Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5 × 10⁻⁹ M; binding site II, 1.2 × 10⁻⁸ M; and for the complete promoter/operator region 1 × 10⁻⁸ M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 × 10⁻⁷ M. Received: 18 August 1998 / Accepted: 5 May 1999     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB⁺ gene of the F plasmid, rifampin, added at 42°C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257–258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784–789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolidigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (M_r 12 000) newly synthesized at 42°C in the presence of rifampin appeared to be the product of the srnB⁺ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30°C, or the addition of Mg²⁺ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB⁺ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB⁺ gene.     

Divalent Cation Interactions with Na,K-ATPase Cytoplasmic Cation Sites: Implications for the <Emphasis Type="Italic">para</Emphasis>-Nitrophenyl Phosphatase Reaction Mechanism

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr²⁺ and Ba²⁺ did not compete with K⁺ for ATPase (an extracellular K⁺ effect). Sr²⁺ and Ba²⁺ did compete with Na⁺ for ATPase (an intracellular Na⁺ effect) and with K⁺ for pNPPase (an intracellular K⁺ effect). These results suggest that Ba²⁺ or Sr²⁺ can bind to the intracellular transport site, yet neither Ba²⁺ nor Sr²⁺ was able to activate pNPPase activity; we confirmed that Ca²⁺ and Mn²⁺ did activate. As another measure of cation binding, we observed that Ca²⁺ and Mn²⁺, but not Ba²⁺, decreased the fluorescence of the FITC-labeled pump; we confirmed that K⁺ substantially decreased the fluorescence. Interestingly, Ba²⁺ did shift the K⁺ dose-response curve. Ethane diamine inhibited Mn²⁺ stimulation of pNPPase (as well as K⁺ and Mg²⁺ stimulation) but did not shift the 50% inhibitory concentration (IC₅₀) for the Mn²⁺-induced fluorescence change of FITC, though it did shift the IC₅₀ for the K⁺-induced change. These results suggest that the Mn²⁺-induced fluorescence change is not due to Mn²⁺ binding at the transport site. The drawbacks of models in which Mn²⁺ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn²⁺ stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl₂ was partially effective as a substitute for MgCl₂ in activating the K⁺-dependent phosphatase reaction catalyzed by a purified (Na⁺ + K⁺)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl₂. Estimates of the concentration of free Mn²⁺ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187–3197). MnCl₂ competed with MgCl₂ as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl₂ appreciable ouabaininhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K⁺ as activator of the reaction and for Na⁺ as inhibitor were both decreased. For the (Na⁺ + K⁺)-ATPase reaction substituting MnCl₂ for MgCl₂ was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K⁺ was increased by the substitution, although that for Na⁺ was decreased as in the phosphatase reaction. Substituting MnCl₂ also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [⁴⁸V]-vanadate to the enzyme; furthermore, binding in the presence of MnCl₂ was, unlike that with MgCl₂, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na⁺ + K⁺)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution.     

Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Ionophorous properties of the 20,000-dalton fragment of (Ca2++Mg2+)-ATPase in phosphatidylcholine: Cholesterol membranes

Summary The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca²⁺+Mg²⁺)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem.251:4147) to have Ca²⁺-selective ionophoric activity. The Ca²⁺-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca²⁺-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same,P _Ba>P _Ca>P _Sr>P _Mg>P _Mn. This selectivity sequence is the same as that for the intact (Ca²⁺ +Mg²⁺)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except thatP _Mn>P _Mg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca²⁺-ionophoric activity of the (Ca²⁺+Mg²⁺)-ATPase.