Effects of Paraquat on Escherichia coli: Differences between B

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JOURNAL OF BATERIOLOGY, Feb. 199, p. 68669 19193/9/6865$./ opyright 199, Amerian Soiety for Mirobiology Vol. 17, No. Effets of Paraquat on Esherihia oli: Differenes between B and K1 Strains JEFFREY W.

1 JOURNAL OF BATERIOLOGY, Feb. 199, p /9/6865$./ opyright 199, Amerian Soiety for Mirobiology Vol. 17, No. Effets of Paraquat on Esherihia oli: Differenes between B and K1 Strains JEFFREY W. KITZLER,t HISANORI MINAKAMI, AND IRWIN FRIDOVIH* Department of Biohemistry, Duke University Medial enter, Durham, North arolina 771 Reeived 3 July 1989/Aepted 7 November 1989 Esherihia oli B and K1 are equally suseptible to the bateriostati effets of aerobi paraquat, but they differed strikingly when the lethality of paraquat was evaluated. E. oli B suffered an apparent loss of viability when briefly exposed to paraquat, whereas E. oli K1 did not. This differene depended on the ability of the B strain, but not the K1 strain, to retain internalized paraquat; the B strain was killed on aerobi trypti soyyeast extrat plates during the inubation whih preeded the ounting of olonies. This differene in retention of paraquat between strains was demonstrated by delayed loss of viability, by growth inhibition, and by yanideresistant respiration after brief exposure to paraquat, washing, and testing in fresh medium. This differene was also shown by using [14]paraquat. This previously unreognized differene between E. oli B and K1 has been the ause of apparently ontraditory reports and should lead to some reevaluation of the pertinent literature. Beause it an inrease the intraellular prodution of,paraquat has been used to explore the biologial effets of this oxygen radial. Several fators influene the toxiity of paraquat to Esherihia oli, inluding uptake (9, 14), dioxygen (5, 7), an NADPH:paraquat oxidoredutase (8), a soure of eletrons (7), onentration of salts (J. W. Kitzler, Ph.D. thesis, Duke University, Durham, N.., 1988), availability of amino aids in the medium (4), and intraellular level of superoxide dismutase (7). The roles of these fators are as follows: the uptake system brings the diation into the ell, the oxidoredutase atalyzes its redution by NADPH, the soure of eletrons serves to maintain the supply of NADPH, salts inhibit the uptake of paraquat, oxygen is redued to by the paraquat monoation radial, and the is dismuted to H plus by the intraellular superoxide dismutase. The bateriostati effet of paraquat is overome by amino aids (4, 1), beause the dihydroxy aid dehydratase on the pathway of biosynthesis of branhedhain amino aids is extremely sensitive to (1,, 13). During the ourse of her thesis work, M. K. Brawn (Ph.D. thesis, Duke University, Durham, N.., 1984) found that K1 strains of E. oli appeared to be resistant to the lethal effet of paraquat, even though this viologen aused the usual inrease in yanideresistant respiration and no more than the usual adaptive inrease in the superoxide dismutase ontent of ells. Sine B and K1 strains of E. oli have been used interhangeably, it seemed essential to larify this differene. We now report that E. oli B and K1 differ strikingly in their retention of paraquat: the B strain retains it for many hours, while the K1 strain allows it to diffuse rapidly from the ell. This has major onsequenes when the assessment of loss of viability depends upon dilution, plating, inubation, and ounting of olonies. MATERIALS AND METHODS Media. TSY medium ontained 3 g of trypti soy broth and 5 g of yeast extrat (both from Difo Laboratories) per liter, adjusted to ph 7. with HI. Petri plates were prepared * orresponding author. t Present address: Department of Biohemistry, University of alifornia, Berkeley, A with TSY medium solidified with 1.5% BatoAgar. Vogel Bonner (VB) salts ontained.8 mm MgSO4, 9.5 mm itri aid, 44 mm KHPO4, and 17 mm Na(NH4)HPO4 (ph 7.). VB medium was VB salts plus.5% gluose and 1,ug of vitamin B1 per ml. Strains and manipulations. E. oli B (AT 968) and E. oli K1 (AT 3716), both of whih are wildtype strains, were ultured for 13 to 16 h at 37 in VB medium aerated at rpm. These overnight ultures were diluted to 1 x 17 to 3 x 17 FU/ml in the speified exposure medium; after inubation in Delong flasks at rpm in air at 37 with a ratio of flask volume to medium volume of 1:1 and under the onditions speified in the figure legends, the ell suspension was diluted by a fator of 14 to 15 with VB salts at 5 and plated onto TSY agar plates. olonies were ounted after 1 to 18 h at 37. The plates were inubated aerobially or in a oy hamber in an atmosphere of 85% N, 1% H, and 5% O. Effets of prior exposure to paraquat on subsequent behavior. (i) Growth effets. Overnight ultures in VB medium were entrifuged at 4, suspended in 1/5 volume of 5 mm potassium phosphate buffer (ph 7.) at 5, and diluted in the speified exposure medium with or without 1,um paraquat at 37 in air at rpm. After 3 min of exposure, the ells were olleted by entrifugation in the old, washed by suspension and entrifugation in the same medium without paraquat and gluose, and suspended in VB medium ontaining 1 jig of thiamine per ml to an A6. of.8 to.1. These ell suspensions were then inubated at 37 in Delong flasks shaken in air at rpm, and the A6 was monitored. (ii) yanideresistant respiration. ells from an overnight ulture in VB medium were suspended in fresh aerobi VB medium1. mm paraquat for 5 min at 37. These exposed ells were washed in VB salts and then suspended in aerobi VB medium at 37. After the intervals speified in the figures, 3.ml samples were taken, entrifuged, and suspended in fresh VB medium. This ell suspension was plaed under a lark eletrode at 37. The density of the ell suspension was adjusted to give 15% onsumption of the total dissolved per min. yanideresistant respiration was measured with 1. mm N in the medium.

VOL. 17, 199 EFFETS OF PARAQUAT ON B AND K STRAINS OF E. OLI 687.5 os 1 8 6 4O 4 3 <.5 l.oqoo B o. 75 _,g3.5 _4 /.5 n3 I I 8 6 4 O I 3 of Growth FIG. 1. Inhibition of ell growth by paraquat.

2 VOL. 17, 199 EFFETS OF PARAQUAT ON B AND K STRAINS OF E. OLI os O 4 3 <.5 l.oqoo B o. 75 _,g3.5 _4 /.5 n3 I I O I 3 of Growth FIG. 1. Inhibition of ell growth by paraquat. Paraquat was added to TSY medium at the following onentrations:. mm (line 1),.5 mm (line ),.1 mm (line 3),.5 mm (line 4), and 1. mm (line 5). E. oli B (A) and E. oli K1 (B) were inoulated, and growth was monitored turbidimetrially at 6 nm. (iii) Retention of ['4]paraquat. [14]paraquat was from Sigma hemial o. Its radiohemial purity was onfirmed by thinlayer hromatography. ells from midlog TSY ultures were olleted by entrifugation ( min in a Fisher miroentrifuge) and loaded with paraquat by suspension for 3 min at 37 with shaking in 5 mm potassium phosphate (ph 7.)1,um [14]paraquat.5% gluose. The ells were then olleted by min of entrifugation in the miroentrifuge and washed by brief suspension in neutral 5 mm potassium phosphate buffer followed by entrifugation. This ell pellet was suspended in neutral 5 mm potassium phosphate.5% gluose at 37. At the intervals speified in Fig. 8, samples were spun down and paraquat retained in the ell pellets was determined by liquid sintillation ounting in 4.5 ml of Aquasol. Paraquat leaked rapidly from E. oli K1, and gluose inreased this rate of leakage. In order to ahieve a reasonable level of retained paraquat in E. oli K1 at the start of the test inubation, we found it neessary to load the E. oli K1 with [14]paraquat in the absene of gluose. RESULTS Bateriostasis versus lethality. We have previously noted that the bateriostati effet of paraquat is at least 1,fold greater in the minimal VB medium than in a rih medium suh as TSY (1). The addition of paraquat to TSY medium inhibited growth to omparable degrees in E. oli B (Fig. 1A) and E. oli K1 (Fig. 1B). In ontrast, exposure to paraquat in VB medium followed by dilution, plating, inubation, and ounting of olonies made it appear that E. oli B was vastly more sensitive to the lethal effet of paraquat than was E. oli K1. olony ounts dropped sharply in h for E. oli B (Fig., line 1), while the K1 strain was unaffeted by 1 h of exposure under the same onditions (Fig., line 3). Sine the toxiity of paraquat is dioxygen dependent, the true situation was made lear when the TSY agar plates, used for ounting the surviving ells, were inubated anaer I I Al I FIG.. Effets of aerobi and anaerobi inubation of plates on the lethality of paraquat to E. oli B and K1. ells were exposed to.5 mm paraquat in aerobi VB medium at 37 for the indiated times. Samples were then diluted and plated on TSY agar plates, whih were inubated overnight either aerobially or anaerobially prior to the enumeration of olonies. Line 1, E. oli B on aerobi plates; line, E. oli B on anaerobi plates; line 3, E. oli K1 on aerobi plates; line 4, E. oli K1 on anaerobi plates. obially rather than in air. It then beame apparent that neither the B nor the K1 strain had lost viability during 1 h of exposure to.5 mm paraquat in the VB medium (Fig., lines 3 and 4). Instead, only the B strain lost viability, after being plated on the aerobi TSY agar. One ould explain these results by supposing that paraquat lethality ould be exerted in TSY but not in VB medium and that paraquat taken up by the ells in VB medium was lost from E. oli K1 but retained by E. oli B during dilution, plating, and inubation on the TSY agar. The previously reported ability of salts to inhibit the uptake of paraquat into E. oli (11) provided one way to test this supposition. E. oli B ells were exposed to.5 mm paraquat in 1. mm phosphate1.,um USO4 with different onentrations of Nal and were then diluted and plated onto TSY agar plates, whih were inubated aerobially or anaerobially. u+ was present during exposure to the paraquat, beause hevion and Kohen (3, 1) have reported that u+ or Fe+ is essential for the expression of the toxiity of paraquat. Raising the onentration of Nal from to 1 mm during the exposure to paraquat diminished the loss of viability whih was apparent on the aerobially inubated TSY plates (Fig. 3, lines 1 to 3). One again, anaerobi inubation of the plates showed that loss of viability ourred on the aerobi plates but not during the exposure to paraquat in the u+ontaining salt solution (Fig. 3, lines 4 to 6). Sine the elevation of the salt onentration dereases the uptake of paraquat (11), we surmise that the paraquat, taken up during the relatively brief exposure of the ells in the salt solution, was retained by E. oli B and exerted its lethal effet during overnight inubation on the aerobi TSY agar. The rate of paraquat uptake by E. oli B was examined as a funtion of the onentration of potassium phosphate in a gluoseontaining medium by using [14]paraquat. The rate of uptake was diminished by inreasing the salt onentra

$688 KITZLER ET AL. J. BATERIOL. o 8 \ 6 4 3 15 3 45 6 FIG. 3. Effets of Nal on the lethality of paraquat to E. oli B. ells were exposed to.5 mm paraquat1. mm potassium phosphate1.$

3 688 KITZLER ET AL. J. BATERIOL. o 8 \ FIG. 3. Effets of Nal on the lethality of paraquat to E. oli B. ells were exposed to.5 mm paraquat1. mm potassium phosphate1.,um us4 and the following onentrations of NaI: 1 mm (lines 1 and 4), 5 mm (lines and 5), and. mm (lines 3 and 6). At the intervals indiated, samples were withdrawn, diluted, and plated onto TSY agar. Plates were inubated aerobially (lines 1 to 3) or anaerobially (lines 4 to 6). tion (Fig. 4). This is in aord with previous results (11) whih were based on indiret measures of paraquat uptake, suh as the level of yanideresistant respiration. Differenes between B and K1 strains in TSY medium. Sine neither the B nor the K1 strain was killed by paraquat in VB medium but the B strain retained paraquat and then lost viability on the TSY plate, it was neessary to ompare the responses of these strains to paraquat in liquid TSY medium. In this experiment, the lethal effets exerted in the liquid medium were distinguished from those exerted on the plates by inubating the plates both anaerobially and 4 6 FIG. 4. Effets of potassium phosphate on the uptake of paraquat. E. oli B ells were exposed to 1 p.m [14]paraquat (ph 7.) at 37 in the following onentrations of potassium phosphate: 5 mm (line 1), 5 mm (line ), 75 mm (line 3), and 1 mm (line 4). Samples were removed at the indiated intervals for washing and sintillation ounting of retained [14]paraquat, as desribed in Materials and Methods. Relative onentration = onentration inside/onentration outside ~~~~~ FIG. 5. Lethality of paraquat in TSY medium. E. oli B (lines 1 and ) and K1 (lines 3 and 4) were inubated aerobially at 37 in TSY ontaining.5 mm paraquat. At the indiated times, samples were removed for dilution and plating on TSY agar. Plates were inubated anaerobially (lines 1 and 3) or aerobially (lines and 4). aerobially. The lethal effet of.5 mm paraquat on E. oli B required more than 4 h of exposure at 37 (Fig. 5, line 1). The B strain retained viability when exposed to paraquat in the liquid medium, when dilution and spreading on the agar surfae were followed by anaerobi inubation of the plates. In ontrast, the B strain rapidly lost viability when the plates were inubated aerobially, allowing the paraquat retained in the ells to exert its toxi effet on the plates (Fig. 5, line ). In the ase of E. oli K1, the lethal effet of paraquat appeared to be the same whether the plates were inubated aerobially or anaerobially (Fig. 5, lines 3 and 4), beause this strain does not retain paraquat and therefore suffered no further damage following dilution and plating. E. oli K1 also differed from the B strain by being more sensitive to the lethal effet of paraquat (Fig. 5, lines 1 and 3). Effets of retained paraquat on growth. The intraellular retention of paraquat ould lead to a lag in growth. Exposure to paraquat, followed by washing and suspension in fresh medium, might therefore impose a growth lag on E. oli B but not E. oli K1. Moreover, sine salts diminish paraquat uptake, loading the ells at a low salt onentration should exert a greater effet than loading at a high salt onentration (11). Prior exposure of E. oli K1 to paraquat in 1, 5, or 1 mm potassium phosphate buffer had no effet on its subsequent growth in VB medium, whereas adding paraquat to only 5,uM slowed growth in that medium (Fig. 6A). In ontrast, prior exposure of E. oli B to paraquat imposed a subsequent growth lag, and the salt onentration during the loading with paraquat was inversely proportional to this growth lag (Fig. 6B). Paraquat added to 5,uM to the VB medium did not immediately inhibit the growth of E. oli B, as it had in the ase of E. oli K1; however, a lear growth inhibition beame apparent over several hours of inubation (Fig. 6, line 5). This probably reflets both the intrinsially greater resistane to paraquat of E. oli B ompared with

VOL. 17, 199 EFFETS OF PARAQUAT ON B AND K STRAINS OF E. OLI 689 a.._ n E _. o ( I.8 1. B ~~~~~~~~~~4 7 1..8.6.4 o~~~~~~or.. 1 3 4 5 6 7 FIG. 6. Effets of retained paraquat on growth in VB medium. E. oli K1 (A) and B (B) were inubated for 3 minl at 37 and at ph 7.

4 VOL. 17, 199 EFFETS OF PARAQUAT ON B AND K STRAINS OF E. OLI 689 a.._ n E _. o ( I.8 1. B ~~~~~~~~~~ o~~~~~~or FIG. 6. Effets of retained paraquat on growth in VB medium. E. oli K1 (A) and B (B) were inubated for 3 minl at 37 and at ph 7. with.5% gluose in the following media: 1 mm potassium phosphate (lines 1 and 5) or 1 p.m paraquat (lines to 4) with 1 mm potassium phosphate (line ), 5 mm potassium phosphate (line 3), 1 mm potassium phosphate (line 4). The ells were then olleted by entrifugation, washed in 5 mm potassium phosphate at ph 7., and transferred to VB medium ontaining 1 p.g of thiamine per ml at 37, and growth was monitored at 6 nm. The data in line 5 were obtained with 5,uM paraquat in this growth medium. that of E. oli K1 and the ability of E. oli B to retain and aumulate paraquat. yanideresistant respiration. Intraellular paraquat an undergo repeated yles of enzymati redution followed by autoxidation. One measure of this redox yling is an inrease in yanideresistant respiration, whih ould be used to further explore the differene between the B and K1 strains with respet to the retention of paraquat. Five minutes of exposure of E. oli B to 1. mm paraquat imposed an inrease in yanideresistant respiration whih was retained for at least 3 h after washing (Fig. 7). E. oli K1, in ontrast, ompletely lost the extra yanideresistant respiration during 9 min of inubation following washing. Diret measurement of paraquat retention. [14]paraquat was used to measure the retention of paraquat in the B and K1 strains. Paraquat was largely retained by E. oli B in a gluosephosphate medium (Fig. 8, line 1), while the K1 strain rapidly lost paraquat under the same onditions (line ). We noted (data not shown) that gluose inreased the rate of leakage of paraquat from E. oli. This effet may be due to the redution of the paraquat diation to the monoation radial in the presene of this gluose and to the relatively rapid loss of the monoation from the ells. In any ase, the loss of [14]paraquat from E. oli K1 was so rapid in the presene of gluose that we found that most of it was lost from the ells during the few minutes needed to wash the ells following loading and prior to the test inubation (Fig. ~.(A L a) l 3 4 FIG. 7. Effets of retained paraquat on yanideresistant respiration. ells were loaded with paraquat by inubation for 5 min at 37 in VB medium ontaining 1. mm paraquat and were olleted by entrifugation, washed in VB salts, and suspended in fresh VB medium at 37. At the indiated times, samples were taken for measurement of yanideresistant respiration, as desribed in Materials and Methods. Line 1, E. oli B; line, E. oli B ontrol not loaded with paraquat; line 3, E. oli K1; line 4, E. oli K1 ontrol not loaded with paraquat. 8). This problem was avoided by loading E. oli K1 in the absene of gluose. DISUSSION In our early studies of the effets of paraquat on E. oli (6, 7), we used E. oli B and ounted surviving ells by plating them on TSY agar following exposure to the viologen. We reognized that the removal of paraquat from E. oli B was diffiult; the ells needed to be washed twie in old TSY medium and then inubated in ieold minimal medium for 9 min prior to further testing to allow the paraquat to diffuse from the ells (6). Furthermore, when demonstrating that dioxygen was essential for the expression of the lethality of paraquat, we used anaerobi onditions throughout the exposure, dilution, plating, and inubation of the plates (7). We did not, however, suspet that the aerobi loss of viability observed following the exposure of E. oli B to i a,o L. S 4 5r 4 3 1IF FIG. 8. Retention of ['4]paraquat. E. oli B (line 1) and K1 (line ) were loaded with paraquat aerobially, washed, and monitored for retention of paraquat, as desribed in Materials and Methods.

69 KITZLER ET AL. J. BATERIOL. paraquat ourred during the many hours of inubation of the plates and not during the relatively brief exposure in the liquid medium.

5 69 KITZLER ET AL. J. BATERIOL. paraquat ourred during the many hours of inubation of the plates and not during the relatively brief exposure in the liquid medium. We were also unaware of the signifiane of having hosen E. oli B rather than E. oli K1. Sine the signifiane of these fators was not known to us, we unknowingly set the stage for ontroversy. Thus, Fee et al. (4) hose to use E. oli K1 and saw a bateriostati effet of paraquat but no loss of viability. This was partially due to their use of a highsalt medium, as previously disussed (11), but was probably mainly due to the rapid loss of paraquat from the K1 strain during dilution and plating, sine Yonei et al. (15) observed the lethality and mutageniity of paraquat with a B strain. We have now demonstrated that E. oli B but not E. oli K1 retains paraquat, and we have done so with several strategems. Although the B and K1 strains were equally suseptible to the bateriostati effets of paraquat, they showed dramati differenes when exposed to paraquat for an hour or two and then washed prior to testing for the effets of that exposure. When tested for its ability to generate olonies on aerobi TSY agar, the K1 strain appeared unaffeted, whereas the B strain appeared to have suffered a loss of viability. The true situation beame apparent only when the agar plates were inubated anaerobially, thus preventing the oxygendependent toxiity of retained paraquat from being expressed on the plates. When tested for its level of yanideresistant respiration, only the B strain showed the inrease, beause of the redox yling of retained paraquat. Similarly, when tested for ability to grow in fresh liquid medium, only the B strain exhibited a growth lag, as a onsequene of its prior exposure to paraquat. The nature of the medium also exerts a striking effet on the lethality of paraquat. Thus, neither E. oli B nor E. oli K1 suffered any loss of viability over 1 h in.5 mm paraquat when the exposure was in VB medium (Fig. ). In ontrast, the K1 and B strains suffered losses of viability after lags of and 4 h, respetively, when the exposure to paraquat was in TSY medium (Fig. 5). Failure to reognize this profound medium effet is another soure of onfusion in the literature dealing with the toxiity of paraquat. The very different abilities of the B and K1 strains of E. oli to retain paraquat should be kept learly in mind when experiments to explore the toxi effets of this viologen are being designed. It may be that similar differenes in the retention of paraquat may be seen when other types of miroorganisms and different types of mammalian ells are ompared. ould the relative seletivity of paraquat for lung ells be a refletion of the retention of paraquat in this tissue? The basis for the retention of paraquat in E. oli B remains to be explored. AKNOWLEDGMENTS This work was supported by researh grants from the National Siene Foundation, The ounil for Tobao ResearhU.S.A., In., and the Amerian aner Soiety. LITERATURE ITED 1. Brown,. R., and R. L. Seither Oxygen and redoxative drugs: shared toxiity sites. Fundam. Appl. Toxiol. 3:914.. Brown,. R., and F. Yein Dihydroxyaid dehydratase: the site of hyperbari oxygen poisoning in branhhain amino aid biosynthesis. Biohem. Biophys. Res. ommun. 85: hevion, M A sitespeifi mehanism for free radial indued biologial damage: the essential role of redoxative transition metals. Free Radial Biol. Med. 5: Fee, J. A., A.. Lees, P. L. Bloh, P. L. Gilliland, and. R. Brown Oxygen stasis of baterial growth: analogy between the stasis of E. oli by hyperbari oxygen and by aerobi paraquat. Biohem. Int. 1: Fisher, H. K., and G. Williams Paraquat is not bateriostati under anaerobi onditions. Life Si. 19: Hassan, H. M., and I. Fridovih Regulation of the synthesis of superoxide dismutase in E. oli: indution by methyl viologen. J. Biol. hem. 5: Hassan, H. M., and I. Fridovih Superoxide radials and the oxygen enhanement of the toxiity of paraquat in Esherihia oli. J. Biol. hem. 53: Hassan, H. M., and I. Fridovih Paraquat and Esherihia oli: mehanism of prodution of extraellular superoxide radial. J. Biol. hem. 54: Kao, S. M., and H. M. Hassan Biohemial haraterization of a paraquattolerant mutant of Esherihia oli. J. Biol. hem. 6: Kitzler, J., and I. Fridovih The effets of paraquat on Esherihia oli: distintion between bateriostasis and lethality. J. Free Radials Biol. Med. : Kitzler, J., and I. Fridovih Effets of salts on the lethality of paraquat. J. Bateriol. 167: Kohen, R., and M. hevion ytoplasmi membrane is the target organelle for transition metal mediated damage indued by paraquat in Esherihia oli. Biohemistry 7: Kuo,. F., T. Mashino, and I. Fridovih a,pdihydroxyisovalerate dehydratase: a superoxide sensitive enzyme. J. Biol. hem. 6: Simons, R. S., P. S. Jakett, M. E. W. arroll, and D. B. Lowrie Superoxide independene of paraquat toxiity in Esherihia oli. Toxiol. Appl. Pharmaol. 37: Yonei, S., A. Noda, A. Tahibana, and S. Akasaka Mutageni and ytotoxi effets of oxygen free radials generated by methylviologen (paraquat) on Esherihia oli with different DNArepair apaities. Mutat. Res. 163:15.

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