DECREASED PERMEABILITY AS THE MECHANISM OF ARSENITE RESISTANCE IN

JOURNAL OF BACTERIOLOGY Vol. 88, No. 1, p. 151-157 July, 1964 Copyright 1964 American Society for Microbiology Printed in U.S.A. DECREASED PERMEABILITY AS THE MECHANISM OF ARSENITE RESISTANCE IN PSEUDOMONAS PSEUDOMALLEI MICHIKO BEPPU AND KEI ARIMA Laboratory of Fermentation, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan Received for publication 2 March 1964 ABSTRACT BEPPU, MICHIKO (University of Tokyo, Tokyo, Japan), AND KEI ARIMA. Decreased permeability as the mechanism of arsenite resistance in Pseudomonas pseudomallei. J. Bacteriol. 88:151-157. 1964.-The mechanism of arsenite resistance of Pseudomonas pseudomallei strain 54, isolated from soil, was studied by use of radioactive arsenite. Arsenite resistance was found to be related to decreased permeation of arsenite into the cells. P. pseudomallei 54 cells can accumulate arsenite, but the organisms grown adaptively in the presence of arsenite accumulate only a small amount of the drug. Arsenite accumulated in the cells can exchange freely with extracellular arsenite. The apparent dissociation constant of the "bacteriumarsenite complex" was calculated as 5.9 X 10-, M for the sensitive cells and 6.3 X 104 M for the resistant ones. No significant difference was observed in the arsenite capacity (maximal uptake) of the cells (2 X 10-3 mmoles per 30 mg of dry cells). The uptake of arsenite by the sensitive cells was markedly dependent on temperature, but it was not inhibited by 2,4-dinitrophenol (5 X 10-3 M) and sodium azide (10-2 M). Omission of the substrate, a-ketoglutarate, from the incubation mixture had no inhibitory effect on arsenite uptake. Treatment of the resistant cells with cetyl-trimethylammonium bromide facilitated the uptake of arsenite by the cells. When the sensitive cells accumulating radioactive arsenite were fractionated by the Schmidt-Thanhauser-Schneider method, the large amount of intracellular arsenite was found in the cold perchloric acid-insoluble hot acid-extractable fraction. The arsenite complex with cellular macromolecular constituents cannot be solubilized by treatment with ribonuclease, deoxyribonuclease, and trypsin. In the preceding paper (Arima and Beppu, 1964), it was shown that Pseudomonas pseudomallei strain 54 isolated from soil can grow adaptively even in the presence of 10-2 M arsenite. The mechanism of arsenite resistance has been suggested to be a specific decrease of arsenite permeation into the resistant cells. In the present report, this point is examined in precise chemical terms. By use of radioactive arsenite, it was shown that induced decrease of drug permeation into cells is an interesting mechanism of drug resistance in microorganisms. MATERIALS AND METHODS Preparation of radioactive arsenite. The nuclear reaction of AS75 (, -y) As76 was used for preparation of radioactive arsenite. The powder of arsenic oxide (As203, 7.6 mg) was irradiated with a neutron beam (1011 neutrons per cm2 per sec) for 2 hr in a JRR-1 reactor (Japan Atomic Energy Research Institute). After irradiation, the arsenic oxide was dissolved in 1 ml of 0.1 N NaOH, and samples of this solution were mixed with carrier arsenite solution and used. Incubation conditions. The media and the source of the strain of P. pseudomallei 54 used were described (Arima and Beppu, 1964). Sensitive organisms were grown on bouillon at 30 C; resistant organisms were grown in the presence of 10-2 M arsenite. For the experiments reported here, bacteria were harvested at the middle of the exponential growth phase. After washing twice 0.8% sodium chloride solution, the sedimented cells were resuspended in 0.2 M phosphate buffer (ph 7.4) to a concentration of 30 to 40 mg (dry weight) per ml. To measure uptake of arsenite, the bacterial suspensions were previously incubated in L-type tubes containing 40 to 50,moles of a-ketoglutarate with gentle shaking at 30 C. Radioactive arsenite was added at a final concentration of 10-4 M, unless otherwise described, and the suspensions were further incubated. Samples (1.5 to 2.0 ml) of the incubation mixtures were taken, cooled rapidly, and centrifuged at 10,000 x g for 5 min at 0 C; 1 ml of the supernatant solutions 151

152 BEPPU AND ARIMA J. BACTERIOL. Minutes FIG. 1. Time course of uptake of radioactive arsenite by the sensitive (0) and the resistant cells (* ). Solid line, radioactivity in supernatant fluids; broken line, radioactivity taken up by the cells after washing with distilled water at 0 C. TABLE 1. Effect of washing on the retention of radioactive arsenite by sensitive cells* Condition Radioactivity in the cellst Control... 471 Washed with cold water... 410 Washed once with phosphate buffer. 459 Washed twice with phosphate buffer.. 419 Washed once with phosphate bufferunlabeled arsenite (10-4 M)... 434 Washed twice with phosphate bufferunlabeled arsenite (10-4 M)... 328 Washed once with 0.8% NaCl... 436 Washed twice with 0.8% NaCl... 450 * Suspensions of sensitive cells were incubated in a defined medium (tris buffer, a-ketoglutarate) containing arsenite (final concentration, 10-4 M) for 20 min at 30 C. t Expressed as counts per minute per sample. was dried and counted. The decrease of radioactivity in the supernatant fluids was used as a measure of arsenite uptake by the cells. Fractionation of the cells. To determine distribution of incorporated arsenite in the cells, two methods were employed: the Schmidt-Thanhauser-Schneider method (Schneider, 1946) and fractional centrifugation. Schmidt-Thanhauser-Schneider method. Both the sensitive and the resistant cells [30 to 40 mg (dry weight)] incubated with radioactive arsenite for 20 min were extracted three times with 4 ml of cold 5% perchloric acid. The precipitates were then extracted successively with 5 ml of ethanolwater (4:1), 7 ml of ethanol, and 7 ml of ethanolether (3:1). These extracts with ethanol and ether were combined as the lipid fraction. The residues were then extracted with 7.5 ml of 10% perchloric acid for 30 min at 90 C. Fractional centrifugation. The harvested cells were washed once with distilled water to remove the extracellular fluid. About 1 g of cell paste was suspended in 10-2 M tris(hydroxymethyl)aminomethane (tris) buffer (ph 7.0) containing 10-3 M MgCl2 to give a final volume of 10 ml. Disruption of the cells was carried out in a Toyo Riko sonic oscillator (10 kc) for 7 min. The disrupted cells were then fractionated into five fractions with a Spinco preparative centrifuge (Fig. 5). Analytical methods. Radioactivity in the cells and supernatant fluids was determined after drying with an end-window Geiger-Mueller counter at infinite thinness. Estimation of extracellular space in the wet packed cells was determined according to the method of Conway and Downey (1950), with dextran as the nonpenetrating solute and centrifugation for 20 min at 26,000 X g. Biochemical reagents. Sephadex G-25 was purchased from the Pharmacia Co., Uppsala, Sweden. Crystalline ribonuclease and deoxyribonuclease were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Trypsin was obtained from the Mochida Pharmaceutical Co., Tokyo, Japan. RESULTS Uptake of radioactive arsenite by P. pseudomallei. When large amounts of the sensitive bacterial cells were incubated with a low concentration (10-4 M) of radioactive arsenite in phosphate buffer, a remarkable decrease of radioactivity in the supernatant fluids was observed (Fig. 1). The decrease was much less with resistant cells than with sensitive cells. At the same time, it was observed that the sedimented cells contained the radioactivity corresponding to that disappearing from the supernatant fluids. These results show

VOL. 88, 1964 ARSENITE RESISTANCE MECHANISM IN P. PSEUDOMALLEI 153 clearly that the sensitive bacteria take up larger amounts of arsenite into the cells than do the resistant bacteria. Maximal uptake was reached within 5 to 10 min and retained in the cells for 104 at least 40 min at 30 C. Arsenite taken up on the sensitive cells could not be removed from the cells by washing with cold water, phosphate buffer, and saline (Table = 1). Repeated washings with phosphate buffer ; Resistant containing carrier arsenite (10-4 M) at 0 C removed some radioactivity from the cells. Exchange between intracellular and extracellu- / lar arsenite was very rapid during incubation at / 30 C. After incubation of the sensitive cells with/ radioactive arsenite for 24 min, 10-3 M "cold" ; 5X103 arsenite was added. Addition of "cold" arsenite / caused a rapid leakage of the accumulated radio- / E activity from the cells (Fig. 2). Effect of external arsenite concentration on the - steady-state level of arsenite accumulated. As the ex- / ternal concentration of arsenite was increased, the amount of arsenite taken up by the bacteria was found to increase. The amounts of radioactivity taken up by the bacteria after 20 min of incubation were measured. It was highly probable that 100 t--'- IW o Sensitive 5 X104 105 1 External Arsenite Conc. (M) FIG. 3. Uptake of radioactive arsenite by sensi- tive and resistant cells as a function of external concentration of arsenite. intracellular arsenite after 20 min of incubation reached equilibrium with extracellular arsenite. C_; A\ / When reciprocals of the 20-min uptake values are plotted as a function of reciprocals of the equilibrium concentrations of arsenite in the medium, a.o=; 50 - \> = */ straight line is obtained with both the sensitive and the resistant organisms (Fig. 3). This suggests that the amount of intracellular arsenite at equilibrium in the presence of increasing external concentrations of arsenite follows quite accurately the Langmuir adsorption isotherm: 1 1+ 0 10 20 30 40 Ain Aex Amax M inutes FIG. 2. Effect of several reagents on uptake and where Aex is the external concentration of arexchange of arsenite by the sensitive cells. Arrow senite, Ain is the amount taken up by the cells, represents the addition of carrier arsenite at the and Ama, is the capacity of the cells (maximal concentration of 103 M. Control, 0; 5 X 1O-4 M amount taken up by the cells at a saturating 2,4-dinitrophenol, 0; 102 M NaN3, A; omission concentration of external arsenite). Of a-ketoglutarate, A. The dissociation constant of the "bacterium-

154 BEPPU AND ARIMA J. BACTERIOL.._ c.1 *- o-c/ cts u - o 0- r z co, 100 -V 10 20 30 Minutes FIG. 4. Effect of temperature on uptake of arsenite by sensitive cells. arsenite complex" (K) is calculated as 5.9 X 10-5 M with the sensitive organisms and 6.3 X 10-4 M with the resistant ones. It is also observed that Amax is not significantly different between two organisms [2 X 10-3 mmoles per 30 mg (dry weight) with both organisms]. When 1 mg (dry weight) of cells is evaluated as equivalent to 0.003 ml of intracellular space, the intracellular concentration of arsenite at the extracellular concentration of 10-4 M is calculated as 1.7 X 10-2 M with the sensitive cells and 4 X 10-3 M with the resistant cells. Effect of some reagents on the ability of the organisms to take up arsenite. As described above, the arsenite concentration attained inside the cells was much greater than that in the external environment. To determine whether energyyielding metabolism of the cells takes some important role in this active accumulation of arsenite, some inhibitors were added to the incubation mixtures with arsenite. As illustrated in Fig. 2, both 2,4-dinitrophenol (5 X 10-3 M) and sodium azide (10-2 M) were ineffective as inhibitors of the uptake of arsenite by the sensitive cells. They also had no effect on the exchange reaction between intracellular radioactive arsenite and external "cold" arsenite added afterwards. When arsenite uptake was measured in the incubation mixtures from which the substrate, a-ketoglutarate, was omitted, no inhibitory effect was observed. The results of these experiments cannot prove the energy requirement of arsenite uptake. Studies were undertaken to determine the effect of the surface-active agent cetyl-trimethylammonium bromide (CTAB) on the uptake of arsenite by resistaint cells. As reported previously (Arima and Beppu, 1964), treatment of the resistant cells with 100,ug/ml of CTAB caused an inhibitory effect by 2 X 10-2 M arsenite on a-ketoglutarate oxidation by the resistant cells, which was not observed before this treatment. It was supposed that the effect of the treatment with CTAB might be due to destruction of the permeability barrier of the cells to arsenite. The results obtained with radioactive arsenite are consistent with this hypothesis. WVhen the resistant cells treated with CTAB at the same concentration were incubated with radioactive arsenite, the amount of arsenite taken up by the cells after 30 min was 6 X 10-7 mmoles/mg (dry weight); the value was 4.6 X 10-7 mmoles/mg (dry weight) with untreated cells. Effect of temperature on the uptake of arsenite by the cells. The sensitive cells were incubated with radioactive arsenite at various temperatures, and the amounts of arsenite taken up by the cells were measured. The uptake of arsenite by the sensitive cells was remarkably depressed at lower temperatures (Fig. 4). It was suggested from the data presented in Table 1 that the rate of the exchange reaction was also depressed at 0 C. Distribution of arsenite in the cells. Studies were then undertaken to determine whether arsenite taken up by the cells existed as the complex form with macromolecular constituents of the cells. Both the sensitive and the resistant organisms were incubated separately with radioactive arsenite for 20 min at 30 C and harvested. After washing twice with cold water, the cells were fractionated as described in the previous section. When the cells were fractionated by the Schmidt- Thanhauser-Schneider method, large amounts of radioactivity appeared in a hot perchloric acidsoluble fraction which contained cellular nucleic

VOL. 88, 1964 ARSENITE RESISTANCE MECHANISM IN P. PSEUDOMALLEI. r wo 2,000 4, 000 F 0. c;s 1, 000 2, 000 F 0 0 04 I o 0 LL 0 rl m. n~~ FIG. 5. Fractionation of radioactive arsenite taken up by the sensitive and resistant cells. Left bar, sensitive; right bar, resistant. acids (Fig. 5). In the case of the sensitive organisms, radioactivity of this fraction reached 63 % of the total. Compared with the distribution in the sensitive cells, an essential feature of the resistant cells is the remarkable decrease of radioactivity in this fraction. Such a distinction between two organisms could not be observed by the fractional centrifugation technique. It will be seen that large amounts of arsenite in the hot perchloric acid-soluble fraction appeared as the supernatant fraction by the fractional centrifugation technique. Although existence of a considerable part of the radioactivity in particulate fractions suggested occurrence of a complex form of arsenite with macromolecular constituents, the large amount of arsenite seems to be in a complex form with nonparticulate constituents. Further evidence for the occurrence of the complex form of arsenite was obtained by means of Sephadex gel filtration. When the supernatant fraction of the sensitive cells was filtered through a Sephadex G-25 gel column, at least 22.7% of the total radioactivity in the supernatant fluids X (0 o> _ (:o oc 0. Co v0 CD ) to x bi o v. TABLE 2. Rernoval of radioactive arsenite from the perchloric acid (PCA)-precipitable fraction of the sensitive cells Total Counts in Per cent Enzyme treatment* radio- fractia solubactivity fract'ion ilized counts/min to -4 counts/min Control. 1,138 167 14.7 Ribonuclease. 1,125 342 30.4 Deoxyribonuclease... 1,198 418 35.0 Trypsin.1,190 390 32.8 All three enzymes 1,117 441 39.4 * Each 1 ml of the homogenates was digested with 0.1 mg of crystalline ribonuclease, 0.1 mg of crystalline deoxyribonuclease, 5,000 units of crystalline trypsin, or all of these enzymes in succession. Incubations were carried out for 30 min at 30 C. appeared in a macromolecular peak before a peak of the free arsenite. The value of 22.7% may be taken as a lower limit. The remaining experiment was designed to de-

156 BEPPU AND ARIMA J. BACTERIOL. termine the nature of macromolecular constituents which act as receptors for arsenite. The homogenates of the sensitive cells were digested with various hydrolyzing enzymes, i.e., trypsin, ribonuclease, and deoxyribonuclease, prior to sedimentation by perchloric acid. Treatment with these enzymes singly or in combination did not increase the radioactive content in a cold perchloric acid-soluble fraction (Table 2). DISCUSSION Recently, it has been recognized that a specific decrease of permeability to the drug may play a role, at least partially, in the mechanism of drug resistance of various microorganisms (Hancock, 1962a, b; Anand, Davis, and Armitage, 1960). To determine that the same mechanism is responsible for arsenite resistance in P. pseudomallei 54, the decrease of the dissociation constant (K) of the "bacterium-arsenite complex," i.e., decrease of arsenite permeability observed in this case, must be sufficient to explain the extent of resistance exhibited by the intact resistant cells. From this consideration, evaluation of the internal concentration of arsenite was undertaken by use of the K value of the sensitive and the resistant cells. As reported previously, (Arima and Beppu, 1964), the external concentration of arsenite required for 50% inhibition of a-ketoglutarate oxidation was much higher in the resistant cells (3.3 X 10-2 M with the resistant cells and 1.6 X 10-3 M with the sensitive cells). The internal concentration of arsenite in each of the two organisms at these respective external concentrations can be calculated from the K value of the respective organisms. Provided that increase of K value is the only mechanism responsible for the arsenite resistance, these two internal concentrations should be the same. The two calculated values were in good agreement, i.e., 2.58 X 10-2 M with the sensitive cells and 2.61 X 10-2 M with the resistant cells. With Bacillus cereus, Mandel and Mayersak (1962) reported that almost all arsenite taken up by the cells could be extracted by cold perchloric acid, but in the present study with P. pseudomallei, a very small part of the intracellular arsenite was contained in this fraction. During the fractionation of the sensitive cells, large amounts of intracellular arsenite fall into a cold perchloric acid-insoluble hot perchloric acid-soluble fraction, and the percentage of radioactivity in this fraction decreases remarkably in the resistant cells. These facts suggest that this fraction contained a receptor of arsenite in the cells. From the experiment on enzymatic digestion, protein, ribonucleic acid, and deoxyribonucleic acid could not be shown to be the receptor. Furthermore, evidence for the existence of the receptor in the cell membrane or particulate structures was not obtained. It was calculated that the internal concentration of arsenite was much higher than the extracellular concentration of arsenite. Formation of a complex with the receptor may account for this remarkable concentration. The mechanism of arsenite resistance cannot be due to a decrease in the contents of receptor in the resistant cell, because Amax values of both organisms calculated from the results shown in Fig. 3 did not differ significantly. Two hypotheses concerning the mechanism of resistance seem to be possible, i.e., (i) change of nature of receptor involving a decrease of affinity to arsenite, and (ii) decreasing of activity of arsenite permeation system. Although dinitrophenol or sodium azide failed to inhibit the uptake of arsenite by Pseudomonas cells, this may not ultimately exclude the possibility that a multienzyme system is involved in this permeation system. To determine these factors, it is necessary to study more precisely the kinetics of arsenite permeation into the cell. As reported previously, resistance to arsenite in Pseudomonas cells is inducible and attained by contact with arsenite for a short period, During this lag period, a remarkable decrease of permeability to arsenite may occur, which results in resistant growth even in the presence of 10-2 M arsenite. It is of interest to determine what chemical events occur with such an inducible decrease of permeability in the cells. LITERATURE CITED ANAND, N., B. D. DAVJS, AND A. K. ARMITAGE. 1960. Effect of streptomycin on Escherichia coli. Nature 185:22-24. ARIMA, K., AND M. BEPPU. 1964. Induction and mechanisms of arsenite resistance in Pseudomonas pseudomallei. J. Bacteriol. 88:143-150. CONWAY, E. J., AND M. DOWNEY. 1950. An outer metabolic region of the yeast cell. Biochem. J. 47:347-355. HANCOCK, R. 1962a. Uptake of 14C-streptomycin by some microorganisms and its relation to

VOL. 88, 1964 ARSENITE RESISTANCE MECHANISM IN P. PSEUDOMALLEI 157 their streptomycin sensitivity. J. Gen. Microbiol. 28:493-501. HANCOCK, R. 1962b. Uptake of 14C-streptomycin by Bacillus megaterium. J. Gen. Microbiol. 28:503-516. MANDEL, H. G., AND J. S. MAYERSAK. 1962. The metabolism and actions of arsenite in microorganisms. Federation Proc. 21 :179p. SCHNEIDER, W. C. 1946. Phosphorous compounds in animal tissues. III. A comparison of methods for the estimation of nucleic acids. J. Biol. Chem. 164:747-751.