Inhibition, but Not Uncoupling, of Respiratory Energy Coupling of Three Bacterial Species by Nitrite

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1980, p /80/ /08$02.00/0 Vol. 144, No. 3 Inhibition, but Not Uncoupling, of Respiratory Energy Coupling of Three Bacterial Species by Nitrite JAMES B. RAKEt AND ROBERT G. EAGON* Department ofmicrobiology, University of Georgia, Athens, Georgia The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitriffying conditions. Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P. denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption. Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed. The lack of enhanced proton permneability due to nitrite was verified via passive proton perneability assays. The H+/O ratio of P. aeruginosa increased when growth conditions were changed from aerobic to denitrifying. This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P. aeruginosa. Nitrite has been a widely used, and recently controversial, additive in the meat processing industry (1). Nitrite functions as a color enhancer and fixative, and it imparts a distinctive flavor to cured meats. It also is considered to have antiputrefactive properties, particularly with respect to Clostridium botulinum and Clostridium perfringens (17, 18, 24). Surprisingly, little is known of the physiological basis for the inhibition of clostridia by nitrite (16). Further, almost no data exist for the physiological basis of the inhibition of aerobic bacteria by nitrite, even though such inhibition has been noted (2, 7, 24, 25). We previously reported the inhibition of active transport, respiration, and oxidative phosphorylation of Pseudomonas aeruginosa by nitrite (21). Comparison of nitrite inhibition of P. aeruginosa, Escherichia coli, and Streptococcus faecalis demonstrated sensitivity of protonlinked active transport to nitrite, but group translocation of sugar via the phosphoenol-pyruvate:phosphotransferase system was resistant to inhibition by nitrite (21, 28). These results indicated an inhibitory action by nitrite at the level of the membrane respiratory system, perhaps by a specific effect on the cytochrome chain (21, 25, 28). Meijer et al. (14) recently reported that the reduced respiratory coupling efficiency of Paracoccus denitrificans noted in the presence of low levels of nitrite was due to an uncoupler-like effect. Uncoupling was expressed as reduced prot Present address: Smith Kline & French Laboratories, Philadelphia, PA ton-to-nitrite stoichiometry, reduced proton-tooxygen stoichiometry, and increased proton resorption rates during pulsed-oxidant proton extrusion assays with P. denitrificans grown under denitrifying conditions. As uncoupling was consistent with our earlier data on nitrite inhibition of membrane-level energy coupling (21, 28), we attempted to confirm the report of Meijer et al. (14) with P. denitrificans and to extend the findings to other oxidative bacteria. Instead, as will be shown herein, we observed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. MATERIALS AND METHODS Organisms and culture conditions. P. aeruginosa PAO (formerly Holloway strain 1), Pseudomonas putida ATCC 12633, and P. denitrificans ATCC were maintained on nutrient agar slants at room temperature. P. aeruginosa was grown on glucose (11 mm) in a broth medium by using the basal salts buffer solution previously described (4). P. putida was grown as above, but with 25 mm sodium succinate as a substrate. P. denitrificans was grown in the same medium as P. putida but enriched with 1% tryptone and 0.1% yeast extract. All carbohydrates were added to autoclaved basal salts as separately filter sterilized, 100- fold concentrated stock solutions. Starter cultures were grown aerobically at 30 C on petri dishes of the appropriate medium solidified with 1.5% agar. The growth from an 18-to-24-h plate was washed off into 20 ml of sterile basal salts buffer, diluted appropriately, and used for inoculation of broth cultures. Aerobic cultures were grown in 200 ml of broth in 1-liter Erlenmeyer flasks shaken at 250 rpm at 30 C. 975

2 976 RAKE AND EAGON Denitrifying cultures of P. aeruginosa and P. denitrificans were grown in completely filled 250-ml flasks of the appropriate medium supplemented with 1% KNO3 and 0.1% yeast extract. The flasks were sealed with rubber stoppers pierced by two sealable ports through which nitrogen was initially bubbled to deoxygenate the medium. The flasks contained a magnetic stirrer and were incubated at 300C with gentle stirring. For proton extrusion experiments, cultures were harvested in late exponential phase by centrifuging at 10,000 x g for 10 min at 250C, washed twice with a solution of 50 mm KCI, 2 mm MgCl2, and 0.5 mm piperazine-n,n'-bis(2-ethanesulfonate) (PIPES) buffer, ph 6.8 (W buffer), and then suspended to 50 mg (wet weight) per ml in W buffer. Cells were aerated by shaking at 30 C until used. For determination of passive proton permeability, cells were harvested as above, but at 40C and with 50 mm KCI, 2 mm MgC12, 100 IU of bovine erythrocyte carbonic anhydrase per ml (EC ; Sigma Chemical Co., St. Louis, Mo.), 1 mm glycylglycine buffer, ph 6.8 (HB buffer) (9) for washing and suspension of cells. Washed cells were kept on ice until used. Proton extrusion assays. Proton extrusion stoichiometry was measured by the technique of Mitchell and Moyle (15) modified after Scholes and Mitchell (22) and Rice and Hempfling (19). The 3.2-ml reaction vessel was water jacketed at 300C and pierced by three ports, two at the sides for an oxygen electrode (model 53; Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio) and a ph probe (model 2885; Markson Science, Inc., Del Mar, Calif.), and a female standard taper joint at the top which was closed during use by a standard taper stopper with a central capillary for additions. The cell was stirred by a miniature Tefloncovered stirrer. For assays, 1.25 ml of the washed cell suspension, 0.75 ml of 1 M KSCN, 0.05 ml of carbonic anhydrase (10,000 IU/ml), and 2.95 ml ofw buffer were added to the reaction chamber and sparged with tank argon to <1% saturation of oxygen, and the top stopper was seated to reduce the cell volume to 3.2 ml. After the ph trace (expanded scale meter with a 10 mv chart recorder set to give 0.15 ph unit full scale, with noise, typically, of <0.005 ph unit) had stabilized, the reaction mixture buffering capacity was calibrated by the addition of 25-nmol samples of HC1. All solutions were deoxygenated before use by bubbling with tank argon. After each addition of oxidant, the ph was titrated back to 6.90 with N HCI or 0.01 N KOH, or both. For determination of proton-to-oxygen stoichiometry, oxygen was added as 5-pl aliquots of buffer saturated with pure 02 at 300C (9.03 nmol of 0). For proton-to-nitrite ratios, nitrite was added as appropriate volumes of 10 mm deoxygenated KNO2. Cell suspensions were routinely recalibrated with HCI after each addition of inhibitor. Carbonyl cyanide-m-chlorophenyl hydrazone (CCCP; Sigma Chemical Co., St. Louis, Mo.), when used, was added from a 10 mm solution in absolute ethanol. Proton pulses were corrected for resorption by extrapolating the resorption phase of the proton pulse back to zero time (19). Proton resorption half-time was also determined directly as the time between addition of the oxidant pulse and the collapse of the J. BACTERIOL. extrusion peak to one-half of its final equilibrium value. Both of these graphical procedures were empirically verified by comparison with the more rigorous semilogarithmic plotting procedures of Scholes and Mitchell (22). The ph electrode used had a response half-time of about 1.7 s. Although this did not significantly effect measurements of proton resorption time (22), the apparent proton extrusion time of the bacteria (4 to 10 s in various experiments) was not readily distinguishable from the electrode response time, and thus is not reported. Passive proton permeability. The permeability of resting cells of the three bacteria to exogenously supplied protons was determined by the technique of Harold and Baarda (9). The apparatus as described above for proton extrusion was used with the recorder scale expansion decreased to 1 ph unit full scale (7.00 to 6.00 ph). For assays, the vessel was filled with 1.25 ml of cell suspension (i.e., 2.5 mg/ml of dry cell mass) plus 3.75 ml of HB buffer. The reaction vessel was sparged with argon which reduced the oxygen content to approximately 0% saturation, and it also removed C02 from solution, which was further facilitated by the action of carbonic anhydrase, thus increasing the ph (22). When the ph of the reaction mixture reached about 6.9, the cell was sealed, and the ph was allowed to equilibrate to a constant ph before the assay. After a stable base line was reached, a sample was pulsed with a quantity of HCI adequate to give a 0.6 to 0.8 ph unit deflection. The quantity of HCI for each day's batch of cells was determined by a preliminary titration and was typically 300 to 500 nmol of HCI. In the range used, there was always a linear relationship between added HCI and ph deflection. Proton permeability was determined by pulsing the cell suspension with HCI and determining the rapidity of equilibration of the added protons with the intracellular buffering capacity as determined by the rate of collapse of the artificially imposed proton gradient. RESULTS Proton extrusion. When pulsed with small quantities (i.e., 9.03 nmol) of oxygen, aerobically grown cells of P. aeruginosa rapidly consumed the oxygen, concomitantly extruding protons. In the absence of inhibitors, the addition of a small quantity of oxygen caused the immediate acidification of the external medium as cellular respiration drove proton extrusion. Thus, a nmol oxygen pulse caused the extrusion of 44.5 nmol of H+, and the proton-to-oxygen ratio was This initial ph differential decayed exponentially with a half-time of 75 s (first-order rate constant, s-'). The addition of small amounts of nitrite caused the proton-to-oxygen ratio to first increase and then to fall, and the proton resorption half-time increased with increasing nitrite concentration. The results from this series of experiments with P. aeruginosa are summarized in Fig. 1. After the sharp peak in the proton-to-oxygen ratio at low (12.5,uM) nitrite concentrations, the

3 VOL. 144, 1980 o -120 I 11tl/ H+/0 U) /M NO2 FIG. 1. Proton-to-oxygen stoichiometry andproton resorption half-time (t4/2) of aerobically grown P. aeruginosa versus nitrite concentration. Proton extrusion was measured after the addition of9.03-nmol oxygen pulses. The assays from which these data were compiled were from the results of a consecutive set of assays on the same cell suspension, which were as follows: control, no nitrite added; 12.5 pm nitrite (added a two 20-nmol additions); 25 M nitrite (preceding plus 40 nmol of KNO2); and 200 pm nitrite (preceding plus 80 and plus 480 nmol of KNO2). At concentrations above 200X pm nitrite, it was not possible to accurately deternine a resorption time. proton-to-oxygen ratio fell smoothly to 1.35 at 1 mm nitrite. The proton resorption time rose erratically up to 200,uM nitrite, above which the extent of resorption was too low to allow accurate determination of a half-time. The response of aerobically grown P. aeruginosa to nitrite was clearly different from that of an authentic uncoupler, CCCP (Fig. 2). Increasing concentrations of CCCP caused the protonto-oxygen ratio to fall, but with concomitant decreases in the proton resorption half-time. Above about 4,uM CCCP the resorption halftime became essentially constant. Thus, 5 AM CCCP was routinely used as a fully uncoupling concentration. The uncoupler caused decreased proton-to-oxygen ratios at higher concentrations due to such fast resorption that large portions of the extruded protons were resorbed during the NITRITE INHIBITION OF BACTERIA 977 response time of the electrode and thus were not measured. Similar results were found when aerobically grown P. putida was used (data not shown). Nitrite caused the proton-to-oxygen ratio to fall, whereas the proton resorption half-time increased nearly proportionately with the nitrite concentration. These responses to nitrite were clearly different from the effect of the uncoupler CCCP and also the uncoupling effect on P. denitrificans as reported by Meijer et al. (14). We therefore examined P. denitrificans to determine if the nitrite effect was specific to that organism. As with the two Pseudomonas species, the respiration-dependent proton extrusion of aerobically grown P. denitrificans was inhibited by nitrite (Fig. 3), but not in a manner consistent with nitrite acting as an uncoupler. We further examined the possibility that the previously reported uncoupling effect of nitrite was a specific feature of cells grown under conditions of anaerobic respiration (denitrifying conditions). Cells of P. denitrificans grown under denitrifying conditions could use either oxygen or nitrogen oxides as terminal electron acceptors driving respiratory proton extrusion (Fig. 4a and b). The added nitrite was consumed by the cells, presumably reduced to N2 gas. Thus, it was not possible to speak of a concentration of nitrite in solution as was done above with aerobically grown cells which, being unin- 0 +' I H/50-40 r 30 t12- resorption 20 --_,~m COOP FIG. 2. Proton-to-oxygen stoichiometry andproton resorption half-time (t112) of aerobically grown P. aeruginosa versus CCCP concentration. Proton extrusion was measured after the addition of 9.03-nmol oxygen pulses to the following cell systems: control, no CCCP; 0.5 p CCCP; 2 pm CCCP; 4 um CCCP; and 16 WM CCCP

4 978 RAKE AND EAGON duced, were unable to use nitrite and other nitrogen oxides as terminal electron acceptors for anaerobic respiration (26). Rather, data are presented as the response of cells which have a given history of nitrite pulses. Nitrite-linked res t 0 H+/O 0 +" t1/ I L200 i LM NO2 FIG. 3. Proton-to-oxygen stoichiometry andproton resorption half-time (t1/2) of aerobically grown P. denitrificans versus nitrite concentration. H+/O 7.29 t12 59S 1/2 H/NO 3.60 ~ ~t12 piration was less efficient than oxygen-linked respiration; the initial H+/NO2- ratio was 3.60 as compared with the initial H+/O ratio of 7.29, but the resorption half-times were comparable. Under these conditions, Meijer et al. (14) had reported that a series of five consecutive nitrite pulses totalling 110 nmol of nitrite (in a reaction volume comparable to that used here) sequentially decreased the H+/NO2 ratio of P. denitrificans and simultaneously dramatically reduced the proton resorption half-time. A single 48-nmol pulse of nitrite similarly depressed the H+/O ratio and its resorption half-time. Our data do not confirm their report. Although subsequent nitrite pulses (Fig. 4c and d) did show a reduced H+/NO2, the proton resorption halftime did not decrease, but rather increased somewhat. Further, a subsequent oxygen pulse (Fig. 4e) which followed eight nitrite pulses (a total of 205 nmol) showed no significant change of proton-to-oxygen stoichiometry or proton resorption half-time relative to the initial control (Fig. 4a). These cells, however, reacted as expected when treated with CCCP, an authentic uncoupler (Fig. 4f and g). Cells of P. aeruginosa grown under denitrifying conditions gave similar results to those above (Fig. 5). Pulses of nitrite did not cause decreased proton resorption half-time of subsequent nitrite or oxygen pulses. The H'/NO2 ratio was depressed in subsequent nitrite pulses (Fig. 5d), but, interestingly, the H+/O ratio rose H+/NO ,2 69S J. BACTERIOL. b lonmiiole 40nmole lonmole NC H+ 9.03nmole 0 NO0 lonmole N02 25nmole NO2 25nmole N0-25 nmole N0- t H+/N0z 1.67 H+/ S 11/2 79S H+/ d 1/2 26 S H+/NO /2 35 S SOnmole 50nmole 9.03nmole nmole 0 20nmole N0- N0- NOi FIG. 4. Proton extrusion of P. denitrificans grown anaerobically with nitrite as the terminal electron acceptor. These cells used both oxygen (a, e, and t) and nitrite (b, c, d, and g) as terminal electron acceptors driving proton extrusion. Traces a through e represent consecutive assays with a single cell suspension. Intervening electron acceptor pulses are indicated by upward arrows without accompanying traces. Traces f and g are consecutive assays with a separate cell suspension prepared from the same cell batch as a through e. CCCP was added to 1 pm 120 s before f. t,12, Half-time.

5 VOL. VNITRITE 144, 1980 INHIBITION OF BACTERIA 979 after the initial 10-nmol nitrite pulse (Fig. 5c), similar to the effect of small nitrite additions previously noted with aerobically grown P. aeruginosa. Table 1 summarizes the uninhibited protonto-oxidant stoichiometries of the organismn studied under both aerobic and denitrifying growth conditions (P. putida does not denitrify). P. putida and P. aeruginosa grown aerobically had similar H'/O ratios of approximately 5.5, but P. denitrificans had a higher ratio of about 7.5. The proton-to-oxygen stoichiometry did not change significantly when P. denitrificans was grown under denitrifying conditions; but, in contrast, the change from aerobic to denitfing growth significantly (P < 0.01, t-test) increased the proton-to-oxygen ratio of P. aeruginosa. The proton-to-nitrite stoichiometry ofboth denitriying bacteria was similar to and less than the corresponding proton-to-oxygen ratio. Passive proton permeability. The cell membrane of intact resting bacteria is relatively impermeable to protons. When an artificial proton gradient is created across the cell membrane by a pulse of exogenously supplied acid, there is only a slow inward equilibration of the protons. Thus, the acidification of the bulk medium due to a 300-nmol HCI pulse was only slowly dissipated by protons permeating the membrane to be consumed by the intracellular buffering capacity of a suspension of aerobically grown P. denitrificans (Fig. 6a). In the presence of CCCP, a proton ionophore, equilibration across the membrane was far more rapid (Fig. 6b). As with the decay of a respiratory proton pulse, the approach to equilibrium was an exponential function. The bulk medium ph did not return completely to its initial value because the internal buffering capacity ofthe cell suspension most likely was not adequate to accommodate the entire proton pulse. When CCCP was added before the HCI pulse (Fig. 6c), the extent of the H ,2 99S 7.77,t1/2 91S lonmole t lonmole) H+/NO t,292S b 9.03nmoe 0 lonole NOj ionmole NO nmole 0 4 min H / lonmole H+ I HOnmoe l 1/2 102 S t- 30nmok NOi 9.03 nmole 0 FIG. 5. Proton extrwion of P. aerugiosa grown anaerobically with nitrite as the terminal electron acceptor. These cells could use both oxygen (a, c, and e) and nitrite (b and d) as terminal electron acceptors drivingproton extrusion. The traces are consecutive assays with a single ceu suspension. Intervening electron acceptor pulses are indicated by upward arrows without accompanying traces. TABLE 1. Uninhibitedproton-to-oxidant stoichiometrya Organism Growth conditions H+/O ratio H /NO2 ratio P. putida ATCC Aerobic 5.40 ± 0.54; n =6 P. aeruginosa PAO Aerobic 5.52 ± 0.94; n - 7 Denitifying 7.25 ± 0.73; n i0.47; - n - 4 P. denitrificans ATCC Aerobic 7.58 ± 0.31; nf-3 Denitrifying 7.47 ± 0.96; n ± 0.18; n - 3 a Mean plus or minus one standard deviation; n - number of trials.

6 980 RAKE AND EAGON J. BACTrERIOL. 0.2pH _ e9 J-HCI 300 nmolehci HCI CCCP HCI CCCP CI KNO * HC 2min f t 1 CCCP 1 t~ KNO2 HCI HCI KNO2 HCI FIG. 6. Passive proton permeability of aerobically grown P. denitrificans. At the time indicated, 300 nmol of HCI was added, and the ph displacement was recorded. (a) Control; (b) 5 um CCCP added 60 s after HCI pulses; (c) 5p CCCP added 150 s before HClpulse; (d) KNO2 (6.25 pmper addition) added 60 and 120 s after HCl pulse; (e) 625 pm KNO2 added 90 s after HCl pulse and 5 p CCCP added 190 s after HCl pulse; (t) 625 p KNO2 added 100 s before HCI; (g) 5 p CCCP added 120 s before HCI and 625 p KNO2 added 90 s after HCI; (h) 625 p KNO2 added 120 s before HCI and 5 p CCCP added 180 s after HCI. ph deflection was reduced, and the medium bulk ph rapidly returned to the same equilibrium seen when CCCP was added after the HCO pulse. In contrast, nitrite in purportedly uncoupling concentrations had no effect on the rate of equilibration of added protons with the cytoplasmic buffering capacity (Fig. 6d). The addition of much larger amounts of nitrite (Fig. 6e), however, gave rise to a rapid, slight alkalinization of the bulk medium, but the external ph did not reach equilibrium with the cytoplasmic buffering capacity, and a significant ph gradient still existed across the membrane, as shown by the subsequent influx of protons when CCCP was added. Addition of 2,000 nmol of nitrite had no effect on an unpulsed cell suspension (Fig. 6f), and the extent and time course of a subsequent HCO pulse in the presence of nitrite were not distinguishable from the control (Fig. 6a). By contrast, the addition of CCCP to an unpulsed cell suspension (Fig. 6g) caused alkalinization of the medium, apparently due to the collapse of the ph of the resting cell suspension (much lower in magnitude than the ph artificially imposed by the HCO pulse). When this CCCP-pretreated cell suspension was pulsed with HCO and subsequently 2,000 nmol of nitrite was added, the rapid alkalinization effect was still noted, indicating that the alkalinization due to large nitrite additions was separable from the presence of a large proton gradient across the membrane. Finally, the pretreatment of the cell suspension with nitrite (Fig. 6h) had no effect on the formation or magnitude of a proton gradient by the subsequent HCO pulse as shown by the effect of CCCP. DISCUSSION Respiratory energy coupling has not been extensively studied in the genus Pseudomonas. We were unable to locate previously published values for the respiratory proton stoichiometry of P. aeruginosa. The current classification of Pseudomonas (3) places P. aeruginosa and P. putida closely within the genus. It was thus not surprising that their proton-to-oxygen ratios were similar. Pseudomonas ovalis has been reported to have a proton-to-oxygen ratio of 6.6 at ph 7.0 (10). Thus, since P. ovalis is considered a synonym of P. putida (3), our data are slightly lower than previously reported. The proton-tonitrite ratio of 3.4 of P. aeruginosa when grown under denitrifying conditions compares well with the H+/NO2 ratio of 3.7 reported for P. denitrificans by Kristjansson et al. (11) as does the H+/O ratio (7.2 versus 7.5). The proton-to-oxygen stoichiometry of P. aeruginosa showed a statistically significant rise when growth conditions were shifted from aerobic to denitrifying. This suggests induction of an additional coupling site in the electron transport

7 VOL. 144, 1980 chain of P. aeruginosa which should be reflected in changes of the cellular cytochrome content during denitrifying growth. Substantial changes in the cytochromes of P. aeruginosa have been noted upon the change of growth conditions from aerobic to denitrifying (20; J. J. Rowe and R. G. Eagon, unpublished observations). Further studies will be required, however, to assign coupling functions to these changes. A number of estimates of the proton-to-oxygen stoichiometry of P. denitrificans are available (5, 11-14, 22). These studies report H+/O ratios (endogenous substrate) of aerobic cultures to be from 6.7 to 8.5. Our currently reported ratio for this organism is in excellent agreement with these previous values. P. denitrificans grown anaerobically with nitrate as terminal oxidant has been reported to have H+/O and H+/NO2 ratios of 7.3 to 7.5 and 3.7 to 6.4, respectively (11, 14). Our values agree well with these previous values, except for the uninhibited proton-to-nitrite stoichiometry reported by Meijer et al. (14). Nitrite anion is toxic to a variety of microorganisms (2, 24, 26, 27). Nitrite toxicity is strongly modulated by ph, with the minimum inhibitory concentration increasing as the antilog of ph in the range of ph 5 to 7 (2); thus, published inhibition values for various microorganisms will vary widely, depending on the ph of the experiment. At a ph near neutrality, nitrite concentrations of 10 to 100 mm generally are inhibitory, In our previous work, active transport and respiration of P. aeruginosa and E. coli were substantially inhibited by 10 mm nitrite, but 50 to 100 mm was required for complete inhibition. By contrast, 10 mm nitrite completely inhibited ATP synthesis and caused rapid loss of intracellular ATP pools (21, 28). The inhibitory effect of nitrite is reflected in its effect on the molar growth yield of bacteria. Nitrite reduced the molar growth yield of Enterobacter aerogenes, C. perfringens, P. denitrificans, and Propionibacterium pentosaceum (23). This inhibition was not a reflection of reduced growth, but of reduced efficiency of conversion of substrate to cell mass. This reduced efficiency was attributed to loss of site 1 phosphorylation activity (25) of P. denitrificans grown in nitrite-limited anaerobic continuous culture. This conclusion was modified (14) to imply uncoupler activity of nitrite rather than a site-specific energy coupling defect. Our present data indicate that nitrite significantly inhibited respiratory proton extrusion at as low as 50,uM and that maximal inhibition of proton extrusion was observed by approximately 1 mm nitrite at ph 6.9. However, the inhibition NITRITE INHIBITION OF BACTERIA 981 due to nitrite was not total. For the three bacteria studied, the H+/O ratio plateaued at approximately 1.3 (P. aeruginosa), 4.4 (P. putida), and 5.1 (P. denitrificans). By contrast, the resorption rate, to the extent that it could be measured, decreased steadily without plateauing. As both ATP synthesis and hydrolysis and active transport are in thermodynamic equilibrium with the transmembrane proton gradient (8), it is likely that the effects which we previously documented are secondary sequelae of a primary defect caused by nitrite upon respiratory proton extrusion. ATP synthesis is completely inhibited at 10 mm nitrite, whereas 50 mm nitrite is required to totally inhibit active transport (21, 28); this may reflect the requirement of ATP synthesis for a more energetic proton gradient than that which will support active transport. Quantitation of the transmembrane proton gradient in the presence of nitrite would be useful to further clarify this point. (A curious phenomenon observed for P. aeruginosa was that a low concentration of nitrite caused the H+/O ratio to increase in contrast to a decreased H+/O ratio at higher concentrations of nitrite. The increase in the H+/O ratio was statistically significant for a series of experiments with single batches of cells. At present the experimental data do not provide us with an explanation for this phenomenon.) It was evident with the bacteria used in this study that the effect of nitrite was not that of an uncoupling agent. Uncoupling refers to an electrochemical event in which the proton permeability of the cytoplasmic membrane is increased, thus dissipating the proton gradient and preventing the coupling of respiration with ATP synthesis. Classical uncoupling agents such as 2,4-dinitrophenol and CCCP act as proton ionophores by being soluble in the membrane as the undissociated weak acid form and as the dissociated base (6). In the data shown here, no evidence for increased membrane permeability was found at nitrite levels which were strongly inhibitory of proton extrusion. Our results, therefore, are not in agreement with the findings of Meijer et al. (14), who concluded that nitrite inhibition was due to uncoupling. However, since the strain of P. denitrificans used in our study was different from that used by Meijer et al. (14), the possibility remains that the effect which they reported is strain specific. In our hands, nitrite caused a decrease in membrane proton permeability. Whether this effect arose by inhibition of membrane-level proton consuming components (e.g., the membrane ATPase, proton-dependent transport carriers, or the flagellar rotor [8]) or by a generalized de-

8 982 RAKE AND EAGON crease in proton permeability of the bulk membrane is unknown. Similarly, we do not now understand the mechanism by which nitrite decreased the respiratory proton stoichiometry. We previously demonstrated that bacterial respiration rate was not inhibited by nitrite below 3 to 5 mm (21; unpublished observations). Thus, inhibition of electron flow per se is unlikely. ACKNOWLEDGMENTS This investigation was supported by National Science Foundation research grant PCM We thank W. R. Finnerty, I. L. Roth, H. B. Howe, and T. J. Kerr for the use of some of the equipment used in this study. We also thank B. D. Ensley for his suggestions concerning the proton extrusion assays. LITERATURE CITED 1. Binkerd, E. F., and 0. E. Kolari The history and use of nitrate and nitrite in the curing of meat. Food Cosmet. Toxicol. 13: Castellani, A. G., and C. F. Niven, Jr Factors affecting the bacteriostatic action of sodium nitrite. Appl. Microbiol. 3: Doudoroff, M., and N. J. Palleroni Pseudomonas, p In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed., The Williams & Wilkins Co., Baltimore. 4. Eagon, R. G., T. W. Hodge, M, J. B. Rake, and J. M. Yarbrough The effect of phenazine methosulfate-ascorbate on bacterial active transport and adenosine triphosphate formation: inhibition ofpseudomonas aeruginosa and stimulation of Escherichia coli. Can. J. Microbiol. 25: Edwards, C., J. A. Spode, and C. W. Jones The growth of Paracoccus denitrificans. FEMS Lett. 1: G6mez-Puyou, A., and C. G6mez-Lojero The use of ionophores and channel formers in the study of the function of biological membranes. Curr. Top. Bioenerg. 6: Hadjipetrou, L. P., and A. H. Stouthamer Energy production during nitrate respiration by Aerobacter aerogenes. J. Gen. Microbiol. 38: Harold, F. M Membranes and energy transduction in bacteria. Curr. Top. Bioenerg. 6: Harold, F. M., and J. R. Baarda Inhibition of membrane transport in Streptococcus faecalis by uncouplers of oxidative phosphorylation and its relationship to proton conduction. J. Bacteriol. 96: Jones, C. W., J. M. Brice, A. J. Downs, and J. W. Drozd Bacterial respiration-linked proton translocation and its relationship to respiratory-chain composition. Eur. J. Biochem. 52: Kristjansson, J. K., B. Walter, and T. C. Hollocher Respiration-dependent proton translocation and the transport of nitrate and nitrite in Paracoccus denitrificans and other denitrifying bacteria. Biochemistry 17: J. BACTERIOL. 12. Lawford, H. G Energy transduction in the mitochondrion-like bacterium Paracoccus denitrificans during carbon- and sulphate-limited aerobic growth in continuous culture. Can. J. Biochem. 56: Lawford, H. G., J. C. Cox, P. B. Garland, and B. A. Haddock Electron transport in aerobicallygrown Paracoccus denitrificans: kinetic characterization of the membrane-bound cytochromes and the stoichiometry of respiration-driven proton translocation. FEBS Lett. 64: Meijer, E. M., J. W. Van Der Zwaan, R. Wever, and A. H. Stouthamer Anaerobic respiration and energy conservation in Paracoccus denitrificans. Eur. J. Biochem. 96: Michell, P., and J. Moyle Respiration-driven proton translocation in rat liver mitochondria. Biochem. J. 105: O'Leary, V., and M. Solberg Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens. Appl. Environ. Microbiol. 31: Perigo, J. A., and T. A. Roberts Inhibition of clostridia by nitrite. J. Food Technol. 3: Pivnick, H., M. A. Johnston, C. Thacher, and R. Loynes Effect of nitrite on destruction and germination of Clostridium botulinum and putrefactive anaerobes 3679 and 3679h in meat and in buffer. Can. Inst. Food Technol. J. 3: Rice, C. W., and W. P. Hempfling Oxygen-limited continuous cultre and respiratory energy conservation in Escherichia coli. J. Bacteriol. 134: Rowe, J. J., B. F. Sherr, W. J. Payne, and IL G. Eagon A unique nitric oxide-binding complex formed by denitrifying Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 77: Rowe, J. J., J. M. Yarbrough, J. B. Rake, and R. G. Eagon Nitrite inhibition of aerobic bacteria. Curr. Microbiol. 2: Scholes, P., and P. Mitchell Respiration-driving proton translocation in Micrococcus denitrificans. J. Bioenerg. 1: Stouthamer, A. H The search for correlation between theoretical and experimental growth yields. Int. Rev. Biochem. 21: Tarr, H. L. A Bacteriostatic action of nitrates. Nature (London) 147: Van Verseveld, H. W., E. M. Meijer, and A. H. Stouthamer Energy conservation during nitrate respiration in Paracoccus denitrificans. Arch. Microbiol. 112: Williams, D. R., J. J. Rowe, P. Romero, and R. G. Eagon Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport. Appl. Environ. Microbiol. 36: Wodzinski, R. S., D. P. Labeda, and M. Alexander Effects of low concentrations of bisulfite-sulfite, and nitrite on microorganisms. Appl. Environ. Microbiol. 35: Yarbrough, J. M., J. B. Rake, and R. G. Eagon Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group tranalocation, and of intracellular enzymes. Appl. Environ. Microbiol. 39: