JOURNAL OF BACTRIOLOGY, Feb. 1984, p. 632-636 21-9193/84/2632-5$2./ Copyright 1984, Amerian Soiety for Mirobiology Vol. 157, No. 2 Pyoin Ri Inhibits Ative Transport in Pseudomonas aeruginosa and Depolarizes Membrane Potential YOSHIHIKO URATANI* AND TOSHIMITSU HOSHINO Mitsubishi-Kasei Institute of Life Sienes, Mahida-shi, Tokyo 194, Japan Reeived 4 Otober 1983/Aepted 27 November 1983 Pyoin Ri, a baterioin of Pseudomonas aeruginosa, inhibited ative transport of proline in the presene of high onentrations of malate and magnesium salt. Pyoin Ri did not affet the respiration of sensitive ells nor indue ell lysis, but it aused a derease in the intraellular ATP level. In addition, a passive inflow of [14C]thioyanate anion, a probe of membrane potential, was indued by pyoin Ri, showing a depolarization of the ytoplasmi membrane. It is onsidered that membrane depolarization is a primary ation of pyoin Ri. Pyoin Ri is a bateriophage tail-like baterioin (with a ontratile sheath) whih is produed by Pseudomonas aeruginosa (7, 8). After the adsorption to the outer membrane of a sensitive ell, a pyoin Ri partile undergoes sheath ontration and ore penetration through the outer membrane (K. Amako, personal ommuniation) and auses an inrease in permeability of the membrane to hydrophobi solutes (17, 19). Inhibition of.ative transport and maromoleular synthesis ours and finally the ell dies (6, 1). On the other hand, the membrane eletron transfer system is not affeted by pyoin Ri treatment, although oxygen uptake for ertain substrates is interfered with as a result of inhibition of ative transport (9, 1). In addition, it has been found that a number of pyoin partiles added to a ell derease the turbidity of the ell suspension beause of ell lysis but that the presene of a high onentration of magnesium represses the pyoin Ri-indued ell lysis (6, 1, 19). The inhibitory ation of pyoin Ri on ative transport has not been investigated in detail under ombined onditions in whih respiration ontinues and ell lysis is repressed. Reently it has been demonstrated that oliins l, Ia, and K, baterioins of sherihia oli, inhibit ative transport in sensitive ells and depolarize the membrane potential formed aross the ytoplasmi membrane (2, 15, 2). In the present paper, we examine the effets of pyoin Ri on membrane potential and ative transport in a sensitive ell under onditions in whih respiration was maintained and ell lysis was not aused. Pyoin Ri aused depolarization of the ytoplasmi membrane in addition to inhibition of ative transport. MATRIALS AND MTHODS Baterial strains and media. P. aeruginosa PML14 and PML15 were used as a pyoin Ri-sensitive strain and a pyoinogeni strain, respetively. Strain PML14 ells were grown aerobially at 37 C in a growth medium ontaining 2 g of sodium glutamate, 5 g of gluose, 5.63 g of Na2HPO4 12H2,.25 g of KH2PO4,.1 g of - MgSO4 7H2, and.5 g of yeast extrat per liter. At early to middle exponential phase the bateria were harvested, washed twie with a solution of.1 M NaCl-5 mm MgCl2-1 mm Tris-hydrohloride (ph 7.4) unless otherwise noted, and resuspended in the same solution. The solution is referred to as the suspension solution. Preparation of pyoin Rl. Pyoin Ri was purified * Corresponding author. from 632 lysates of strain PML15 ells treated with mitomyin C by the method of Kageyama (8). Killing ativity of pyoin Ri was determined by ounting the survival of ells after pyoin treatment. Preparation of membrane vesiles. Membrane vesiles were prepared from pyoin Ri-treated ells by the proedure desribed by Hoshino and Kageyama (4). The preparation of membrane vesiles was started within 15 min after the addition of pyoin Ri. The vesiles were suspended in.1 M potassium phosphate, ph 6.8, ontaining 1 mm MgSO4. Transport measurement. The transport of amino aids by strain PML14 ells was measured at 3 C in the presene of malate as a respiratory substrate. Cells were aerated by stirring during the experiment. After 4 min of preinubation of the ells with 2 mm potassium malate, [U-14C]proline, [U-14C]asparagine, or [U-14C]leuine (45,uCi/,umol) was mixed with the ells to give a final onentration of 2,uM. At intervals, 5-ixl samples were diluted with 5 ml of the suspension solution, filtered through Millipore filters (HA type,.45-,um pore size), and washed one with the same solution. The transport ativity of pyoin Ri-treated ells was assayed 5 or 1 min after pyoin Ri was added to the ell suspension preinubated with malate. The transport assay of membrane vesiles was arried out at 26 C by the method of Hoshino and Kageyama (4), using 2 mm sodium malate as an eletron donor. To determine the amount of proline inorporated into a trihloroaeti aid-insoluble fration, a 5-,I portion of the ell suspension was pipetted out into 5 ml of 1% old trihloroaeti aid at the indiated time and left on ie for 1 min. Preipitates were entrapped by a membrane filter and washed one with 5 ml of 5% trihloroaeti aid. The radioativity entrapped by the membrane filters was determined after drying them with a gas flow Geiger ounter, type LBC-451 (Aloka, Tokyo, Japan). Respiration measurement. Oxygen onsumption was measured polarographially with a Clark-type oxygen eletrode YSI-5331 (Yellow Springs Instrument Co., Yellow Springs, Ohio). The ell suspension (2.5 ml) was stirred in a thermostated vessel at 3 C during the measurement. ATP determination. ATP ontent of ell suspensions was determined by luminesene reation with a highly purified luiferin-luiferase reagent, using the photon ounter of a Luma bioounter M21 (Luma B.V., The Netherlands). Washed ells were aerated at 3 C for 6 min after the addition of 2 mm potassium malate, and then pyoin Ri was added. At intervals, 25-,ul samples were transferred Downloaded from http://jb.asm.org/ on September 4, 218 by guest
VOL. 157, 1984 into 1 ml of boiling water, inubated for 1 min, and ooled in an ie bath. For the determination of ATP ontent, 1 RI1 of the ell extrat was diluted with 39,ul of distilled water, and 1,u1 of the freshly prepared solution of the luiferinluiferase reagent Lumit was added to the above mixture. Immediately, luminesene was ounted for 1 s at room temperature. Measurement of membrane potential. The membrane potential of ells was measured by downhill influx of [14C]thioyanate anion into the ells (5). Cells were onentrated up to a. 2 x 11 ells per ml by entrifugation. Cell suspension (2 ml) was preinubated in the presene of 2 mm malate for 2 min at 3 C with vigorous stirring, and then 1. mm potassium [14C]thioyanate (.25 p.ci/ml) and [3H]inulin (2.5,uCi/ml) were added. After 5 min of inubation, pyoin Rl was added to the ell mixture. At the indiated time, tripliate 25-,ul samples were sedimented in ppendorf mirotubes for 5 s at 1, rpm at room temperature. After suspension of the ell pellet in 5 p.1 of distilled water, the radioativity in the ell suspension was ounted by a Bekman sintillation ounter with NCS solubilizer (Amersham Corp., Arlington Heights, Ill.). Chemials. L-[U-14C]proline (25 mci/mmol), L-[U-14C]- asparagine (2 mci/mmol), L-[U- 4C]leuine (3 mci/ mmol), potassium [14C]thioyanate (3 mci/mmol), and [3H]inulin (5 mci/mmol) were purhased from Amersham. Luiferin-luiferase reagent (Lumit) was supplied by Luma B.V. RSULTS ffet of pyoin Rl on respiration. The effet of pyoin Rl on the respiration of sensitive ells was first examined in the presene of 5 mm MgCl2 but without the addition of respiratory substrate (Fig. 1A). The ells harvested from the growth medium ontinued to respire in the suspension solution laking nutrients for growth and substrates for respiration. The addition of pyoin Rl to the ell suspension repressed respiration, and the respiration rate after the pyoin Rl addition dereased with inreasing onentrations of pyoin Rl. The effet of pyoin Rl on respiration was.2.2 MMBRAN DPOLARIZATION BY PYOCIN Ri 633 examined in the presene of 2 mm potassium malate as a respiratory substrate (Fig. 1B). The respiration of harvested ells was enhaned several times by the addition of malate. The enhaned respiration was not affeted by the pyoin Rl addition. On the other hand, the respiration was stopped within a few minutes after the addition of 2 mm KCN. Inhibition of proline uptake by pyoin R1. The effet of pyoin Rl on ellular ative transport was examined by using radioatively labeled proline (Fig. 2A). Whole ells aumulated proline to form a fairly large pool, although a portion of proline was inorporated into an aid-insoluble fration orresponding to maromoleules suh as proteins. The addition of pyoin Rl or 2 mm KCN aused a rapid efflux of the aumulated proline and an inhibition of maromoleular synthesis. Cells pretreated with pyoin Rl for 5 min did not transport proline into the intraellular spae (Fig. 2B). Membrane vesiles were prepared from the pyoin Ritreated ells whose ative transport of solutes was inhibited, and then their residual transport ativities were assayed (Fig. 3). Asparagine and leuine were used as transported solutes instead of proline, sine these amino aids were muh more effetively aumulated into membrane vesiles than proline (4). The uptake of asparagine and leuine by whole ells was inhibited almost ompletely by the pyoin Rl treatment (Fig. 3A and C), whereas membrane vesiles prepared from the same preparation of the pyoin Ri-treated ells aumulated these amino aids approximately one third as muh as did vesiles from untreated ells (Fig. 3B and D). Membrane vesiulation of pyoin Ri-treated ells thus restores lost ativity of ative transport to a onsiderable extent. ffet of pyoin Rl on membrane potential. The membrane potential, interior negative, was measured by passive inflow of lipophili anion, thioyanate ion (SCN-). The measurement of membrane potential with lipophili tetraphenylmethylphosphonium ation, whih was used widely as a probe of membrane potential, was unsuessful in P. aeruginosa, sine the intat ell envelope of sensitive ells seemed to be impermeable to the ation and the ells were killed by Tris-.3-.2-2~ ~ ~ ~ ~ 1 ~ - ~~~ Downloaded from http://jb.asm.org/ on September 4, 218 by guest &.1 l.1 5 1 15 2.5 5. 7.5 Time, min FIG. 1. ffet of pyoin Rl on respiration in (A) the absene and (B) presene of 2 mm potassium malate. (A) After sensitive ells were onentrated by entrifugation,.1 ml of the ell suspension was added to 2.3 ml of the suspension solution in a thermostated vessel. The ell suspension was inubated for 4 min, and then.1 ml of pyoin Rl solution was added at the time indiated by the arrow. Curve 1, no pyoin Rl added; urve 2, pyoin Rl added. Cell survival after pyoin Rl treatment was.3 to.4%. (B) In the presene of malate, pyoin Rl (urve 2) or 2 mm KCN (urve 3) was added after 2.5 min of preinubation of the ell suspension with malate. The ell onentration was 2.3 x 19 ells per ml in (A) and 9.2 x 18 ells per ml in (B). Moleular oxygen ontent in the hamber was alulated by using an oxygen solubility value of 5.24 m3 ( C, 76 mmhg [a. 11,325 Pa]) per liter of water at 3 C (14).
634 URATANI AND HOSHINO DTA treatment, whih made the outer membrane permeable. Intat respiring ells sarely aumulated SCN- anion in the ytoplasmi spae, but the addition of pyoin Ri aused a rapid influx of the anion whih ended within a few minutes (Fig. 4). The intraellular spae was estimated to be 1.6,ul per 11 ells, assuming that the whole ytoplasmi spae of the pyoin Ri-treated ells was equilibrated with the extraellular SCN- anion at the measured ell survival of.1%. The membrane potential, A+, of the untreated ell was alulated to be -9 mv, aording to the Nernst equation: A = -(RT/F)ln([SCN-]J[SCN-]j), where [SCN-jo and [SCN-]i are the onentration of thioyanate at the outside and the inside of the ell, respetively, and R, T, and F have their usual meanings. The above result indiates that pyoin Ri dissipates the membrane potential from -9 mv to mv. On the other hand, the addition of KCN aused a slow inflow of SCN anion (Fig. 4). In the absene of magnesium hloride, a rapid inflow of the anion was indued by the addition of 5,uM arbonyl yanide m-hlorophenyl hydrazone, an unoupler of oxidative phosphorylation (data not shown). ffet of pyoin Rt on intraellular ATP level. The intraellular ATP pool is formed in a onstant flow from synthesis to onsumption. The effet of pyoin Ri on the intraellular ATP level was examined. The addition of malate to untreated ells inreased the ontent of intraellular ATP by 1.6 times during inubation. The ATP level reahed a new stationary state within 5 min after malate was added. A rapid derease in ATP ontent was aused by the addition of pyoin Ri or KCN to the ell suspension (Fig. 5). The derease in ATP ontent present in the ell suspension represents a redution of ATP amount ontained in the intraellular spae rather than a leakage from the inside of the ell to the medium. DISCUSSION In the presene of malate and high onentrations of magnesium hloride, the addition of pyoin Ri to sensitive ells did not ause the inhibition of ellular respiration, but it inhibited ative transport of amino aids and efflux of the aumulated ones. The present results show that the inhibition of ative transport aused by pyoin Ri is not attributed 4-9 C -K of._ a m L a 'I.. 2 2 4 Time, min FIG. 3. Transport ativity of ells and membrane vesiles with or without pyoin Ri treatment. Initial ell onentration was 8.1 x 18 ells.per ml. Cell survival after pyoin RI treatment was 2.7%. Transport of asparagihe and leuine in whole ells (A and C) and membrane vesiles (B and D), respetively. Open and losed symbols orrespond to the absene and presene of pyoin Ri treatment, respetively. Triangles in (B) and (D) represent the ontrol level of amino aid aumulation without malate. to respiration inhibition and ell lysis and onfirm the onlusion of Kageyama that pyoin Ri treatment does not interfere with the eletron transfer system (9). The mehanism of ation of pyoin Ri that results in transport inhibition should be different from that of potassium yanide, sine KCN inhibited not only the ative transport but also respiration. In the absene of malate, harvested ells ontinued to respire, probably onsuming respiratory substrates present in the ytoplasmi spae, but respiration was inhibited by the addition of pyoin Ri. The inhibition of respiration may be due to a release of the respiratory substrates to the 4- to. ID Go & J. BACTRIOL. Ii& m.2 to Downloaded from http://jb.asm.org/ on September 4, 218 by guest 2 4 6 8 2 3 time, min FIG. 2. ffet of pyoin Rl on uptake of proline and maromoleular synthesis in the presene of 5 mm magnesium hloride and 2 mm potassium malate. (A) fflux of aumulated proline after pyoin Rl addition. Open and losed symbols represent inorporation of proline into total and trihloroaeti aid-insoluble frations, respetively. No addition (, ) or addition of pyoin Rl (O, *) or 2 mm KCN (A, A) was made to ell suspensions at the time indiated by the arrow. (B) Proline transport 5 min after no addition (), pyoin Rl addition (), or addition of 2 mm KCN (A).
VOL. 157, 1984 z u ur) 1..5 1.. 5 Time, FIG. 4. Influx of thioyanate anion after the addition of pyoin Rl. ntry of SCN- into the interior of the ell was alibrated by an extraellular volume oupied with [3H]inulin and estimated as a ounting ratio of [14C]SCN- to [3H]inulin. Initial ell onentration was 1.9 x 11 ells per ml. No addition () or addition of pyoin Rl () or 2 mm KCN (A) was made to the ell suspension at the time indiated by the arrow. Cell survival was.1% with pyoin Rl. extraellular medium, sine pyoin Ri aused the efflux of the aumulated solutes. Membrane vesiles prepared from pyoin Ri-treated ells, whih had lost the transport ativity, aumulated solutes, although the transport ativity of suh vesiles was lower than that of membrane vesiles from untreated ells. This result suggests that pyoin Ri may not damage ative transport systems integrated in the ytoplasmi membrane. Pyoin Ri aused the dissipation of membrane potential in the presene of malate and magnesium salt. The membrane potential in an intat ell was measured by passive inflow of thioyanate and estimated to be a. -9 mv at neutral ph. It has been reently reported that whole P. aeruginosa ells generate the membrane potential of -165 to -19 mv at the neutral ph region (21). The underestimation of the membrane potential alulated by us may be due to an inauray of the estimated intraellular volume. However, it is ertain that pyoin Ri aused a depolarization of the ytoplasmi -6 C a. 8 4 min 1 2 FIG. 5. ffet of pyoin Rl on intraellular ATP level. Total ontent of ATP in the ell suspension was measured after no addition () or addition of pyoin Rl(O) or 2 mm KCN (A) was made at time zero. Initial ell onentration was 7.1 x 1' ells per ml. Cell survival after the addition of pyoin Rl was.9%.. I~~~~ 1. Fillingame, R. H. 198. The proton-transloating pumps of oxidative phosphorylation. Annu. Rev. Biohem. 49:179-1113. 2. Gould, J. M., and W. A. Cramer. 1977. Studies on the depolarization of sherihia oli ell membrane by oliin l. J. Biol. Chem. 252:5491-5497. 3. Harold, F. M. 1972. Conservation and transformation of energy by baterial membranes. Bateriol. Rev. 36:172-23. 4. Hoshino, T., and M. Kageyama. 1979. Sodium-dependent transportof L-leuine in membrane vesiles prepared from Pseudomonas aeruginosa. J. Bateriol. 137:73-81. 5. Hsung, J. C., and A. Haug. 1977. Membrane potential of Thermoplasma aidophila. FBS Lett. 73:47-5. 6. Igima, M. 1978. Mode of ation of pyoin Rl. J. Biohem. 83:395-42. 7. Ishii, S., Y. Nishi, and F. gami. 1965. The fine struture of a pyoin. J. Mol. Biol. 13;428-431. 8. Kageyama, M. 1964. Studies of a pyoin. I. Physipal and hemial properties. J. Biohem. 55:49-53. 9. Kageyama, M. 1978. ffet of pyoin Rl on the gluose metabo-.-5-8 - 1 MMBRAN DPOLARIZATION BY PYOCIN Rl 635 membrane of at least 9 mv. Aording to the hemiosmoti theory of energy transdution and (baterial) ative transport (1, 3, 11), a driving fore for ative transport and ATP synthesis is proton motive fore, whih is omposed of proton hemial potential and membrane eletrial potential. The proton motive fore in an aerobi baterium like P. aeruginosa is generated aross the ytoplasmi membrane by funtioning of respiratory hain system (1, 3, 13). Inversely, the dissipation of the proton motive fore results in inhibition of ative transport and ATP synthesis. The membrane depolarization indued by pyoin Ri strongly suggests that the proton motive fore should be dissipated by pyoin Ri. Therefore, it is onsidered that membrane depolarization ours before the inhibition of ative transport. In this respet, the ation of pyoin Ri that auses the depolarization of the ytoplasmi membrane is similar to that of membrane-depolarizing oliins l, K, and Ia (2, 15, 2). The result of SCN- inflow experiments for the measurement of membrane depolarization showed rapid entry of the anion after the treatment with pyoin Ri and slow influx after the addition of KCN. The latter result seems to suggest that the membrane depolarization indued by KCN may our slowly. However, this respiratory inhibitor rapidly aused not only the omplete inhibition of respiration and ative transport but also the efflux of aumulated solutes. In addition, the generation of proton motive fore for ative transport and ATP synthesis in P. aeruginosa ells is solely dependent on the respiratory hain system (1, 3, 13). It is therefore onsidered that KCN should also depolarize the membrane at a faster rate than that observed. Reent studies of oliins l, K, and Ia, whih ause membrane depolarization, have provided evidene that these oliin moleules possess an ion hannel-like funtion (12, 16, 18). From the above result, it is likely that pyoin Ri may also form the ion hannel aross the ytoplasmi membrane. An intraellular ATP level is maintained in a onstant flow from synthesis to onsumption of ATP. The ATP level was dereased by the addition of pyoin Ri as well as KCN. As desribed above, pyoin Ri or KCN appears to dissipate the proton motive fore, resulting in the inhibition of ATP synthesis. Therefore, the derease in ellular ATP pool shows that ATP onsumption proeeds after ATP synthesis is inhibited by pyoin Ri or KCN. ACKNOWLDGMNT We thank M. Kageyama for valuable advie. LITRATUR CITD Downloaded from http://jb.asm.org/ on September 4, 218 by guest
636 URATANI AND HOSHINO lism of sensitive ells of Pseudomonas aeruginosa. J. Biohem. 84:1373-1379. 1. Kaziro, Y., and M. Tanaka. 1965. Studies on the mode of ation of pyoin. I. Inhibition of maromoleular synthesis in sensitive ells. J. Biohem. 57:689-695. 11. Mithell, P. 1961. Coupling of phosphorylation to eletron and hydrogen transfer by a hemi-osmoti type. Nature (London) 191:144-148. 12. Shein, S. J., B. L. Kagan, and A. Finkelstein. 1978. Coliin K ats by forming voltage-dependent hannels in phospholipid bilayer membranes. Nature (London) 276:159-163. 13. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobi pseudomonads: a taxonomi study. J. Gen. Mirobiol. 43:159-271. 14. Stephen, H., and T. Stephen. 1963. Air-water, p. 88. In H. Stephen and T. Stephen (ed.), Solubilities of inorgani and organi ompounds, vol. 1. Pergamon Press, Oxford. 15. Tokuda, H., and J. Konisky. 1978. Mode of ation of oliin Ia: effet of oliin on the sherihia oli proton eletrohemial J. BACTRIOL. gradient. Pro. Natl. Aad. Si. U.S.A. 75:2579-2583. 16. Tokuda, H., and J. Konisky. 1979. ffet of oliin Ta and l on ion permeability of liposomes. Pro. Natl. Aad. Si. U.S.A. 76:6167-6171. 17. Uratani, Y. 1982. Dansyl hloride labeling of Pseudomonas aeruginosa treated with pyoin Rl: hange in permeability of the ell envelope. J. Bateriol. 149:523-528. 18. Uratani, Y., and W. A. Cramer. 1981. Reonstitution of oliin l into dimyristoylphosphatidylholine membrane vesiles. J. Biol. Chem. 256:417-423. 19. Uratani, Y., and M. Kageyama. 1977. A fluoresent probe response to the interation of pyoin Rl with sensitive ells. J. Biohem. 81:333-341. 2. Weiss, M. J., and S.. Luria. 1978. Redution of membrane potential, an immediate effet of oliin K. Pro. Natl. Aad. Si. U.S.A. 75:2483-2487. 21. Whooley, M. A., and A. J. MLoughlin. 1983. The protonmotive fore in Pseudomonas aeruginosa and its relationship to exoprotease prodution. J. Gen. Mirobiol. 129:986-996. Downloaded from http://jb.asm.org/ on September 4, 218 by guest