has been purified 17,000-fold to near homogeneity from a 5-fold overproducing strain. One major band of Mr = 92,000 and several minor bands are seen on sodium dodecyl sulfate-polyacrylamide electrophoresis of the purest fractions. Identification of the enzyme with the 92,000-Da protein is based on the correlation of this band with activity when highly purified enzyme is eluted from ADP-sepharose columns. The native enzyme has a molecular weight of 95,000 determined by gel filtration, showing that the enzyme is active as a monomer. The purest enzyme has a specific activity of 700 nmol min-1 mg-1, indicating a turnover number of about 100 min-1. Our data indicate that there are only about 15 molecules of the enzyme in wild type cells of E. coli. In crude extracts, over 80% of the activity is soluble after centrifugation at 100,000 x g, indicating the enzyme is soluble or, at most, loosely membrane bound. The enzyme is only moderately stable in crude extracts and becomes more unstable as purification proceeds. Activity is stabilized by ATP, or at -20 degrees C as an ammonium sulfate precipitate or in 50% glycerol. The enzyme has an absolute requirement for divalent cations. Maximum activity with Mg2+ is reached at 30 mM. Mn2+ is a good substitute; Co2+ activates well at low concentrations but becomes inhibitory at high concentrations; and Ca2+ is a potent inhibitor in the presence of Mg2+. The isoelectric point of the enzyme is 6.1, and its pH optimum is 8.5. The enzyme is inhibited by its substrate, with a Km of about 1 mM and a Ki of about 1.5 mM, and is noncompetitively inhibited by PPi, ADP, GTP, and a number of other compounds. The data suggest that dissociation of PPi from the first enzyme-product complex is the rate-limiting step in the reaction. Activation of the enzyme, inferred to occur in vivo, could be produced by a postulated regulatory effector which speeds release of PPi from the enzyme-product complex.     

Purification and characterization of catalase HPII from Escherichia coli K12

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90,000 and 92,000. Only a single 92,000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532,000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4-11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.     

Purification and properties of the sn-glycerol 3-phosphate-binding protein of Escherichia coli.

A binding protein for sn-glycerol 3-phosphate was isolated from the cell envelope of Escherichia coli by the cold osmotic shock procedure. The protein was purified to homogeneity. It has a molecular weight of 45,000 and binds sn-glycerol 3-phosphate with a KD of 0.2 microM. The protein is monomeric and has L-leucine as NH2-terminal amino acid. The intrinsic fluorescence of the protein is altered upon binding of substrate. At an excitation of 285 nm, the emission maximum at 340 nm is quenched and shifted to 330 nm. Binding of sn-glycerol 3-phosphate is reversible and no chemical alteration occurs with the substrate. The appearance of the binding protein in the periplasm is the result of a mutation that renders the cells constitutive for sn-glycerol 3-phosphate transport. Simultaneously, two other proteins appear in the periplasm. These proteins were also purified. They do not bind sn-glycerol 3-phosphate and do not cross-react with antibodies against the pure binding protein.     

Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli

Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function. Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive. Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification. Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping. Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined. Unexpectedly, disproportionalities occurred during the purification. However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide. This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E. coli strain and from plasmid-containing strains which express the plsB gene product to different extents. The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional. In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution. The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.     

Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.     

Purification and characterization of the deoR repressor of Escherichia coli.

The deoR gene, which encodes the deor repressor protein in Escherichia coli, was fused to the strong Ptrc promoter in plasmid pKK233-2. The Ptrc promoter is kept repressed by lacI repressor to prevent cell killing. Induction of the Ptrc--deoR fusion plasmid resulted in the accumulation of 4% of the soluble protein as deoR protein. The deoR repressor protein was purified to 80% purity using conventional techniques; it has a mass of 28.5 kd and appears to exist as an octamer in solution. The deoR repressor is shown by DNase I footprinting to bind to the 16 bp palindromic sequence in the Pribnow box region of the deoP1 promoter. Also, the deoR repressor binds cooperatively in vitro to a DNA template with two deoR binding sites separated by 224 bp in keeping with the conclusion from genetic experiments that more than one operator is required for efficient repression of the deo operon.     

Phenethyl alcohol inhibition of sn-glycerol 3-phosphate acylation in Escherichia coli.

In vivo and in vitro experiments were performed to determine how phenethyl alcohol (PEA) inhibits phospholipid synthesis in Escherichia coli. This drug drastically reduced the rate of incorporation of sn-glycerol 3-phosphate into the phospholipids of an sn-glycerol 3-phosphate auxotroph. PEA also reduced the rate of fatty acid incorporation into the phospholipids of a fatty acid auxotroph. The kinetics of PEA inhibition of the rate of incorporation of sn-glycerol 3-phosphate were almost identical to those of PEA inhibition of the rate of fatty acid incorporation into phospholipids. The in vivo experiments suggested that the rate-limiting step(s) in phospholipid biosynthesis inhibited by PEA is at the level of the acylation of sn-glycerol 3-phosphate or beyond this step. PEA inhibited the sn-glycerol 3-phosphate acyltransferase with either palmitoyl coenzyme A or palmitoyl-acyl carrier protein as the acyl donor. This drug, however, had no effect on the cytidine 5'-diphosphate-diglyceride:glycerol 3-phosphate phosphatidyl transferase, cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase, and acyl coenzyme A:lysophatidic acid acyltransferase. The in vitro findings suggested that PEA inhibits phospholipid synthesis primarily at the level of sn-glycerol 3-phosphate acyltransferase.     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.     

Purification and characterization of glpX-encoded fructose 1, 6-bisphosphatase, a new enzyme of the glycerol 3-phosphate regulon of Escherichia coli

In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn(2+) for activity, and exhibited an apparent K(m) of 35 microM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.     

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.     

Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).     

Genetic analysis of Escherichia coli mutants defective in adenylate kinase and sn-glycerol 3-phosphate acyltransferase.

Complementation analysis with independently isolated plA and adk (adenylate kinase) mutants of Escherichia coli showed that all the mutants belong to the same complementation group. The results suggest that the adk (plsA) locus is the structural gene for adenylate kinase.     

Repressor for the sn-glycerol-3-phosphate regulon of Escherichia coli K-12: cloning of the glpR gene and identification of its product

The glpR gene encoding the repressor for the glp regulon of Escherichia coli was cloned from a library of HindIII DNA fragments established in bacteriophage lambda. Phages harboring glpR were isolated by selection for sn-glycerol-3-phosphate dehydrogenase function encoded by glpD, which is adjacent to glpR on the E. coli linkage map. Restriction endonuclease analysis and recloning of DNA fragments localized glpR to a 3-kilobase-pair EcoRI-SalI segment of DNA. Strains exhibiting constitutive expression of the glp operons were strongly repressed after introduction of multicopy plasmids containing the glpR gene. Analysis of proteins labeled in minicells harboring either glpR+ recombinant plasmids or a glpR::Tn5 derivative showed that the glpR gene product is a protein with an apparent molecular weight of 33,000.     

Purification of the NusB gene product of Escherichia coli K12

Summary The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al. 1984; Swindle et al. 1984). The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite. Purification of the protein was monitored using ¹⁴C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker. The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3. Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.Abbreviations SDS sodium dodecyl sulphate - kDa kilodaltons - bp base pair(s) - kbp kilobase pair(s)     

Regulation of Escherichia coli pyrC by the purine regulon repressor protein.

The purine regulon repressor, PurR, was identified as a component of the Escherichia coli regulatory system for pyrC, the gene that encodes dihydroorotase, an enzyme in de novo pyrimidine nucleotide synthesis. PurR binds to a pyrC control site that resembles a pur regulon operator and represses expression by twofold. Mutations that increase binding of PurR to the control site in vitro concomitantly increase in vivo regulation. There are completely independent mechanisms for regulation of pyrC by purine and pyrimidine nucleotides. Cross pathway regulation of pyrC by PurR may provide one mechanism to coordinate synthesis of purine and pyrimidine nucleotides.