DIHYDROSTREPTOMYCIN, VITAMIN K2-COUPLED

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1 JOURNAL OF BACTERIOLOGY Vol. 88, No. 4, p October, 1964 Copyright 1964 American Society for Microbiology Printed in U.S.A. DIHYDROSTREPTOMYCIN, VITAMIN K2-COUPLED TETRAZOLIUM REDUCTION, AND OXIDATIVE PHOSPHORYLATION IN ESCHERICHIA COLI P. D. BRAGG Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada Received for publication 8 June 1964 ABSTRACT BRAGG, P. D. (University of British Columbia, Vancouver, B.C., Canada). Dihydrostreptomycin, vitamin K2-coupled tetrazolium reduction, and oxidative phosphorylation in Escherichia coli. J. Bacteriol. 88: Dihydrostreptomycin inhibited the vitamin K2-coupled pyruvate-triphenyltetrazolium (TTC) reductase system in extracts from antibiotic-sensitive cells, but stimulated this reaction with extracts from a dependent mutant. The effects of nucleotides, phosphate, and uncoupling agents on the vitamin K2-TTC system are consistent with the linkage of reduction of the dye through intermediates of oxidative phosphorylation. Previous publications from this laboratory (Bragg and Polglase, 1963a, c) have indicated that, in cell extracts of antibiotic-sensitive Escherichia coli, dihydrostreptomycin (DHSM) inhibits at least two processes, namely, succinatetriphenyltetrazolium (TTC) reductase and oxidative phosphorylation. In both processes, extracts prepared from resistant and dependent mutants were indifferent to the antibiotic. When the effect of DHSM on the reduced nicotinamide adenine dinucleotide (NADH)-TTC reductase system was measured, reduction of the dye was either unaffected or generally slightly stimulated in extracts of all mutants. The present report describes the marked stimulation of TTC reduction which is produced by addition of vitamin K2 or menadione to the system, and the sensitivity to DHSM conferred upon the reductase system by these substances. Asano and Brodie (1963) suggested that tetrazolium dye reduction might be related to oxidative phosphorylation. The effect of adenosine diphosphate (ADP), phosphate, and uncoupling agents upon the vitamin K2-mediated NADH- TTC reductase system supports this hypothesis, and links together the observed effects of DHSM 1019 on tetrazolium dye reduction and on bacterial oxidative phosphorylation. MATERIALS AND METHODS The E. coli cultures used in this work and the method of culture were described previously (Bragg and Polglase, 1962). SB refers to streptomycin-sensitive and DA to streptomycin-dependent strains. DA was depleted of DHSM as described before (Bragg and Polglase, 1963b). Cell-free extracts were prepared from cells harvested towards the end of the logarithmic phase (SB) or from depleted DA cells. The cells were washed once by suspension and sedimentation from 0.05 M potassium phosphate buffer (ph 7.0), and were finally suspended in 0.01 M tris(hydroxymethyl)aminomethane buffer containing 0.01 M MgCl2 at ph 7.4. The cell suspension (0.2 g of packed cells per ml of buffer) was treated for 3 min in a 20-kc Bronwill sonic oscillator. Large cell fragments were removed by centrifuging at 20,000 X g; the residue was discarded. The supernatant fluid was centrifuged at 100,000 X g for 2.5 hr, and the upper half of the supernatant fluid was removed for use in the experiments described below. The reduction of TTC was measured in Thunberg tubes by use of the following system: 0.05 M potassium phosphate (ph 7.6), 0.25 ml; 0.1 M glycylglycine (ph 8.0), 0.8 ml; 0.05 M MgC92, 0.2 ml; M nicotinamide adenine dinucleotide, 0.05 ml; 6.5 X 10-4 M thiamine pyrophosphate, 0.1 ml; water, 0.75 ml; and extract, 0.5 ml. For some experiments, 0.02 ml of 2 X 10-2 M menadione or 0.04 ml of 2.6 X 10-4M vitamin K2 were present. Inhibitors were added in or made up to 0.1 ml. The stopper contained 0.1 ml of 0.1 M sodium pyruvate and 0.5 ml of 102 M TTC. After evacuation, the tube was preincubated at 37 C for 5 min prior to the addition of the contents of the stopper. The reaction was terminated after 5 min by adding 4 ml of acetone;

2 1020 BRAGG O K V 5 J. BACTERIOL. all dye reduction was inhibited. Vitamin K2, the natural quinone of E. coli, gave a linear stimulation of the system. When DHSM was added to the TTC-reductase system of the sensitive strain in the presence of vitamin K2, a variable degree of inhibition was observed among different preparations of the extract. However, if the effect of different concentrations of DHSM was studied both in the presence and absence of added naphthoquinone (Fig. 2), it was observed that 0.5 X 10-4 to 1.5 X 10-4 M DHSM gave substantial inhibition of the quinone-stimulated increase in dye reduction in extracts from sensitive cells. The effect of r- dicoumarol closely resembled that of DHSM, control and gave only slight inhibition of TTC reduction E t Q4 in the absence of added quinone (Table 1), which suggested that the antibiotic- and dicoumarol-insensitive dye reduction was not naphthoquinone-mediated. Similar results were obtained if vitamin K2 was replaced by menadione.,, Dye reduction with extracts prepared from DHSM-depleted dependent cells was also stimulated by vitamin K2. DHSM slightly TI ME (minutes) stimulated formazan formation (Fig. 2), in contrast with the FIG. 1. Effect of menadione and vitamin K2 on inhibition obtained with sensitive extracts. pyruvate succinate triphenyltetrazolium rreductase of sensitive extract. See Materials and Mrethods for details. Extract protein, 8.1 mg per tube n^ 5.0_ 0 after centrifuging, the optical densiity of the x supernatant fluid was measured at 48l5 m,u (Bril, c ). The rate of dye reduction in ti he presence E / of vitamin K2 was linear under these conditions. N. In some experiments, the rate of dye reduction X at 485 m,u was followed in anaerob: ic cuvettes C.) which had been evacuated and re,filled with X oxygen-free nitrogen several times. Vitamin K2 was kindly supplied by 0. Isler, _ Hoffman-La Roche and Co., Ltd., Bas;le, Switzer- (Bragg _ land, or was extracted from E. coli cbells and Polglase, 1963b). REsuuTs :. Effect of DHSM on naphthoquinruo e-stimulated 0.01 tetrazolium reduction. Figure 1 shows the effect of adding vitamin K2 and menadicne to the pyruvate-ttc system in extracts of cells of the HSM (x103m) streptomycin-sensitive strain. The reiaction was FIG. 2. Effect of dihydrostreptomycin on pyrufollowed in anaerobic cuvettes. IWith both vate-succinate triphenyltetrazolium reductase of naphthoquinones, reduction eventualliy exceeded sensitive (0) and dependent (A) extracts. Vitamin K2 that of the control, although with menadione present as indicated. See Materials and Methods for this was preceded by a lag phase du ring which details. Extract protein, 1.9 mg per tube.

3 VOL. 88, 1964 DIHYDROSTREPTOMYCIN INHIBITION OF E. COLI 1021 However, if vitamin K2 was replaced by menadione, 0.35 X 10-3 M DHSM inhibited the system from dependent cells to a plateau value of 67% of the initial reduction. Both naphthoquinonemediated systems of extracts from DHSMdependent cells were inhibited by dicoumarol. Action of nucleotides on quinone-stimulated TTC reduction. Table 2 shows the effect on formazan production of various nucleotides and inhibitors. Of the nucleotides tested, only ADP and, to a lesser extent, adenosine monophosphate effected the reaction. When the concentration of ADP was varied, a plateau of maximal inhibition TABLE 1. Effect of dicoumarol on pyruvate-succinate triphenyltetrazolium (TTC) reductase of SB in the presence and absence of vitamin K2* Concn of dicoumarol (X 104) M TTC reducedt Vitamin K2 absent Vitamin K2 present * See Materials and Methods for details. Extract protein, 3.1 mg per tube. t Results expressed as micromoles per minute X 102. TABLE 2. Effect of nucleotides and inhibitors on pyruvate-succinate triphenyltetrazolium (TTC) reductase of SB* Addition Concn (X 10-4) reductiont M None Adenosine diphosphate Adenosine monophosphate Uridine diphosphate Guanosine... diphosphate p-chloromecuribenzoate Heptyl-4-hydroxyquinoline N- oxide Pentachlorophenol Sodium cyanide * Vitamin K2 was added. See Materials and Methods for details. Extract protein, 2.6 mg per tube. t Expressed as percentage of control ( ugmoles of TTC reduced per min). TABL,E 3. Effect of dihydrostreptomycin (DHSM) and adenosine diphosphate (ADP) on pyruvatesuccinate triphenyltetrazolium (TTC) reductase* Concn of inhibitor TTC reductiont DHSM (X 108 m) ADP (X 103 M) SB DA O * Vitamin K2 was added. See Materials and Methods for details. Extract protein, 2.6 mg (SB) or 1.55 mg per tube (DA). t Expressed as percentage of control [(SB) or (DA) ,moles of TTC reduced per min]. was obtained at 0.33 X 10-3 to 0.65 X 10-3 M ADP. The extent of inhibition was of similar magnitude to that produced by DHSM in the same system (Table 3). Extracts of both mutants were inhibited by ADP. Effect of dicoumarol and DHSM in the presence of ADP. Table 4 summarizes the results of experiments in which the action of DHSM and of dicoumarol on the vitamin K2-stimulated reduction of TTC was measured with or without the addition of ADP or inorganic phosphate, or both, to the system. When phosphate was added to the basic mixture from which it had been omitted, there was an inhibition of dye reduction. If ADP was also added, the maximal inhibition which was obtained was equivalent to that produced by the ADP alone. Both DHSM and dicoumarol increased the inhibition produced by ADP (or phosphate). DIscussION The failure of DHSM to stimulate TTC reduction in cell extracts from dependent cells in the presence of menadione, compared with the stimulation produced with vitamin K2, together with the nonlinear stimulation of dye reduction produced by menadione with extracts of sensitive cells, indicates that menadione cannot act in the same manner as vitamin K2. This type of response was obtained by Brodie (1961) with

4 1022 BRAGG J. BACTERIOL. TABLE 4. Effect of comibinations of inhibitors on pyruvate-s24ccinate triphenyltetrazolium)n (TTC) red?ictase of SB* Expt no. Inhibitort ductiont 1 None 100 DHSM (1.7 X 10-4 M) 69 Phosphate (3.1 X 10-3 M) 83 ADP (6.5 X 10-4 M) 59 ADP + DHSM 44 ADP + phosphate CO0 Phosphate + DHSM 57 ADP, phosphate, DHSM 44 2 None 100 Dicoumarol (3.1 X 10-3 M) 48 Phosphate (3.1 X 10-3 M) 74 ADP (6.5 X 10-4 M) 64 ADP + dicoumarol 40 ADP + phosphate 62 Phosphate + dicoumarol 25 Phosphate, ADP, dicoumarol 34 * Vitamin K2 was added to, and phosphate was omitted from, basic system; otherwise, conditions were as in Materials and Methods. Extract protein, 2.6 mg per tube. t Abbreviations: DHSM, dihydrostreptomycin; ADP, adenosine diphosphate. t Expressed as percentage of control (0.0105,umoles of TTC reduced per min). MIycobacteriumtt phlei, by the use of irradiated cell extracts in which restoration of oxidative phosphorylation was obtained on addition of vitamin K1 ; menadione restored electron transport only. These results. together with the finding (Brodie, 1961) that vitamin K plays a central role in bacterial oxidative phosphorylation, suggest that the vitamin K2-mediated TTC reductase of E. coli may involve the linkage of dye reduction through an intermediate of oxidative phosphorylation. The inhibition by DHSM of tetrazolium dye reduction would then be in agreement with the previously reported (Bragg and Polglase, 1963c) effect of the antibiotic on oxidative phosphorylation. Support for the concept that vitamin K-mediated tetrazolium dye reduction involves intermediates of oxidative phosphorylation was recently given by Asano and Brodie (1963), who were unable to separate malate-vitamin K,-3 (4, 5-dimethyl thiazolyl 1-2)2, 5-diphenyltetrazolium reductase activity from the coupling factor activity for oxidative phosphorylation. Both ADP and inorganic phosphate, when added separately, inhibited the reduction of the dye in the vitamin K2-stimulated system. A mixture of both inhibited only to the maximal value given by ADP. The inhibition produced by phosphate could be due to competition with TTC at an intermediate, presumably a reduced vitamin K derivative (Brodie, 1961), of oxidative phosphorylation. Since small amounts of phosphate from the cell-free extract are present in the system, the effect of added ADP alone would be to stimulate incorporation of phosphate into the intermediate, with subsequent formation of adenosine triphosphate (ATP), and thus increase the inhibition of dye reduction produced by phosphate alone. Since the adenosine triphosphatase activity of the extracts is high (0.48,umoles of phosphate liberated per min per mg of protein), ADP would be rapidly regenerated and a steady level of inhibition would result, the magnitude of which would depend upon the extent of coupling of the system. Addition of more inorganic phosphate would thus not increase the degree of inhibition produced by ADP in the presence of small amounts of phosphate. However, excess phosphate was routinely added to the assay mixture to ensure reproducible coupling of the system. Of the nucleotides tested, only ADP (and, to a lesser extent, adenosine monophosphate) inhibited dve reduction. This is consistent with a specific effect of ADP on oxidative phosphorylation. Both dicoumarol and DHSM gave additional inhibition with both ADP and phosphate, and therefore must interact with the system at a different locus than the latter compounds. Kashket and Brodie (1963) found that dicoumarol and p-chloromercuribenzoate uncoupled oxidative phosphorylation in E. coli extracts, and Ishikawa and Lehninger (1962) noted the same effect with pentachlorophenol and extracts from Mllicrococcus lysodeikticus. All these compounds inhibit the vitamin K2-stimulated tetrazolium reductase system, and thus support the hypothesis that intermediates of oxidative phosphorylation are involved in this system. The stimulation of dye reduction produced by 2-heptyl-4-hydroxyquinoline N-oxide, compared with the inhibition given by DHSM in extracts from streptomycin-sensitive cells, is of interest in view of the observations of Lightbown (1954) that the former compound is an antagonist of streptomycin.

5 VOL. 88, 1964 DIHYDROSTREPTOMYCIN INHIBITION OF E. COLI 1023 ACKNOWLEDGMENTS The author is indebted to W. J. Polglase for stimulating discussion and advice, and to J. Withaar for expert technical assistance. This investigation was supported by a scholarship from the Medical Research Council of Canada. LITERATURE CITED ASANO, A., AND A. F. BRODIE Oxidative phosphorylation in fractionated bacterial systems. XI. Separation of soluble factors necessary for oxidative phosphorylation. Biochem. Biophys. Res. Communs. 13: BRAGG, P. D., AND W. J. POLGLASE Extracellular metabolites of streptomycin mutants of Escherichia coli. J. Bacteriol. 84: BRAGG, P. D., AND W. J. POLGLASE. 1963a. Effect of dihydrostreptomycin on tetrazolium dye reduction in Escherichia coli. J. Bacteriol. 85: BRAGG, P. D., AND W. J. POLGLASE. 1963b. Electron-transport components of streptomycindependent Escherichia coli. J. Bacteriol. 86: BRAGG, P. D., AND W. J. POLGLASE. 1963c. Inhibition of oxidative phosphorylation in Escherichia coli by dihydrostreptomycin. J. Bacteriol. 86: BRIL, C Enzymic microdetermination of succinate and fumarate in tissue homogenates. Biochim. Biophys. Acta 15: BRODIE, A. F Vitamin K and other quinones as coenzymes in oxidative phosphorylation in bacterial systems. Federation Proc. 20: ISHIKAWA, S., AND A. L. LEHNINGER Reconstitution of oxidative phosphorylation in preparations from Micrococcus lysodeikticus. J. Biol. Chem. 237: KASHKET, E. R., AND A. F. BRODIE Oxidative phosphorylation in fractionated bacterial systems. VIII. Role of particulate and soluble fractions from Escherichia coli. Biochim. Biophys. Acta 78: LIGHTBOWN, J. W An antagonist of streptomycin and dihydrostreptomycin produced by Pseudomonas aeruginosa. J. Gen. Microbiol. 11: Downloaded from on December 1, 2018 by guest