Plasmid R68.45 and S-a transduction by the temperate bacteriophage 59 of Erwinia carotovora 268

Temperature bacteriophage 59 of Erwinia carotovera 268 had transduced extrachromosomal DNA: plasmids of R68.45 and S-a. Before plasmid transduction experiments the suitable donor strains of indicator culture Erwinia horticola 450 harbouring R68.45 and S-a were created. The frequency of plasmid R68.45 transfer from Pseudomonas putida to E. horticola 450-8 by conjugation was equal to 5 x 10(-8) per a donor cell and in the case of S-a--from E. coli C600 for the same recipient cells--was 2 x 10(-6). Bacteriophage 59 has transduced only separate markers of plasmid R68.45, since plasmid S-a is probably transduced by the phage as an intact unit.     

Kinetic studies on the adsorption of Cd2+, Cu2+ and Zn2+ ions from aqueous solutions by cassava (Manihot sculenta Cranz) tuber bark waste

The kinetics of Cd2+, Cu2+ and Zn2+ adsorption onto pure and thioglycolic acid treated cassava tuber bark wastes (CTBW) were investigated using a batch sorption technique at 30 degrees C. Kinetic data suggested that the adsorption process was exothermic, the rate limiting sorption step was physisorption and adsorption rates could be best described by a pseudo-second order model. Rate coefficients were determined to range between 1.39x10(-2)min(-1) and 5.94x10(-2)min(-1), 1.46x10(-3)min(-1) and 5.76x10(-3)min(-1) and 0.69x10(-3)min(-1) and 5.8x10(-3)min(-1) for Cd2+, Cu2+ and Zn2+, respectively. The results from these studies indicated that the sorption process is fast and stable. The adsorption equilibria were evaluated using the Langmuir equation and the monolayer sorption capacity was found to range between 5.88-26.3mg/g, 33.3-90.9 mg/g and 22.2-83.3mg/g for Cd2+, Cu2+ and Zn2+, respectively. Negative values of DeltaG(ads)(0) indicated that the adsorption process was spontaneous and exothermic in nature.     

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sorption of lead, cadmium, and zinc on sulfur-containing chemically modified wastes of fluted pumpkin (Telfairia occidentalis Hook f.)

We have tested the applicability of regular as well as sulfanylacetic acid (SA) modified fluted pumpkin waste biomass as adsorbents for Pb2+, Cd2+, and Zn2+ aqueous solutions by means of the batch-sorption technique. The data revealed that SA modification produces a larger surface area, enhancing the metal-ion binding capacity of the biomass. The sorption process was examined by means of Freundlich and Langmuir models. The kinetic study showed that the sorption rates can be described by a pseudo-second-order process. The rate constants for the control biomass (CB) were 2.2x10(-2), 4.4x10(-2), and 1.6x10(-2) mg g(-1) min(-1) for Pb2+, Cd2+, and Zn2+, respectively; and the corresponding rate constants for the SA-modified biomass were 4.0x10(-2), 4.7x10(-2), and 1.7x10(-2) mg g(-1) min(-1), respectively. Thermodynamic considerations indicated a spontaneous exothermic process, which implies that physisorption is the main mechanism in the sorption process.     

Post-chemiluminescence behaviour of Ni2+, Mg2+, Cd2+ and Zn2+ in the potassium ferricyanide-luminol reaction.

A new chemiluminescence (CL) reaction was observed when Ni2+, Mg2+, Cd2+ or Zn2+ was injected into the reaction mixture after the finish of the CL reaction of alkaline luminol and potassium ferricyanide. This reaction is described as a post-chemiluminescence (PCL) reaction. The possible mechanism for the PCL was proposed based on studies of the CL kinetic characteristic and the CL spectra. The experimental conditions of the CL reactions were optimized and the feasibility of using the reaction to analyse these metal ions was evaluated. The PCL reaction method operates in the ranges: 1 x 10(-7)-8 x 10(-6) g/L Ni2+; 3 x 10(-6)-2 x 10(-4) g/L Mg2+; 8 x 10(-7)-1 x 10(-4) g/L Cd2+; and 2 x 10(-4)-2 x 10(-3) g/L Zn2+, with detection limits of 4 x 10(-8) g/mL, 1 x 10(-6) g/mL, 3 x 10(-7) g/mL, 8 x 10(-5) g/mL, respectively.     

Production of Bacteriophage T7

Bacteriophage T7 was grown with Escherichia coli B as the host organism in 3- and 20-liter vessels. Under the best growth conditions devised, the yields of T7 in the culture lysates averaged 1.33 x 10(12) and 0.95 x 10(12) plaque-forming units per ml, respectively, compared with the best previously reported yields of 10(11) to 3 x 10(11) plaque-forming units per ml in 1-liter batches grown in the presence of air, or double this in similar batches grown in the presence of oxygen. The bacteriophage was purified by a simple method which gave average yields of 143 mg/liter and 131 mg/liter from the 3- and 20-liter batches, respectively. The efficiency of plating of the final material ranged from 18 to 42%. The purified bacteriophage is a convenient source of monodisperse deoxyribonucleic acid, the molecular weight of which is about 25 x 10(6).     

Transduction in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The virulent bacteriophage psi M1 of Methanobacterium thermoautotrophicum Marburg mediated transduction of a resistance marker and of three biosynthesis markers. Transductants were observed at frequencies of 6 x 10(-4) to 5 x 10(-6)/PFU.     

Guanylate cyclase of isolated bovine retinal rod axonemes.

The guanylate cyclase activity of axoneme--basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexes of GTP4- serve as substrates. Binding of an additional mole of Mg2+ or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+, Ni2+, Fe2+, Fe3+, Zn2+, and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+ or Mn2+ to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mg--enzyme complex and the Mn--enzyme complex are 9.5 x 10(-4) and 1.1 x 10(-4) M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex of the enzyme with the same metal are 7.9 x 10(-4) and 1.4 x 10(-4) M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+ and high concentrations of Mg2+ and Mn2+ are inhibitory. Ca2+ inhibition appears to require the binding of 2 mol of Ca2+ per mol of enzyme. The dissociation constant of the Ca2--enzyme complex is estimated to be 1.4 x 10(-6) M2. The axoneme--basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1--10% that of the guanylate cyclase activity.     

Transduction of Escherichia coli in soil

Bacteriophage P1-mediated generalized transduction of Escherichia coli K-12 was assessed in nonsterile soil. Auxotrophic recipient cells (thr- leu- thi- rpsL) were incubated in a sandy and a silty clay loam soil, and the transducing phage lysates from prototrophic strains carrying transposon 10(Tn10) in either purE or aroL regions were added. At intervals, the bacterial populations derived from the soils were plated on selective-differential media to enumerate prototrophic (thr+, leu+, or Tcr) transductants. Of 100 bacterial isolates obtained on the selective-differential media, 58 (14 thr+; 11, leu+; 33 Tcr) were confirmed E. coli transductants. The frequency of transduction in soil was ca. 10(-6). These data demonstrate the potential use of bacteriophage P1 to genetically manipulate E. coli in situ.     

Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity

ZntA, a P1B-type ATPase, confers resistance specifically to Pb2+, Zn2+, and Cd2 in Escherichia coli. Inductively coupled plasma mass spectrometry measurements show that ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain. Both sites can bind monovalent and divalent metal ions. Two proteins, deltaN-ZntA, in which the N-terminal domain is deleted, and C59A/C62A-ZntA, in which the N-terminal metal-binding site is disabled by site-specific mutagenesis, can only bind one metal ion. Because C59A/C62A-ZntA can bind a metal ion at the transmembrane site, the N-terminal domain does not block direct access of metal ions to it from the cytosol. A third mutant protein, C392A/C394A-ZntA, in which cysteines from the conserved CPC motif in transmembrane helix 6 are altered, binds metal ions only at the N-terminal site, indicating that both these cysteines form part of the transmembrane site. The metal affinity of the transmembrane site was determined in deltaN-ZntA and C59A/C62A-ZntA by competition titration using a metal ion indicator and by tryptophan fluorescence quenching. The binding affinity for the physiological substrates, Zn2+, Pb2+, and Cd2+, as well as for the extremely poor substrates, Cu2+, Ni2+, and Co2+, range from 10(6)-10(10) M(-1), and does not correlate with the metal selectivity shown by ZntA. Selectivity in ZntA possibly results from differences in metal-binding geometry that produce different structural responses. The affinity of the transmembrane site for metal ions is of similar magnitude to that of the N-terminal site [Liu J. et al. (2005) Biochemistry 44, 5159-5167]; thus, metal transfer between them would be facile.     

Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin

Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+. The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain. For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M. The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M).     

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Isolation and characterization of Caulobacter crecentus bacteriophage phi Cd1.

A DNA-containing bacteriophage, phiCd1, was isolated from sewage and shown to infect both stalked and swarmer cells of Caulobacter crescentus strain CB13B1a. phiCd1 is a small, icosohedral bacteriophage, 60 nm in diameter, which possesses a short, noncontractile tail, 10 to 12 nm in length. The bacteriophage particle is composed of at least eight structural proteins. phiCd1 nucleic acid exists as a linear duplex of DNA as judged by: (i) thermal denaturation (Tm), (ii) CsCl density gradient centrifugation, and (iii) chemical analysis of its base composition. The DNA is 61% guanosine plus cytosine, has a buoyant density in CsCl of 1.721 +/- 0.001 g/cm3, and denatures sharply at 78.5 C in 0.1 SSC (standard saline citrate) buffer. The S20, w value for the DNA is 34.3 +/- 0.1S as compared with T7 DNA, indicating a molecular weight of about 29 x 10(6).     

Low-affinity signature of the rat beta-parvalbumin CD site. Evidence for remote determinants

Although rat beta-parvalbumin and chicken parvalbumin 3 (CPV3) are identical at 74 of 108 residues, rat beta exhibits perceptibly lower Ca2+ and Mg2+ affinities. At 25 degrees C, in Hepes-buffered saline, at pH 7.4, the overall deltadeltaG degrees ' values are 2.0 and 3.9 kcal/mol, respectively. These differences primarily reflect the disparate behavior of the CD sites in the two proteins. Their respective binding constants for Ca2+, for example, are 1.5 x 10(6) and 2.4 x 10(7) M-1. The extent to which this differential behavior is dictated by local and remote sequence differences is unknown. To explore this question, we performed mutagenesis on rat beta, substituting the corresponding CPV3 codon for residues 49, 50, 57, 58, 59, and 60. The resulting CD site is identical to CPV3 at 27 of 30 positions. The mutations were introduced in four stages, replacing residues 49 and 50 (yielding beta 49/50), then 57 and 58 (beta 49/50/57/58), then 59 (beta 49/50/57/58/59), and finally 60 (beta 49/50/57/58/59/60). Apoprotein stability was examined by scanning calorimetry and chemical denaturation and divalent ion affinity by titration calorimetry. All four variants exhibit elevated Tm values and are between 0.13 and 0.39 kcal/mol more stable at 25 degrees C. Although all four proteins display heightened divalent ion affinity, the increases are small. The maximal deltadeltaG degrees ' values, observed for 49/50/57/58/59/60, are just -0.56 and -0.96 kcal/mol for Ca2+ and Mg2+, respectively. Evidently, structural features beyond the metal ion-binding motif contribute to the unusual divalent ion-binding behavior associated with the rat beta CD site.     

Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).     

Study of the catalytic effect of PAR on the luminol-potassium ferricyanide reaction using a flow-injection chemiluminescence method.

We discovered that 4-(2-pyridylazo) resorcinol (PAR) has a strong catalytic effect on luminol-potassium ferricyanide chemiluminescence (CL). Results indicated that the chemiluminescence intensities at maximum light emission were linearly corrected with the concentration of PAR over the range 1.0 x 10(-5)-1.0 x 10(-7) mol/L. A detection limit of 5.7 x 10(-8) mol/L for PAR was achieved. It was found that some metal ions strongly affected this catalytic reaction. Based on this finding, the luminol-potassium ferricyanide-PAR reaction was developed for the determination of metal ions. The detection limits (S/N = 3) for Ni2+, Cr3+, Zn2+, Co2+ and Mn2+ were determined to be 1.0 x 10(-9) mol/L, 5.0 x 10(-9) mol/L, 5.0 x 10(-8) mol/L, 1.0 x 10(-9) mol/L and 1.0 x 10(-8) mol/L, respectively. In addition, the relative standard deviation values for these metal ion assays were in the range 0.82-2.72% (n = 6).     

Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis

Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The cotransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7 x 10(3) to approximately 2.7 x 10(6) per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7 x 10(3) protoplasts per ml is about 100 times lower than 3.2 x 10(5) competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.     

The effect of ouabain on sensory neuron neurite growth in organotypic culture

Ouabain, a known inhibitor of Na+, K(+)-ATP, taken in a wide range of concentrations, was investigated in organotypic tissue culture of dorsal root ganglia cells of 10-11 day old chick embryos. Ouabain inhibited neurite growth in a dose-dependent manner. The Hill coefficient was defined as 1, and the Kp value was estimated as 1 x 10(-10) M. At inhibitor concentrations exceeding 1 x 10(-9) M, the growth of neurites was totally inhibited. It is assumed that Na+, K(+)-ATPase may play an important role in regulation of the process of neurite growth in sensory neurones.     

A new class of cardiotonic steroids]

A series of 23-oxosteroid derivatives have been synthesized and tested for their inhibiting Na+, K(+)-dependent ATPase from rat brain in the 1 x 10(-6)-1 x 10(-4) M concentrations. Natural 23-oxogenins from sea star Asterias amurensis and synthetic monoesters showed the inhibiting activity upto 50-55%. These compounds caused heart contraction in frogs at the level of the known cardiotonic strophanthin G, and inotropic activity on isolated heart of mollusk Spisula sachalinensis.