APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, p. 257-263 0099-2240/78/0036-0257$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No.2
Printed in U.S.A.
Denitrifying Pseudomonas aeruginosa: Some Parameters of Growth and Active Transport D. R. WILLIAMS, J. J. ROWE,t P. ROMERO,4 AND R. G. EAGON* Department of Microbiology, University of Georgia, Athens, Georgia 30602
Received for publication 10 February 1978
Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maimal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruguiosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity. The anaerobic reduction of nitrate and nitrite processes similar to aerobic respiration. It is to nitrous oxide or elemental nitrogen is termed clear, however, that different spectra of cytodenitrification, and the physiological process is chromes and enzymes are required for anaerobic called anaerobic respiration. Denitrification is respiration (16). The process of denitrification is important because of its role in the regeneration thought to occur in a stepwise manner as follows: of fixed nitrogen. It has become of particular N03 -- N02 -- NO - N20 -- N2. As would be interest in recent years due to increased cost of expected, many of the enzymes of the pathway nitrogen fertilizer and potential reduction of crop are closely associated with cytochromes in yields because of this microbiological phenome- Pseudomonas aeruginosa. Nitrate reductase renon (16). In addition, the generation of gaseous quires association with cytochrome c (4), nitrite nitrogen oxides has become a concern because reductase is analogous to cytochrome cd, and of the potential effect on the ozone layer of the nitric oxide reduction appears to be associated upper atmosphere (16). With these factors as with a 570-nm-absorbing pigment (18). Several themes run constant. The absence of impetus, basic knowledge in the area of denitrification has expanded rapidly in a relatively oxygen derepresses the enzymes necessary for denitrification (3, 22). Once derepressed, the short time. The most predominant denitifying bacteria quantity of these enzymes is directly affected by in our environment have been reported to belong the initial nitrate concentration in the culture to the genus Pseudomonas (6). (It should be (3). Anaerobiosis diminishes the a-type cytonoted, however, that in other instances Akcali- chromes drastically and stimulates production genes has been found to be the dominant deni- of c-type cytochromes in most denitifying bactrifler [5].) Species of Pseudomonas are nonfer- teria (16). Finally, nitrite accumulates in the menting organisms capable of generating energy culture media, usually before the onset of visible only by respiration. The mechanism(s) and reg- gas production. Nitrite may or may not inhibit ulation of electron flow and ATP synthesis un- further reduction of nitrogen oxides, depending der denirifying conditions are considered to be on the species of bacteria and the culture conditions (1, 21). t Present addres: Department of Biology, University of One important denitrification aspect which Dayton, Dayton, OH 45469. t Permanent addreu Department of Microbiology, Faculty has been largely ignored is the transport of nutrients by denitrifiers during anaerobic respiraof Pharmacy, University of Granada, Granada, Spain. 257
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tion and its relationship to the enzymes and the cytochrome system. Evidence has been presented to demonstrate coupling between anaerobic transport of lactose and amino acids and anaerobic electron transfer in isolated membrane vesicles of Escherichia coli (12). More recently, it has been shown that amino acid transport can be coupled to electron transfer by using nitrate as an electron acceptor in the obligate anaerobe Veillonella alcalescens (11). Several energy donors and components of the membrane-bound anaerobic electron transport chain of V. alcalescens were also identified. Virtually no information is available, however, on the active transport of solutes in bacteria growing under denitrifying conditions. P. aeruginosa possesses an inducible active transport system for glucose as well as inducible enzymes for the Enter-Doudoroff pathway and the oxidative portion of the hexose monophosphate-pentose cycle (7, 17). Guymon and Eagon (7), moreover, demonstrated that in P. aeruginosa glucose and gluconate were actively transported as the free sugars. Evidence has been found, however, which implicates the phosphoenolpyruvate-phosphotransferase system as the mechanism by which fructose is transported by Pseudomonas species (19). Thus, with the sole exception of fructose, sugars are considered to be accumulated by a carrier-mediated active transport system in P. aeruginosa. In the present study we characterized aspects of the transport of glucose and gluconate by denitrifying P. aeruginosa. These results were compared with those of the aerobically respiring organism. We also determined the optimal concentrations of nitrate and nitrite for denitifying growth and for optimal rates of anaerobic transport of glucose and gluconate. MATERIALS AND METHODS Organism and culture conditions. P. aerugunosa PAO (formerly Holloway strain 1) was maintained on agar slants prepared by using the basal salts medium previously described (2) containing 11 mM glucose. Starter cultures for all transport and enzyme assays were grown aerobically as follows. Erlenmeyer flasks (500 ml) containing 200 ml of minimal salts supplemented with 11 mM glucose were inoculated from 18h agar slants and incubated with rotary shaking (200 rpm) at 30°C for 6 to 8 h to exponential phase before introduction into experimental culture flasks. Inocula for anaerobic experiments were aseptically harvested from starter cultures by centrifuging during early exponential growth. The whole-cell pellet from 200 ml of starter culture was used to initiate anaerobic cultivation in 1-liter stoppered Erlenmeyer flasks filled to capacity and fitted with two ports to allow gas evolution and sampling of the culture. Bacteria were grown anaerobically at 300C in basal salts medium (2)
APPL. ENVIRON. MICROBIOL.
containing 1.0% KNO3 and 0.1% yeast extract sterilized by autoclaving. Concentrated, filter-sterilized solutions of D-glucose or D-gluconate were added in final concentrations of 11 mM. Oxygen was excluded from the flasks by bubbling nitrogen through the culture for 5 min before incubation. The culture was stirred slowly with a magnetic bar and stirrer to prevent clumping. Modifications to the media during initial growth characterization are described below when appropriate. P. aeruginosa used for aerobic transport experiments were grown aerobically in 1-liter Erlenmeyer flasks containing 200-ml of basal salts medium containing 11 mM glucose. Addition of the carbon source and incubation were accomplished as with anaerobic growth experiments. The cultures were inoculated with 20 ml from early-exponential-growth-phase starter cultures and incubated with rotary shaking (200 rpm) at 300C to midexponential phase. In certain experiments, as indicated below, P. aeruginosa was grown in basal salts-glucose medium containing yeast extract or in tryptic soy broth (Difco Laboratories, Detroit, Mich.). Nitrite determinations. Samples (5 ml) were taken from anaerobically growing cultures at timed intervals, the absorbance of the culture was determined at 540 nm with a Spectronic 20 spectrophotometer (Bausch & Lomb Inc., Rochester, N.Y.), and the cells were removed by centrifuging. Nitrite in the medium was determined by the method of Strickland and Parsons (20) with an Hitachi-Perkin-Elmer 139 UV-VIS spectrophotometer (Perkin-Elmer Coleman Instruments Div., Oak Brook, Ill.). Active transport assays. Cells were harvested by centrifuging at 250C, washed in glucose-free basal salts solution containing 50 ,ug of chloramphenicol, per ml, and suspended in glucose-free basal salts solution with 50 jg of chloramphenicol per ml at a density of 1 g (wet weight) per 20 ml. This cell suspension was used for transport assays. Transport of ['4C]glucose and ['4C]gluconate by P. aeruginosa was determined under aerobic conditions at 30°C on a reciprocal shaking water bath. Each 10ml Erlenmeyer reaction flask contained in a final concentration 0.2 ml of cell suspension and 0.1 mM ['4C]glucose (6.54 pCi/iLmol) or 0.1 mM ['4C]gluconate (39 uCi/pmol). Basal salts solution was added to give a final volume of 1 ml. The reaction was initiated by the addition of labeled substrates. Identical volumes, concentrations, and specific activities of radioactive substrates were used in anaerobic transport experiments. Uptake was determined at 300C in stoppered tubes fitted with two ports to allow constant flushing with nitrogen gas and sample removal. Potassium nitrate or sodium nitrite was added in a final concentration of 1 mM. Addition of the cell suspension, preincubated anaerobically, was used to initiate the reaction. In aerobic and anaerobic assays, 0.05-ml samples were withdrawn at specific time intervals and delivered over membrane filters (25-mm diameter, 0.45-pm pore size; Amicon Corp., Lexington, Mass.) previously overlaid with 1 ml of 0.1 M LiCl, filtered rapidly, and washed immediately with an additional 5 ml of 0.1 M LiCl. The washed membrane filters bearing the cells were transfenred immediately to vials containing 10 ml
VOL. 36, 1978
of scintillation fluid and counted in a Packard liquid scintillation spectrometer, (model 527; Packard Instrument Co., Inc., Downers Grove, Ill.) as previously described (17). Metabolizable substrates and intact, wild-type cells of P. aeruginosa were used for our transport studies in spite of obvious objections. We have shown previously that initial rates of transport of metabolizable substrates by intact, wild-type cells were identical to those of membrane vesicles or glucose-negative mutants and, that the glucose transport system had such low affinity for non-metabolizable glucose analogs that the results gained from the use of analogs did not reflect true kinetics of glucose transport (7). In all except one experiment reported herein, initial rates of transport were measured. The results thus gained, therefore, are considered to be an accurate measure of transport. Nitrate and nitrite reductase (dissimilatory) assays. Cells were collected by centrifuging at 25°C, washed in minimal salts medium containing 50 ,ug of chloramphenicol per ml, and suspended at a density of 1 g (wet weight) per ml in ice-cold basal salts containing 50 ug of benzyl viologen per ml. Cell extracts were prepared by two passages through an icecold French pressure cell. The nitrate and nitrite reductases in extracts were protected from oxygen by the dropwise addition of a 1.6% Na2S204-1.6% NaHCO3 mixte, with the purple benzyl viologen serving an an indicator of the reduced state (14). Whole cells were removed with minimal loss of membrane fragments from the broken-cell suspension by low-speed centrifuging at room temperature. The protein concentration was adjusted to 0.5 to 1.0 mg/ml with basal salts, and nitrate or nitrite reductase activity was determined. The nitrate and nitrite reductases were stable for 2 h when extracts were stored on ice. Enzyme assay mixtures contained in a final concentration: 0.5 ml of cell extract, 20 mM potassium phosphate buffer (pH 7.0), 10 mM KNO3, and 0.1 mM benzyl viologen or 10 mM NaNO2, 0.2 mM methylene blue, and a mixture of 32% Na3S305 and 3.2% NaHCO3 (final concentration) in a final volume of 2.5 ml. The extracts were incubated at 30°C for 5 min and flushed with nitrogen gas before initiation of the reaction by addition of substrate. Excess reducing agent ensured reduced conditions throughout the assay. Samples (0.5 ml) were removed at timed intervals, added to an equal volume of ice-cold ethanol, and mixed vigorously with a Vortex mixer until oxidized to stop the reaction (13). Nitrite concentration was then determined by the method of Strickland and Parsons (20). Oxygen trap. Nitrogen gas used in anaerobic experiments was sparged through a stoppered tube (250 ml) containing 0.2% methyl viologen reduced with powdered zinc to remove all traces of oxygen. Protein determinations. Protein determinations in whole cells were made by a modified biuret procedure (9). Urea (60%) was used to facilitate cell lysis. A semimicro-biuret method was employed for protein determinations in broken-cell extracts (15). Reagents. D-[U-14C]glucose (327 mCi/mmol) and D-[U-_4C]gluconate (3.9 mCi/mmol) were purchased from Amersham/Searle, Arlington Heights, M. Viologen dyes were purchased from Schwarz/Mann, Or-
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angeburg, N.Y.; sulfanilamide was purchased from Eastman Organic Chemical Div., Eastman-Kodak Co., Rochester, N.Y.; and N-(l-naphthyl)ethylenediamine dihydrochloride and chloramphenicol were purchased from Sigma Chemical Co., St. Louis, Mo. All other materials were purchased from commercial sources in the highest state of purity. RESULTS
Optimal concentrations of nitrate and nitrite to support anaerobic growth. Experiments were carried out to determine the optimal concentration of nitrate required to support maximal growth of P. aeruginosa when cultivated anerobically under denitrifying conditions. Three different media were used: glucose-basal salts, glucose-yeast extract-basal salts, and tryptic soy broth. In each case, 100 mM nitrate supported the maximal yields of cells (Fig. 1). Thus, for all subsequent experiments a final concentration of 100 mM nitrate was used in media for the anaerobic cultivation of P. aeruginosa.
Nitrite was not as satisfactory as an electron acceptor to support the growth of P. aeruginosa under anaerobic denitrifying conditions as was nitrate. Only slow growth resulted when the final concentration of nitrite was kept below 15 mM, and no growth occurred at higher concentrations of nitrite (data not shown). Optimal concentrations of nitrate and nitrite to support active transport under anaerobic conditions. P. aeruginosa transported glucose under anaerobic conditions when either nitrate or nitrite was used as the terminal electron acceptor. The concentration of each nitrogen oxide that supported the greatest rate of transport was 1 mM (Fig. 2). The rate of transport rapidly decreased as higher concentrations of the nitrogen oxides were used. However, nitrate supported transport of glucose more effectively than did nitrite at all concentrations. Finally, whereas 1 mM nitrate was optimal for transport of glucose, 100 mM nitrate was optimal for maximnal cell yield under growing conditions. Nitrite accumulation in media containing nitrate as the terminal electron acceptor under denitrifying conditions. Nitrite determinations were performed on samples of culture media taken at various time intervals throughout the growth cycle. Nitrite accumulated rapidly during the early exponential growth phase to concentrations of 1.6 to 3.0 mM in simple media and 8 mM in complex media and then declined rapidly to extinction at the midexponential phase (Fig. 3). Significantly, an increased growth rate occurred upon disappearance of nitrite from the medium. Nitrate and nitrite reductase activity and
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NITRATE CONCENTRATION (mM)
FIG. 1. Effect of nitrate concentration on denitrifying growth ofP. aeruginosa in three media: basal saltsglucose (A), basal salts-glucose-yeast extract, (B), and tryptic soy broth (C).
rate of growth increased. The assays were linear over time and with respect to protein concentration. Saturating concentrations of both substrates were employed. Parallel experiments conducted in the absence of substrate or enzyme demonstrated little or no endogenous or nonspecific reduction of nitrate or nitrite during the assays. The maximal rate of glucose uptake under denitrifying conditions corresponded closely to the maximal levels of nitrate and nitrite reductase activities observed during growth (Fig. 3). All three of these maxima were observed within 2 h after the peak of maximal accumulation of nitrite was seen in the medium and coincided NITROGEN OXIDE (mM)
FIG. 2. Support of anaerobic glucose uptake in P. aeruginosa by varying concentrations of nitrate and nitrite as terminal electron acceptors. Initial transport rates were approximated by measuring glucose uptake after incubation for 15 s. Symbols: 0, nitrate; 0, nitrite.
glucose transport activity in cells harvested at different growth phases. Nitrate and nitrite reductases were assayed in cell samples taken at various times during anaerobic cultivation of P. aeruginosa from lag through late exponential growth phases. Nitrate reductase activity was detected at approximately 3 h after inoculation of the anaerobic cultures, increased to maximal activity in midexponential growth, and then sharply declined in the late exponential phase (Fig. 3). Nitrite reductase activity followed a similar course, and maximum activity was demonstrated at approximately the same point in midgrowth (Fig. 3). It was not possible, however, to detect nitrite reductase activity earlier than 7 h after inoculation, which coincided with the beginning of exponential growth. The maxima of both enzyme activities closely coincided with the point at which the
with its disappearance from the medium and with the initiation of an increased growth rate. Transport of glucose in cells grown aerobically versus anaerobically and harvested at different growth phases. The rate of anaerobic glucose transport (with nitrate as the terminal electron acceptor) by cells harvested at various stages of anaerobic growth was determined. Aerobic uptake rates were also measured for glucose in cells collected at various stages of aerobic growth. The rate at which P. aeruginosa actively transported glucose under denitrifying conditions increased slowly during the early exponential phase until an absorbance at 540 nm of 0.3 was attained (Fig. 4). At this point, the rate of uptake increased sharply, more than doubling during the time in which the culture increased to an absorbance at 540 nm of 0.4. Uptake then dropped off slightly and maintained a steady rate (about 75% of the maximal) throughout the late exponential growth phase. With aerobically grown cells the rate of aerobic glucose transport was characterized by a gradual increase to the maximal rate during late exponential growth followed by a sharp decline thereafter (Fig. 4). In comparison, moreover, it was noted that the maximal rate of glucose
261
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VOL. 36, 1978
transport occurred later in the exponential phase acceptor and that the rate of transport was under aerobic conditions than under anaerobic greater under aerobic conditions than under anconditions with nitrate as the terminal electron aerobic conditions. a c)
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P TIME (h) FIG. 3. Production of nitrate and nitrite reductases, accumulation of nitrite in the medium, and glucose uptake rate by cells of P. aeruginosa during denitrifying growth. Initial glucose uptake rates were approximated by measuring glucose taken up after incubation for 15 s. Symbols: A, growth curve during denitrifying growth; 0, nitrite accumulation in the medium; A, anaerobic glucose uptake rate with 1 mM nitrate as the terminal electron acceptor; 0, nitrate reductase activity; 0, nitrite reductase activity.
TIME (h)
FIG. 4. Aerobic glucose uptake by aerobically grown P. aeruginosa and anaerobic glucose uptake by P. aeruginosa grown under denitrifying conditions. Initial transport rates were approximated by measuring glucose uptake after incubation for 15 Symbols: A, aerobic growth curve; 0, aerobic glucose uptake; A, anaerobic (denitrifying) growth curve; 0, anaerobic glucose uptake with 1 mM nitrate as the terminal electron s.
acceptor.
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WILLIAMS ET AL.
Comparison of glucose and gluconate transport rates of cells grown aerobically versus anaerobically. P. aeruginosa was cultured aerobically and anaerobically with glucose or gluconate as the major carbon and energy sources. Yeast extract (0.1%) was added to the anaerobic cultures. Bacteria were assayed for uptake of the substrate on which they were grown under aerobic and anaerobic conditions. Nitrate (1 mM) was provided as the terminal electron acceptor for anaerobic assays. Cells grown anaerobically carried out transport of glucose under both aerobic and anaerobic conditions (Fig. 5). The aerobic uptake rate of glucose by anaerobically grown cells was significantly higher than seen under anaerobic conditions. Aerobically grown cells, however, could not transport glucose anaerobically with nitrate as an electron acceptor. In all cases, transport rates were greater under aerobic conditions than under anaerobic conditions. When gluconate was used instead of glucose, similar patterns of transport were seen (data not shown). DISCUSSION Optimal growth of P. aeruginosa (defined on the basis of greatest cell yield) was supported by 100 mM nitrate in a variety of media under anaerobic (i.e., denitrifying) conditions. When nitrate was used as the terminal electron acceptor under anaerobic conditions, however, it was found that 1 mM was the optimal concentration to support the transport of glucose. The optimal concentration of nitrite as the terminal electron acceptor to support the anaerobic transport of glucose was also 1 mM. The rate of transport was less, however, when nitrite was used as the terminal electron acceptor than when nitrate was used. Nitrite, on the other hand, supported only slow growth when added to media in concentrations of less than 15 mM. We previously determined that nitrite in concentrations greater than 10 mM exerted toxicity toward cells of P. aeruginosa by inhibiting respiration, apparently by preventing the flow of electrons through the terminal electron transport chain (J. J. Rowe, T. W. Hodge III, and R. G. Eagon, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, K226, p. 223). Moreover, in the presence of 10 mM or higher concentrations of nitrite, P. aeruginosa was inhibited in the ability to carry out active transport and oxidative phosphorylation. Thus, it is not surprising that nitrite was not an electron acceptor of choice to support anaerobic (denitrifying) growth except at low
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60
120
180
240
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FIG. 5. Glucose uptake measured under aerobic and anaerobic conditions by P. aeruginosa grown aerobically and under anaerobic (denitrifying) conditions. Symbols: U, aerobically grown cells, aerobic glucose uptake; 0, anaerobically grown cells, aerobic glucose uptake; A, anaerobically grown cells, anaerobic glucose uptake with I mM nitrate as the terninal electron acceptor: A, anaerobically grown cells, anaerobic glucose uptake with no added terninal electron acceptor; El aerobically grown cells, anaerobic glucose uptake with 1 mM nitrate as the terminal electron acceptor.
quential induction (or perhaps to sequential derepression [16]) of nitrate and nitrite reductases. Evidence for this is that nitrate reductase activity could be detected 4 h before nitrite reductase activity was observed. Thus, these data confirm the observation of van Hartingsveldt and Stouthamer (21) of sequential synthesis of nitrate and nitrite reductases by P. aeruginosa when subjected to denitrifying conditions of growth. The maximal rate of glucose uptake under denitrifying conditions corresponded to the maximal levels of nitrate and nitrite reductase activities and coincided with the disappearance of nitrite from the medium. We interpret this observation to indicate that active transport rates under denitrifying conditions are directly related to the presence and quantity of nitrate and nitrite reductases. The latter are required for the mediation of such physiological phenomena as electron acceptance and energy generation. The rate of glucose transport by P. aeruginosa, however, was less when nitrate was used concentrations. Transient accumulation of nitrite was ob- as the terminal electron acceptor as compared served when P. aeruginosa was grown under with when oxygen was used as the terminal denitrifying conditions, apparently owing to se- electron acceptor. John and Whatley (8) re-
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ported that in the case of Micrococcus (Paracoccus) denitrificans ATP synthesis coupled with oxygen was about 70% more efficient than that coupled with nitrate reduction to nitrite. Similarly, Koike and Hattori (10) reported that the growth yield per mole of electrons transported by Pseudomonas denitrificans under denitrifying conditions was about one-half that under aerobic conditions. Thus, our present observations further support the idea that anaerobic respiration is less efficient in energy generation than are aerobic systems. ACKNOWLEDGMENIS This study was supported by National Science Foundation research grants BMS74-14819 and PCM77-02928.
LITERATURE CITED 1. Bovell, C. 1967. The effect of sodium nitrite on the growth
of Micrococcus denitrificans. Arch. Mikrobiol. 59:13-19. 2. Eagon, R. G., and P. V. Phibbs, Jr. 1971. Kinetics of transport of glucose, fructose and mannitol by Pseudomonas aeruginosa. Can. J. Biochem. 49:1031-1041. 3. Elliott, L F., and C. IL Gilmour. 1971. Growth of Pseudomonas stutzeri with nitrate and oxygen as terminal electron acceptors Soil Biol. Biochem. 3:331-335. 4. Fewson, C. A., and D. J. D. Nicholas. 1961. Nitrate reductase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 49:335-349. 5. Focht, D. D., and W. Verstraete. 1977. Biochemical ecology of nitrification and denitrification. Adv. Microb. Ecol. 1:135-214. 6. Gamble, T. N., ML R. Betlach, and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. 33:926-939. 7. Guymon, L. F., and R. G. Eagon. 1974. Transport of glucose, gluconate, and methyl a-D-glucoside by Pseudomonas aeruginosa. J. Bacteriol. 117:1261-1269. 8. John, R., and F. R. Whatley. 1970. Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans. Biochim. Biophys. Acta 216:342-352. 9. King, T. E. 1966. Glucose dehydrogenase-particulate. II.
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Acetobacter suboxydans. Methods Enzymol. 9:98-103. 10. Koike, I., and A. Hattori. 1975. Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions. J. Gen. Microbiol. 88:1-10. 11. Konings, W. N., J. Boonstra, and W. de Vries. 1975. Amino acid transport in membrane vesicles of obligately anaerobic Veillonella akalescens. J. Bacteriol.
L22:245-249. 12. Konings, W. N., and H. R. Kaback. 1973. Anaerobic transport in Escherichia coli membrane vesicles. Proc. Natl. Acad. Sci. U.S.A. 70:3376-3381. 13. Losada, M., and A. Paneque. 1971. Nitrate reductase. Methods Enzymol. 23:487-491. 14. Lowe, R. H., and H. J. Evans. 1964. Preparation and some properties of a soluble nitrate reductase from Rhizobium japonicum. Biochim. Biophys. Acta
85:377-389. 15. Mokrasch, L C., W. D. Davidson, and R. W. McGilvery. 1956. Purification properties of fructose-1,6diphosphatase. J. Biol. Chem. 221:909-919. 16. Payne, W. J. 1976. Denitrification. Trends Biochem. Sci. 1:220-222. 17. Phibbs, P. V., Jr., and R. C. Eagon. 1970. Transport and phosphorylation of glucose, fructose and mannitol by Pseudomonas aeruginosa. Arch. Biochem. Biophys. 138:470-482. 18. Rowe, J. J., B. F. Sherr, W. J. Payne, and R. G. Eagon. 1977. A unique nitric oxide-binding complex formed by denitifying Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 77:253-258. 19. Sawyer, M. H., P. Baumann, L Baumann, S. M. Berman, J. L Canovas, and R. H. Berman. 1977. Pathways ofD-fructose catabolism in species of Pseudomonas. Arch. Microbiol. 112:49-55. 20. Strickland, J. D. H., and T. R. Parsons. 1968. A practical handbook of seawater analysis. Bull. 167. Fisheries Research Board of Canada, Ottawa. 21. van Hartingsveldt, J., and A. H. Stouthamer. 1973. Mapping and characterization of mutants of Pseudomonas aeruginosa affected in nitrate respiration in aerobic or anaerobic growth. J. Gen. Microbiol. 74:97-106. 22. Van't Riet, J., A. H. Stouthamer, and R. J. Planta. 1968. Regulation of nitrate assimilation and nitrate respiration in Aerobacter aerogenes. J. Bacteriol. 96:1455-1464.
Vol. 36, No.2
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Denitrifying Pseudomonas aeruginosa: Some Parameters of Growth and Active Transport D. R. WILLIAMS, J. J. ROWE,t P. ROMERO,4 AND R. G. EAGON* Department of Microbiology, University of Georgia, Athens, Georgia 30602
Received for publication 10 February 1978
Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maimal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruguiosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity. The anaerobic reduction of nitrate and nitrite processes similar to aerobic respiration. It is to nitrous oxide or elemental nitrogen is termed clear, however, that different spectra of cytodenitrification, and the physiological process is chromes and enzymes are required for anaerobic called anaerobic respiration. Denitrification is respiration (16). The process of denitrification is important because of its role in the regeneration thought to occur in a stepwise manner as follows: of fixed nitrogen. It has become of particular N03 -- N02 -- NO - N20 -- N2. As would be interest in recent years due to increased cost of expected, many of the enzymes of the pathway nitrogen fertilizer and potential reduction of crop are closely associated with cytochromes in yields because of this microbiological phenome- Pseudomonas aeruginosa. Nitrate reductase renon (16). In addition, the generation of gaseous quires association with cytochrome c (4), nitrite nitrogen oxides has become a concern because reductase is analogous to cytochrome cd, and of the potential effect on the ozone layer of the nitric oxide reduction appears to be associated upper atmosphere (16). With these factors as with a 570-nm-absorbing pigment (18). Several themes run constant. The absence of impetus, basic knowledge in the area of denitrification has expanded rapidly in a relatively oxygen derepresses the enzymes necessary for denitrification (3, 22). Once derepressed, the short time. The most predominant denitifying bacteria quantity of these enzymes is directly affected by in our environment have been reported to belong the initial nitrate concentration in the culture to the genus Pseudomonas (6). (It should be (3). Anaerobiosis diminishes the a-type cytonoted, however, that in other instances Akcali- chromes drastically and stimulates production genes has been found to be the dominant deni- of c-type cytochromes in most denitifying bactrifler [5].) Species of Pseudomonas are nonfer- teria (16). Finally, nitrite accumulates in the menting organisms capable of generating energy culture media, usually before the onset of visible only by respiration. The mechanism(s) and reg- gas production. Nitrite may or may not inhibit ulation of electron flow and ATP synthesis un- further reduction of nitrogen oxides, depending der denirifying conditions are considered to be on the species of bacteria and the culture conditions (1, 21). t Present addres: Department of Biology, University of One important denitrification aspect which Dayton, Dayton, OH 45469. t Permanent addreu Department of Microbiology, Faculty has been largely ignored is the transport of nutrients by denitrifiers during anaerobic respiraof Pharmacy, University of Granada, Granada, Spain. 257
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tion and its relationship to the enzymes and the cytochrome system. Evidence has been presented to demonstrate coupling between anaerobic transport of lactose and amino acids and anaerobic electron transfer in isolated membrane vesicles of Escherichia coli (12). More recently, it has been shown that amino acid transport can be coupled to electron transfer by using nitrate as an electron acceptor in the obligate anaerobe Veillonella alcalescens (11). Several energy donors and components of the membrane-bound anaerobic electron transport chain of V. alcalescens were also identified. Virtually no information is available, however, on the active transport of solutes in bacteria growing under denitrifying conditions. P. aeruginosa possesses an inducible active transport system for glucose as well as inducible enzymes for the Enter-Doudoroff pathway and the oxidative portion of the hexose monophosphate-pentose cycle (7, 17). Guymon and Eagon (7), moreover, demonstrated that in P. aeruginosa glucose and gluconate were actively transported as the free sugars. Evidence has been found, however, which implicates the phosphoenolpyruvate-phosphotransferase system as the mechanism by which fructose is transported by Pseudomonas species (19). Thus, with the sole exception of fructose, sugars are considered to be accumulated by a carrier-mediated active transport system in P. aeruginosa. In the present study we characterized aspects of the transport of glucose and gluconate by denitrifying P. aeruginosa. These results were compared with those of the aerobically respiring organism. We also determined the optimal concentrations of nitrate and nitrite for denitifying growth and for optimal rates of anaerobic transport of glucose and gluconate. MATERIALS AND METHODS Organism and culture conditions. P. aerugunosa PAO (formerly Holloway strain 1) was maintained on agar slants prepared by using the basal salts medium previously described (2) containing 11 mM glucose. Starter cultures for all transport and enzyme assays were grown aerobically as follows. Erlenmeyer flasks (500 ml) containing 200 ml of minimal salts supplemented with 11 mM glucose were inoculated from 18h agar slants and incubated with rotary shaking (200 rpm) at 30°C for 6 to 8 h to exponential phase before introduction into experimental culture flasks. Inocula for anaerobic experiments were aseptically harvested from starter cultures by centrifuging during early exponential growth. The whole-cell pellet from 200 ml of starter culture was used to initiate anaerobic cultivation in 1-liter stoppered Erlenmeyer flasks filled to capacity and fitted with two ports to allow gas evolution and sampling of the culture. Bacteria were grown anaerobically at 300C in basal salts medium (2)
APPL. ENVIRON. MICROBIOL.
containing 1.0% KNO3 and 0.1% yeast extract sterilized by autoclaving. Concentrated, filter-sterilized solutions of D-glucose or D-gluconate were added in final concentrations of 11 mM. Oxygen was excluded from the flasks by bubbling nitrogen through the culture for 5 min before incubation. The culture was stirred slowly with a magnetic bar and stirrer to prevent clumping. Modifications to the media during initial growth characterization are described below when appropriate. P. aeruginosa used for aerobic transport experiments were grown aerobically in 1-liter Erlenmeyer flasks containing 200-ml of basal salts medium containing 11 mM glucose. Addition of the carbon source and incubation were accomplished as with anaerobic growth experiments. The cultures were inoculated with 20 ml from early-exponential-growth-phase starter cultures and incubated with rotary shaking (200 rpm) at 300C to midexponential phase. In certain experiments, as indicated below, P. aeruginosa was grown in basal salts-glucose medium containing yeast extract or in tryptic soy broth (Difco Laboratories, Detroit, Mich.). Nitrite determinations. Samples (5 ml) were taken from anaerobically growing cultures at timed intervals, the absorbance of the culture was determined at 540 nm with a Spectronic 20 spectrophotometer (Bausch & Lomb Inc., Rochester, N.Y.), and the cells were removed by centrifuging. Nitrite in the medium was determined by the method of Strickland and Parsons (20) with an Hitachi-Perkin-Elmer 139 UV-VIS spectrophotometer (Perkin-Elmer Coleman Instruments Div., Oak Brook, Ill.). Active transport assays. Cells were harvested by centrifuging at 250C, washed in glucose-free basal salts solution containing 50 ,ug of chloramphenicol, per ml, and suspended in glucose-free basal salts solution with 50 jg of chloramphenicol per ml at a density of 1 g (wet weight) per 20 ml. This cell suspension was used for transport assays. Transport of ['4C]glucose and ['4C]gluconate by P. aeruginosa was determined under aerobic conditions at 30°C on a reciprocal shaking water bath. Each 10ml Erlenmeyer reaction flask contained in a final concentration 0.2 ml of cell suspension and 0.1 mM ['4C]glucose (6.54 pCi/iLmol) or 0.1 mM ['4C]gluconate (39 uCi/pmol). Basal salts solution was added to give a final volume of 1 ml. The reaction was initiated by the addition of labeled substrates. Identical volumes, concentrations, and specific activities of radioactive substrates were used in anaerobic transport experiments. Uptake was determined at 300C in stoppered tubes fitted with two ports to allow constant flushing with nitrogen gas and sample removal. Potassium nitrate or sodium nitrite was added in a final concentration of 1 mM. Addition of the cell suspension, preincubated anaerobically, was used to initiate the reaction. In aerobic and anaerobic assays, 0.05-ml samples were withdrawn at specific time intervals and delivered over membrane filters (25-mm diameter, 0.45-pm pore size; Amicon Corp., Lexington, Mass.) previously overlaid with 1 ml of 0.1 M LiCl, filtered rapidly, and washed immediately with an additional 5 ml of 0.1 M LiCl. The washed membrane filters bearing the cells were transfenred immediately to vials containing 10 ml
VOL. 36, 1978
of scintillation fluid and counted in a Packard liquid scintillation spectrometer, (model 527; Packard Instrument Co., Inc., Downers Grove, Ill.) as previously described (17). Metabolizable substrates and intact, wild-type cells of P. aeruginosa were used for our transport studies in spite of obvious objections. We have shown previously that initial rates of transport of metabolizable substrates by intact, wild-type cells were identical to those of membrane vesicles or glucose-negative mutants and, that the glucose transport system had such low affinity for non-metabolizable glucose analogs that the results gained from the use of analogs did not reflect true kinetics of glucose transport (7). In all except one experiment reported herein, initial rates of transport were measured. The results thus gained, therefore, are considered to be an accurate measure of transport. Nitrate and nitrite reductase (dissimilatory) assays. Cells were collected by centrifuging at 25°C, washed in minimal salts medium containing 50 ,ug of chloramphenicol per ml, and suspended at a density of 1 g (wet weight) per ml in ice-cold basal salts containing 50 ug of benzyl viologen per ml. Cell extracts were prepared by two passages through an icecold French pressure cell. The nitrate and nitrite reductases in extracts were protected from oxygen by the dropwise addition of a 1.6% Na2S204-1.6% NaHCO3 mixte, with the purple benzyl viologen serving an an indicator of the reduced state (14). Whole cells were removed with minimal loss of membrane fragments from the broken-cell suspension by low-speed centrifuging at room temperature. The protein concentration was adjusted to 0.5 to 1.0 mg/ml with basal salts, and nitrate or nitrite reductase activity was determined. The nitrate and nitrite reductases were stable for 2 h when extracts were stored on ice. Enzyme assay mixtures contained in a final concentration: 0.5 ml of cell extract, 20 mM potassium phosphate buffer (pH 7.0), 10 mM KNO3, and 0.1 mM benzyl viologen or 10 mM NaNO2, 0.2 mM methylene blue, and a mixture of 32% Na3S305 and 3.2% NaHCO3 (final concentration) in a final volume of 2.5 ml. The extracts were incubated at 30°C for 5 min and flushed with nitrogen gas before initiation of the reaction by addition of substrate. Excess reducing agent ensured reduced conditions throughout the assay. Samples (0.5 ml) were removed at timed intervals, added to an equal volume of ice-cold ethanol, and mixed vigorously with a Vortex mixer until oxidized to stop the reaction (13). Nitrite concentration was then determined by the method of Strickland and Parsons (20). Oxygen trap. Nitrogen gas used in anaerobic experiments was sparged through a stoppered tube (250 ml) containing 0.2% methyl viologen reduced with powdered zinc to remove all traces of oxygen. Protein determinations. Protein determinations in whole cells were made by a modified biuret procedure (9). Urea (60%) was used to facilitate cell lysis. A semimicro-biuret method was employed for protein determinations in broken-cell extracts (15). Reagents. D-[U-14C]glucose (327 mCi/mmol) and D-[U-_4C]gluconate (3.9 mCi/mmol) were purchased from Amersham/Searle, Arlington Heights, M. Viologen dyes were purchased from Schwarz/Mann, Or-
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angeburg, N.Y.; sulfanilamide was purchased from Eastman Organic Chemical Div., Eastman-Kodak Co., Rochester, N.Y.; and N-(l-naphthyl)ethylenediamine dihydrochloride and chloramphenicol were purchased from Sigma Chemical Co., St. Louis, Mo. All other materials were purchased from commercial sources in the highest state of purity. RESULTS
Optimal concentrations of nitrate and nitrite to support anaerobic growth. Experiments were carried out to determine the optimal concentration of nitrate required to support maximal growth of P. aeruginosa when cultivated anerobically under denitrifying conditions. Three different media were used: glucose-basal salts, glucose-yeast extract-basal salts, and tryptic soy broth. In each case, 100 mM nitrate supported the maximal yields of cells (Fig. 1). Thus, for all subsequent experiments a final concentration of 100 mM nitrate was used in media for the anaerobic cultivation of P. aeruginosa.
Nitrite was not as satisfactory as an electron acceptor to support the growth of P. aeruginosa under anaerobic denitrifying conditions as was nitrate. Only slow growth resulted when the final concentration of nitrite was kept below 15 mM, and no growth occurred at higher concentrations of nitrite (data not shown). Optimal concentrations of nitrate and nitrite to support active transport under anaerobic conditions. P. aeruginosa transported glucose under anaerobic conditions when either nitrate or nitrite was used as the terminal electron acceptor. The concentration of each nitrogen oxide that supported the greatest rate of transport was 1 mM (Fig. 2). The rate of transport rapidly decreased as higher concentrations of the nitrogen oxides were used. However, nitrate supported transport of glucose more effectively than did nitrite at all concentrations. Finally, whereas 1 mM nitrate was optimal for transport of glucose, 100 mM nitrate was optimal for maximnal cell yield under growing conditions. Nitrite accumulation in media containing nitrate as the terminal electron acceptor under denitrifying conditions. Nitrite determinations were performed on samples of culture media taken at various time intervals throughout the growth cycle. Nitrite accumulated rapidly during the early exponential growth phase to concentrations of 1.6 to 3.0 mM in simple media and 8 mM in complex media and then declined rapidly to extinction at the midexponential phase (Fig. 3). Significantly, an increased growth rate occurred upon disappearance of nitrite from the medium. Nitrate and nitrite reductase activity and
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APPL. ENVIRON. MICROBIOL.
NITRATE CONCENTRATION (mM)
FIG. 1. Effect of nitrate concentration on denitrifying growth ofP. aeruginosa in three media: basal saltsglucose (A), basal salts-glucose-yeast extract, (B), and tryptic soy broth (C).
rate of growth increased. The assays were linear over time and with respect to protein concentration. Saturating concentrations of both substrates were employed. Parallel experiments conducted in the absence of substrate or enzyme demonstrated little or no endogenous or nonspecific reduction of nitrate or nitrite during the assays. The maximal rate of glucose uptake under denitrifying conditions corresponded closely to the maximal levels of nitrate and nitrite reductase activities observed during growth (Fig. 3). All three of these maxima were observed within 2 h after the peak of maximal accumulation of nitrite was seen in the medium and coincided NITROGEN OXIDE (mM)
FIG. 2. Support of anaerobic glucose uptake in P. aeruginosa by varying concentrations of nitrate and nitrite as terminal electron acceptors. Initial transport rates were approximated by measuring glucose uptake after incubation for 15 s. Symbols: 0, nitrate; 0, nitrite.
glucose transport activity in cells harvested at different growth phases. Nitrate and nitrite reductases were assayed in cell samples taken at various times during anaerobic cultivation of P. aeruginosa from lag through late exponential growth phases. Nitrate reductase activity was detected at approximately 3 h after inoculation of the anaerobic cultures, increased to maximal activity in midexponential growth, and then sharply declined in the late exponential phase (Fig. 3). Nitrite reductase activity followed a similar course, and maximum activity was demonstrated at approximately the same point in midgrowth (Fig. 3). It was not possible, however, to detect nitrite reductase activity earlier than 7 h after inoculation, which coincided with the beginning of exponential growth. The maxima of both enzyme activities closely coincided with the point at which the
with its disappearance from the medium and with the initiation of an increased growth rate. Transport of glucose in cells grown aerobically versus anaerobically and harvested at different growth phases. The rate of anaerobic glucose transport (with nitrate as the terminal electron acceptor) by cells harvested at various stages of anaerobic growth was determined. Aerobic uptake rates were also measured for glucose in cells collected at various stages of aerobic growth. The rate at which P. aeruginosa actively transported glucose under denitrifying conditions increased slowly during the early exponential phase until an absorbance at 540 nm of 0.3 was attained (Fig. 4). At this point, the rate of uptake increased sharply, more than doubling during the time in which the culture increased to an absorbance at 540 nm of 0.4. Uptake then dropped off slightly and maintained a steady rate (about 75% of the maximal) throughout the late exponential growth phase. With aerobically grown cells the rate of aerobic glucose transport was characterized by a gradual increase to the maximal rate during late exponential growth followed by a sharp decline thereafter (Fig. 4). In comparison, moreover, it was noted that the maximal rate of glucose
261
DENITRIFYING P. AERUGINOSA
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transport occurred later in the exponential phase acceptor and that the rate of transport was under aerobic conditions than under anaerobic greater under aerobic conditions than under anconditions with nitrate as the terminal electron aerobic conditions. a c)
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P TIME (h) FIG. 3. Production of nitrate and nitrite reductases, accumulation of nitrite in the medium, and glucose uptake rate by cells of P. aeruginosa during denitrifying growth. Initial glucose uptake rates were approximated by measuring glucose taken up after incubation for 15 s. Symbols: A, growth curve during denitrifying growth; 0, nitrite accumulation in the medium; A, anaerobic glucose uptake rate with 1 mM nitrate as the terminal electron acceptor; 0, nitrate reductase activity; 0, nitrite reductase activity.
TIME (h)
FIG. 4. Aerobic glucose uptake by aerobically grown P. aeruginosa and anaerobic glucose uptake by P. aeruginosa grown under denitrifying conditions. Initial transport rates were approximated by measuring glucose uptake after incubation for 15 Symbols: A, aerobic growth curve; 0, aerobic glucose uptake; A, anaerobic (denitrifying) growth curve; 0, anaerobic glucose uptake with 1 mM nitrate as the terminal electron s.
acceptor.
262
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Comparison of glucose and gluconate transport rates of cells grown aerobically versus anaerobically. P. aeruginosa was cultured aerobically and anaerobically with glucose or gluconate as the major carbon and energy sources. Yeast extract (0.1%) was added to the anaerobic cultures. Bacteria were assayed for uptake of the substrate on which they were grown under aerobic and anaerobic conditions. Nitrate (1 mM) was provided as the terminal electron acceptor for anaerobic assays. Cells grown anaerobically carried out transport of glucose under both aerobic and anaerobic conditions (Fig. 5). The aerobic uptake rate of glucose by anaerobically grown cells was significantly higher than seen under anaerobic conditions. Aerobically grown cells, however, could not transport glucose anaerobically with nitrate as an electron acceptor. In all cases, transport rates were greater under aerobic conditions than under anaerobic conditions. When gluconate was used instead of glucose, similar patterns of transport were seen (data not shown). DISCUSSION Optimal growth of P. aeruginosa (defined on the basis of greatest cell yield) was supported by 100 mM nitrate in a variety of media under anaerobic (i.e., denitrifying) conditions. When nitrate was used as the terminal electron acceptor under anaerobic conditions, however, it was found that 1 mM was the optimal concentration to support the transport of glucose. The optimal concentration of nitrite as the terminal electron acceptor to support the anaerobic transport of glucose was also 1 mM. The rate of transport was less, however, when nitrite was used as the terminal electron acceptor than when nitrate was used. Nitrite, on the other hand, supported only slow growth when added to media in concentrations of less than 15 mM. We previously determined that nitrite in concentrations greater than 10 mM exerted toxicity toward cells of P. aeruginosa by inhibiting respiration, apparently by preventing the flow of electrons through the terminal electron transport chain (J. J. Rowe, T. W. Hodge III, and R. G. Eagon, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, K226, p. 223). Moreover, in the presence of 10 mM or higher concentrations of nitrite, P. aeruginosa was inhibited in the ability to carry out active transport and oxidative phosphorylation. Thus, it is not surprising that nitrite was not an electron acceptor of choice to support anaerobic (denitrifying) growth except at low
EC
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60
120
180
240
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TIME (s)
FIG. 5. Glucose uptake measured under aerobic and anaerobic conditions by P. aeruginosa grown aerobically and under anaerobic (denitrifying) conditions. Symbols: U, aerobically grown cells, aerobic glucose uptake; 0, anaerobically grown cells, aerobic glucose uptake; A, anaerobically grown cells, anaerobic glucose uptake with I mM nitrate as the terninal electron acceptor: A, anaerobically grown cells, anaerobic glucose uptake with no added terninal electron acceptor; El aerobically grown cells, anaerobic glucose uptake with 1 mM nitrate as the terminal electron acceptor.
quential induction (or perhaps to sequential derepression [16]) of nitrate and nitrite reductases. Evidence for this is that nitrate reductase activity could be detected 4 h before nitrite reductase activity was observed. Thus, these data confirm the observation of van Hartingsveldt and Stouthamer (21) of sequential synthesis of nitrate and nitrite reductases by P. aeruginosa when subjected to denitrifying conditions of growth. The maximal rate of glucose uptake under denitrifying conditions corresponded to the maximal levels of nitrate and nitrite reductase activities and coincided with the disappearance of nitrite from the medium. We interpret this observation to indicate that active transport rates under denitrifying conditions are directly related to the presence and quantity of nitrate and nitrite reductases. The latter are required for the mediation of such physiological phenomena as electron acceptance and energy generation. The rate of glucose transport by P. aeruginosa, however, was less when nitrate was used concentrations. Transient accumulation of nitrite was ob- as the terminal electron acceptor as compared served when P. aeruginosa was grown under with when oxygen was used as the terminal denitrifying conditions, apparently owing to se- electron acceptor. John and Whatley (8) re-
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ported that in the case of Micrococcus (Paracoccus) denitrificans ATP synthesis coupled with oxygen was about 70% more efficient than that coupled with nitrate reduction to nitrite. Similarly, Koike and Hattori (10) reported that the growth yield per mole of electrons transported by Pseudomonas denitrificans under denitrifying conditions was about one-half that under aerobic conditions. Thus, our present observations further support the idea that anaerobic respiration is less efficient in energy generation than are aerobic systems. ACKNOWLEDGMENIS This study was supported by National Science Foundation research grants BMS74-14819 and PCM77-02928.
LITERATURE CITED 1. Bovell, C. 1967. The effect of sodium nitrite on the growth
of Micrococcus denitrificans. Arch. Mikrobiol. 59:13-19. 2. Eagon, R. G., and P. V. Phibbs, Jr. 1971. Kinetics of transport of glucose, fructose and mannitol by Pseudomonas aeruginosa. Can. J. Biochem. 49:1031-1041. 3. Elliott, L F., and C. IL Gilmour. 1971. Growth of Pseudomonas stutzeri with nitrate and oxygen as terminal electron acceptors Soil Biol. Biochem. 3:331-335. 4. Fewson, C. A., and D. J. D. Nicholas. 1961. Nitrate reductase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 49:335-349. 5. Focht, D. D., and W. Verstraete. 1977. Biochemical ecology of nitrification and denitrification. Adv. Microb. Ecol. 1:135-214. 6. Gamble, T. N., ML R. Betlach, and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. 33:926-939. 7. Guymon, L. F., and R. G. Eagon. 1974. Transport of glucose, gluconate, and methyl a-D-glucoside by Pseudomonas aeruginosa. J. Bacteriol. 117:1261-1269. 8. John, R., and F. R. Whatley. 1970. Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans. Biochim. Biophys. Acta 216:342-352. 9. King, T. E. 1966. Glucose dehydrogenase-particulate. II.
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Acetobacter suboxydans. Methods Enzymol. 9:98-103. 10. Koike, I., and A. Hattori. 1975. Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions. J. Gen. Microbiol. 88:1-10. 11. Konings, W. N., J. Boonstra, and W. de Vries. 1975. Amino acid transport in membrane vesicles of obligately anaerobic Veillonella akalescens. J. Bacteriol.
L22:245-249. 12. Konings, W. N., and H. R. Kaback. 1973. Anaerobic transport in Escherichia coli membrane vesicles. Proc. Natl. Acad. Sci. U.S.A. 70:3376-3381. 13. Losada, M., and A. Paneque. 1971. Nitrate reductase. Methods Enzymol. 23:487-491. 14. Lowe, R. H., and H. J. Evans. 1964. Preparation and some properties of a soluble nitrate reductase from Rhizobium japonicum. Biochim. Biophys. Acta
85:377-389. 15. Mokrasch, L C., W. D. Davidson, and R. W. McGilvery. 1956. Purification properties of fructose-1,6diphosphatase. J. Biol. Chem. 221:909-919. 16. Payne, W. J. 1976. Denitrification. Trends Biochem. Sci. 1:220-222. 17. Phibbs, P. V., Jr., and R. C. Eagon. 1970. Transport and phosphorylation of glucose, fructose and mannitol by Pseudomonas aeruginosa. Arch. Biochem. Biophys. 138:470-482. 18. Rowe, J. J., B. F. Sherr, W. J. Payne, and R. G. Eagon. 1977. A unique nitric oxide-binding complex formed by denitifying Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 77:253-258. 19. Sawyer, M. H., P. Baumann, L Baumann, S. M. Berman, J. L Canovas, and R. H. Berman. 1977. Pathways ofD-fructose catabolism in species of Pseudomonas. Arch. Microbiol. 112:49-55. 20. Strickland, J. D. H., and T. R. Parsons. 1968. A practical handbook of seawater analysis. Bull. 167. Fisheries Research Board of Canada, Ottawa. 21. van Hartingsveldt, J., and A. H. Stouthamer. 1973. Mapping and characterization of mutants of Pseudomonas aeruginosa affected in nitrate respiration in aerobic or anaerobic growth. J. Gen. Microbiol. 74:97-106. 22. Van't Riet, J., A. H. Stouthamer, and R. J. Planta. 1968. Regulation of nitrate assimilation and nitrate respiration in Aerobacter aerogenes. J. Bacteriol. 96:1455-1464.
