APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, p. 257-2630099-2240/78/0036-0257$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 36, No.2

Printed in U.S.A.

Denitrifying Pseudomonas aeruginosa: Some Parameters ofGrowth and Active Transport

D. R. WILLIAMS, J. J. ROWE,t P. ROMERO,4 AND R. G. EAGON*Department ofMicrobiology, University of Georgia, Athens, Georgia 30602

Received for publication 10 February 1978

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying con-ditions was obtained with 100 mM nitrate as the terminal electron acceptor,irrespective of the medium used. Nitrite as the terminal electron acceptor sup-ported poor denitrifying growth when concentrations of less than 15 mM, but nothigher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accu-mulated in the medium during early exponential phase when nitrate was theterminal electron acceptor and then decreased to extinction before midexponentialphase. The maimal rate of glucose and gluconate transport was supported by 1mM nitrate or nitrite as the terminal electron acceptor under anaerobic condi-tions. The transport rate was greater with nitrate than with nitrite as the terminalelectron acceptor, but the greatest transport rate was observed under aerobicconditions with oxygen as the terminal electron acceptor. When P. aeruguiosawas inoculated into a denitrifying environment, nitrate reductase was detectedafter 3 h of incubation, nitrite reductase was detected after another 4 h ofincubation, and maximal nitrate and nitrite reductase activities peaked togetherduring midexponential phase. The latter coincided with maximal glucose trans-port activity.

The anaerobic reduction of nitrate and nitriteto nitrous oxide or elemental nitrogen is termeddenitrification, and the physiological process iscalled anaerobic respiration. Denitrification isimportant because of its role in the regenerationof fixed nitrogen. It has become of particularinterest in recent years due to increased cost ofnitrogen fertilizer and potential reduction ofcropyields because of this microbiological phenome-non (16). In addition, the generation of gaseousnitrogen oxides has become a concern becauseof the potential effect on the ozone layer of theupper atmosphere (16). With these factors asimpetus, basic knowledge in the area of denitri-fication has expanded rapidly in a relativelyshort time.The most predominant denitifying bacteria

in our environment have been reported to belongto the genus Pseudomonas (6). (It should benoted, however, that in other instances Akcali-genes has been found to be the dominant deni-trifler [5].) Species of Pseudomonas are nonfer-menting organisms capable of generating energyonly by respiration. The mechanism(s) and reg-ulation of electron flow and ATP synthesis un-der denirifying conditions are considered to be

t Present addres: Department of Biology, University ofDayton, Dayton, OH 45469.

t Permanent addreu Department ofMicrobiology, Facultyof Pharmacy, University of Granada, Granada, Spain.

processes similar to aerobic respiration. It isclear, however, that different spectra of cyto-chromes and enzymes are required for anaerobicrespiration (16). The process of denitrification isthought to occur in a stepwise manner as follows:N03 -- N02 -- NO - N20 -- N2. As would beexpected, many of the enzymes of the pathwayare closely associated with cytochromes inPseudomonas aeruginosa. Nitrate reductase re-quires association with cytochrome c (4), nitritereductase is analogous to cytochrome cd, andnitric oxide reduction appears to be associatedwith a 570-nm-absorbing pigment (18).

Several themes run constant. The absence ofoxygen derepresses the enzymes necessary fordenitrification (3, 22). Once derepressed, thequantity of these enzymes is directly affected bythe initial nitrate concentration in the culture(3). Anaerobiosis diminishes the a-type cyto-chromes drastically and stimulates productionof c-type cytochromes in most denitifying bac-teria (16). Finally, nitrite accumulates in theculture media, usually before the onset of visiblegas production. Nitrite may or may not inhibitfurther reduction of nitrogen oxides, dependingon the species of bacteria and the culture con-ditions (1, 21).One important denitrification aspect which

has been largely ignored is the transport of nu-trients by denitrifiers during anaerobic respira-

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258 WILLIAMS ET AL.

tion and its relationship to the enzymes and thecytochrome system. Evidence has been pre-sented to demonstrate coupling between anaer-obic transport of lactose and amino acids andanaerobic electron transfer in isolated mem-brane vesicles of Escherichia coli (12). Morerecently, it has been shown that amino acidtransport can be coupled to electron transfer byusing nitrate as an electron acceptor in the ob-ligate anaerobe Veillonella alcalescens (11).Several energy donors and components of themembrane-bound anaerobic electron transportchain of V. alcalescens were also identified.Virtually no information is available, however,on the active transport of solutes in bacteriagrowing under denitrifying conditions.

P. aeruginosa possesses an inducible activetransport system for glucose as well as inducibleenzymes for the Enter-Doudoroff pathway andthe oxidative portion of the hexose monophos-phate-pentose cycle (7, 17). Guymon and Eagon(7), moreover, demonstrated that in P. aerugi-nosa glucose and gluconate were actively trans-ported as the free sugars. Evidence has beenfound, however, which implicates the phospho-enolpyruvate-phosphotransferase system as themechanism by which fructose is transported byPseudomonas species (19). Thus, with the soleexception of fructose, sugars are considered tobe accumulated by a carrier-mediated activetransport system in P. aeruginosa.

In the present study we characterized aspectsof the transport of glucose and gluconate bydenitrifying P. aeruginosa. These results werecompared with those of the aerobically respiringorganism. We also determined the optimal con-centrations of nitrate and nitrite for denitifyinggrowth and for optimal rates of anaerobic trans-port of glucose and gluconate.

MATERIALS AND METHODSOrganism and culture conditions. P. aerugunosa

PAO (formerly Holloway strain 1) was maintained onagar slants prepared by using the basal salts mediumpreviously described (2) containing 11 mM glucose.Starter cultures for all transport and enzyme assayswere grown aerobically as follows. Erlenmeyer flasks(500 ml) containing 200 ml of minimal salts supple-mented with 11 mM glucose were inoculated from 18-h agar slants and incubated with rotary shaking (200rpm) at 30°C for 6 to 8 h to exponential phase beforeintroduction into experimental culture flasks.

Inocula for anaerobic experiments were asepticallyharvested from starter cultures by centrifuging duringearly exponential growth. The whole-cell pellet from200 ml of starter culture was used to initiate anaerobiccultivation in 1-liter stoppered Erlenmeyer flasks filledto capacity and fitted with two ports to allow gasevolution and sampling of the culture. Bacteria weregrown anaerobically at 300C in basal salts medium (2)

containing 1.0% KNO3 and 0.1% yeast extract sterilizedby autoclaving. Concentrated, filter-sterilized solu-tions of D-glucose or D-gluconate were added in finalconcentrations of 11 mM. Oxygen was excluded fromthe flasks by bubbling nitrogen through the culture for5 min before incubation. The culture was stirred slowlywith a magnetic bar and stirrer to prevent clumping.Modifications to the media during initial growth char-acterization are described below when appropriate.

P. aeruginosa used for aerobic transport experi-ments were grown aerobically in 1-liter Erlenmeyerflasks containing 200-ml of basal salts medium con-taining 11 mM glucose. Addition of the carbon sourceand incubation were accomplished as with anaerobicgrowth experiments. The cultures were inoculatedwith 20 ml from early-exponential-growth-phasestarter 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 mediumcontaining yeast extract or in tryptic soy broth (DifcoLaboratories, Detroit, Mich.).

Nitrite determinations. Samples (5 ml) weretaken from anaerobically growing cultures at timedintervals, the absorbance of the culture was deter-mined at 540 nm with a Spectronic 20 spectrophotom-eter (Bausch & Lomb Inc., Rochester, N.Y.), and thecells were removed by centrifuging. Nitrite in themedium was determined by the method of Stricklandand Parsons (20) with an Hitachi-Perkin-Elmer 139UV-VIS spectrophotometer (Perkin-Elmer ColemanInstruments Div., Oak Brook, Ill.).Active transport assays. Cells were harvested by

centrifuging at 250C, washed in glucose-free basal saltssolution containing 50 ,ug of chloramphenicol, per ml,and suspended in glucose-free basal salts solution with50 jg of chloramphenicol per ml at a density of 1 g(wet weight) per 20 ml. This cell suspension was usedfor transport assays.

Transport of ['4C]glucose and ['4C]gluconate by P.aeruginosa was determined under aerobic conditionsat 30°C on a reciprocal shaking water bath. Each 10-ml Erlenmeyer reaction flask contained in a finalconcentration 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 givea final volume of 1 ml. The reaction was initiated bythe addition of labeled substrates.

Identical volumes, concentrations, and specific ac-tivities of radioactive substrates were used in anaero-bic transport experiments. Uptake was determined at300C in stoppered tubes fitted with two ports to allowconstant flushing with nitrogen gas and sample re-moval. Potassium nitrate or sodium nitrite was addedin a final concentration of 1 mM. Addition of the cellsuspension, preincubated anaerobically, was used toinitiate the reaction.

In aerobic and anaerobic assays, 0.05-ml sampleswere withdrawn at specific time intervals and deliv-ered over membrane filters (25-mm diameter, 0.45-pmpore size; Amicon Corp., Lexington, Mass.) previouslyoverlaid with 1 ml of 0.1 M LiCl, filtered rapidly, andwashed immediately with an additional 5 ml of 0.1 MLiCl. The washed membrane filters bearing the cellswere transfenred immediately to vials containing 10 ml

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DENITRIFYING P. AERUGINOSA 259

of scintillation fluid and counted in a Packard liquidscintillation spectrometer, (model 527; Packard Instru-ment Co., Inc., Downers Grove, Ill.) as previouslydescribed (17).

Metabolizable substrates and intact, wild-type cellsof P. aeruginosa were used for our transport studiesin spite of obvious objections. We have shown previ-ously that initial rates of transport of metabolizablesubstrates by intact, wild-type cells were identical tothose of membrane vesicles or glucose-negative mu-tants and, that the glucose transport system had suchlow affinity for non-metabolizable glucose analogs thatthe results gained from the use of analogs did notreflect true kinetics of glucose transport (7). In allexcept one experiment reported herein, initial rates oftransport were measured. The results thus gained,therefore, are considered to be an accurate measure oftransport.

Nitrate and nitrite reductase (dissimilatory)assays. Cells were collected by centrifuging at 25°C,washed in minimal salts medium containing 50 ,ug ofchloramphenicol per ml, and suspended at a densityof 1 g (wet weight) per ml in ice-cold basal saltscontaining 50 ug of benzyl viologen per ml. Cell ex-tracts were prepared by two passages through an ice-cold French pressure cell. The nitrate and nitrite re-ductases in extracts were protected from oxygen bythe dropwise addition of a 1.6% Na2S204-1.6%NaHCO3 mixte, with the purple benzyl viologenserving an an indicator of the reduced state (14).Whole cells were removed with minimal loss of mem-brane fragments from the broken-cell suspension bylow-speed centrifuging at room temperature. The pro-tein concentration was adjusted to 0.5 to 1.0 mg/mlwith basal salts, and nitrate or nitrite reductase activ-ity was determined. The nitrate and nitrite reductaseswere stable for 2 h when extracts were stored on ice.Enzyme assay mixtures contained in a final concen-

tration: 0.5 ml of cell extract, 20 mM potassium phos-phate buffer (pH 7.0), 10 mM KNO3, and 0.1 mMbenzyl viologen or 10 mM NaNO2, 0.2 mM methyleneblue, and a mixture of 32% Na3S305 and 3.2% NaHCO3(final concentration) in a final volume of 2.5 ml. Theextracts were incubated at 30°C for 5 min and flushedwith nitrogen gas before initiation of the reaction byaddition of substrate. Excess reducing agent ensuredreduced conditions throughout the assay. Samples (0.5ml) were removed at timed intervals, added to anequal volume ofice-cold ethanol, and mixed vigorouslywith a Vortex mixer until oxidized to stop the reaction(13). Nitrite concentration was then determined bythe method of Strickland and Parsons (20).Oxygen trap. Nitrogen gas used in anaerobic ex-

periments was sparged through a stoppered tube (250ml) containing 0.2% methyl viologen reduced withpowdered zinc to remove all traces of oxygen.Protein determinations. Protein determinations

in whole cells were made by a modified biuret proce-dure (9). Urea (60%) was used to facilitate cell lysis. Asemimicro-biuret method was employed for proteindeterminations in broken-cell extracts (15).

Reagents. D-[U-14C]glucose (327 mCi/mmol) andD-[U-_4C]gluconate (3.9 mCi/mmol) were purchasedfrom Amersham/Searle, Arlington Heights, M. Violo-gen dyes were purchased from Schwarz/Mann, Or-

angeburg, N.Y.; sulfanilamide was purchased fromEastman Organic Chemical Div., Eastman-Kodak Co.,Rochester, N.Y.; and N-(l-naphthyl)ethylenediaminedihydrochloride and chloramphenicol were purchasedfrom Sigma Chemical Co., St. Louis, Mo. All othermaterials were purchased from commercial sources inthe highest state of purity.

RESULTS

Optimal concentrations of nitrate and ni-trite to support anaerobic growth. Experi-ments were carried out to determine the optimalconcentration of nitrate required to supportmaximal growth of P. aeruginosa when culti-vated anerobically under denitrifying conditions.Three different media were used: glucose-basalsalts, glucose-yeast extract-basal salts, and tryp-tic soy broth. In each case, 100 mM nitratesupported the maximal yields of cells (Fig. 1).Thus, for all subsequent experiments a finalconcentration of 100 mM nitrate was used inmedia for the anaerobic cultivation of P. aerugi-nosa.

Nitrite was not as satisfactory as an electronacceptor to support the growth of P. aeruginosaunder anaerobic denitrifying conditions as wasnitrate. Only slow growth resulted when the finalconcentration of nitrite was kept below 15 mM,and no growth occurred at higher concentrationsof nitrite (data not shown).Optimal concentrations of nitrate and ni-

trite to support active transport under an-aerobic conditions. P. aeruginosa transportedglucose under anaerobic conditions when eithernitrate or nitrite was used as the terminal elec-tron acceptor. The concentration of each nitro-gen oxide that supported the greatest rate oftransport was 1 mM (Fig. 2). The rate of trans-port rapidly decreased as higher concentrationsof the nitrogen oxides were used. However, ni-trate supported transport of glucose more effec-tively than did nitrite at all concentrations. Fi-nally, whereas 1 mM nitrate was optimal fortransport ofglucose, 100mM nitrate was optimalfor maximnal cell yield under growing conditions.

Nitrite accumulation in media containingnitrate as the terminal electron acceptorunder denitrifying conditions. Nitrite deter-minations were performed on samples of culturemedia taken at various time intervals through-out the growth cycle. Nitrite accumulated rap-idly during the early exponential growth phaseto concentrations of 1.6 to 3.0 mM in simplemedia and 8 mM in complex media and thendeclined rapidly to extinction at the midexpo-nential phase (Fig. 3). Significantly, an increasedgrowth rate occurred upon disappearance of ni-trite 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 salts-

glucose (A), basal salts-glucose-yeast extract, (B), and tryptic soy broth (C).

NITROGEN OXIDE (mM)FIG. 2. Support of anaerobic glucose uptake in P.

aeruginosa by varying concentrations of nitrate andnitrite as terminal electron acceptors. Initial trans-port rates were approximated by measuring glucoseuptake after incubation for 15 s. Symbols: 0, nitrate;0, nitrite.

glucose transport activity in cells har-vested at different growth phases. Nitrateand nitrite reductases were assayed in cell sam-ples taken at various times during anaerobiccultivation of P. aeruginosa from lag throughlate exponential growth phases. Nitrate reduc-tase activity was detected at approximately 3 hafter inoculation of the anaerobic cultures, in-creased to maximal activity in midexponentialgrowth, and then sharply declined in the lateexponential phase (Fig. 3). Nitrite reductase ac-tivity followed a similar course, and maximum

activity was demonstrated at approximately thesame point in midgrowth (Fig. 3). It was notpossible, however, to detect nitrite reductaseactivity earlier than 7 h after inoculation, whichcoincided with the beginning of exponentialgrowth. The maxima of both enzyme activitiesclosely coincided with the point at which the

rate of growth increased. The assays were linearover time and with respect to protein concentra-tion. Saturating concentrations of both sub-strates were employed. Parallel experimentsconducted in the absence of substrate or enzymedemonstrated little or no endogenous or nonspe-cific reduction of nitrate or nitrite during theassays.The maximal rate of glucose uptake under

denitrifying conditions corresponded closely tothe maximal levels of nitrate and nitrite reduc-tase activities observed during growth (Fig. 3).All three of these maxima were observed within2 h after the peak of maximal accumulation ofnitrite was seen in the medium and coincidedwith its disappearance from the medium andwith the initiation of an increased growth rate.Transport of glucose in cells grown aer-

obically versus anaerobically and har-vested at different growth phases. The rateof anaerobic glucose transport (with nitrate asthe terminal electron acceptor) by cells har-vested at various stages of anaerobic growth wasdetermined. Aerobic uptake rates were also mea-sured for glucose in cells collected at variousstages of aerobic growth. The rate at which P.aeruginosa actively transported glucose underdenitrifying conditions increased slowly duringthe early exponential phase until an absorbanceat 540 nm of 0.3 was attained (Fig. 4). At thispoint, the rate ofuptake increased sharply, morethan doubling during the time in which theculture increased to an absorbance at 540 nm of0.4. Uptake then dropped off slightly and main-tained a steady rate (about 75% of the maximal)throughout the late exponential growth phase.With aerobically grown cells the rate of aero-

bic glucose transport was characterized by agradual increase to the maximal rate during lateexponential growth followed by a sharp declinethereafter (Fig. 4). In comparison, moreover, itwas noted that the maximal rate of glucose

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DENITRIFYING P. AERUGINOSA 261

transport occurred later in the exponential phaseunder aerobic conditions than under anaerobicconditions with nitrate as the terminal electron

acceptor and that the rate of transport was

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FIG. 3. Production of nitrate and nitrite reductases, accumulation of nitrite in the medium, and glucoseuptake rate by cells of P. aeruginosa during denitrifying growth. Initial glucose uptake rates were approxi-mated by measuring glucose taken up after incubation for 15 s. Symbols: A, growth curve during denitrifyinggrowth; 0, nitrite accumulation in the medium; A, anaerobic glucose uptake rate with 1 mM nitrate as theterminal 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 measuringglucose uptake after incubation for 15 s. 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 electronacceptor.

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262 WILLIAMS ET AL.

Comparison of glucose and gluconatetransport rates of cells grown aerobicallyversus anaerobically. P. aeruginosa was cul-tured aerobically and anaerobically with glucoseor gluconate as the major carbon and energysources. Yeast extract (0.1%) was added to theanaerobic cultures. Bacteria were assayed foruptake of the substrate on which they weregrown under aerobic and anaerobic conditions.Nitrate (1 mM) was provided as the terminalelectron acceptor for anaerobic assays. Cellsgrown anaerobically carried out transport of glu-cose under both aerobic and anaerobic condi-tions (Fig. 5). The aerobic uptake rate of glucoseby anaerobically grown cells was significantlyhigher than seen under anaerobic conditions.Aerobically grown cells, however, could nottransport glucose anaerobically with nitrate asan electron acceptor. In all cases, transport rateswere greater under aerobic conditions than un-der anaerobic conditions. When gluconate wasused instead of glucose, similar patterns of trans-port were seen (data not shown).

DISCUSSIONOptimal growth of P. aeruginosa (defined on

the basis of greatest cell yield) was supported by100 mM nitrate in a variety of media underanaerobic (i.e., denitrifying) conditions. Whennitrate was used as the terminal electron accep-tor under anaerobic conditions, however, it wasfound that 1 mM was the optimal concentrationto support the transport of glucose.The optimal concentration of nitrite as the

terminal electron acceptor to support the anaer-obic transport of glucose was also 1 mM. Therate of transport was less, however, when nitritewas used as the terminal electron acceptor thanwhen nitrate was used. Nitrite, on the otherhand, supported only slow growth when addedto media in concentrations of less than 15 mM.We previously determined that nitrite in concen-trations greater than 10 mM exerted toxicitytoward cells of P. aeruginosa by inhibiting res-piration, apparently by preventing the flow ofelectrons through the terminal electron trans-port chain (J. J. Rowe, T. W. Hodge III, and R.G. Eagon, Abstr. Annu. Meet. Am. Soc. Micro-biol. 1977, K226, p. 223). Moreover, in the pres-ence of 10 mM or higher concentrations of ni-trite, P. aeruginosa was inhibited in the abilityto carry out active transport and oxidative phos-phorylation. Thus, it is not surprising that nitritewas not an electron acceptor of choice to supportanaerobic (denitrifying) growth except at lowconcentrations.

Transient accumulation of nitrite was ob-served when P. aeruginosa was grown underdenitrifying conditions, apparently owing to se-

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TIME (s)FIG. 5. Glucose uptake measured under aerobic

and anaerobic conditions by P. aeruginosa grownaerobically and under anaerobic (denitrifying) con-ditions. Symbols: U, aerobically grown cells, aerobicglucose uptake; 0, anaerobicallygrown cells, aerobicglucose uptake; A, anaerobically grown cells, anaer-obic glucose uptake with ImM nitrate as the terninalelectron acceptor: A, anaerobically grown cells, an-aerobic glucose uptake with no added terninal elec-tron acceptor; El aerobically grown cells, anaerobicglucose uptake with 1 mM nitrate as the terminalelectron acceptor.

quential induction (or perhaps to sequential de-repression [16]) of nitrate and nitrite reductases.Evidence for this is that nitrate reductase activ-ity could be detected 4 h before nitrite reductaseactivity was observed. Thus, these data confirmthe observation of van Hartingsveldt and Stou-thamer (21) of sequential synthesis of nitrateand nitrite reductases by P. aeruginosa whensubjected to denitrifying conditions of growth.The maximal rate of glucose uptake under

denitrifying conditions corresponded to the max-imal levels of nitrate and nitrite reductase activ-ities and coincided with the disappearance ofnitrite from the medium. We interpret this ob-servation to indicate that active transport ratesunder denitrifying conditions are directly relatedto the presence and quantity of nitrate andnitrite reductases. The latter are required forthe mediation of such physiological phenomenaas electron acceptance and energy generation.The rate of glucose transport by P. aerugi-

nosa, however, was less when nitrate was usedas the terminal electron acceptor as comparedwith when oxygen was used as the terminalelectron acceptor. John and Whatley (8) re-

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VOL. 36, 1978

ported that in the case of Micrococcus (Para-coccus) denitrificans ATP synthesis coupledwith oxygen was about 70% more efficient thanthat coupled with nitrate reduction to nitrite.Similarly, Koike and Hattori (10) reported thatthe growth yield per mole of electrons trans-ported by Pseudomonas denitrificans underdenitrifying conditions was about one-half thatunder aerobic conditions. Thus, our present ob-servations further support the idea that anaer-obic respiration is less efficient in energy gener-ation than are aerobic systems.

ACKNOWLEDGMENISThis study was supported by National Science Foundation

research grants BMS74-14819 and PCM77-02928.

LITERATURE CITED1. Bovell, C. 1967. The effect ofsodium nitrite on the growth

of Micrococcus denitrificans. Arch. Mikrobiol.59:13-19.

2. Eagon, R. G., and P. V. Phibbs, Jr. 1971. Kinetics oftransport of glucose, fructose and mannitol by Pseudo-monas aeruginosa. Can. J. Biochem. 49:1031-1041.

3. Elliott, L F., and C. IL Gilmour. 1971. Growth ofPseudomonas stutzeri with nitrate and oxygen as ter-minal electron acceptors Soil Biol. Biochem. 3:331-335.

4. Fewson, C. A., and D. J. D. Nicholas. 1961. Nitratereductase from Pseudomonas aeruginosa. Biochim.Biophys. Acta 49:335-349.

5. Focht, D. D., and W. Verstraete. 1977. Biochemicalecology 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 worldsoils. Appl. Environ. Microbiol. 33:926-939.

7. Guymon, L. F., and R. G. Eagon. 1974. Transport ofglucose, gluconate, and methyl a-D-glucoside byPseudomonas aeruginosa. J. Bacteriol. 117:1261-1269.

8. John, R., and F. R. Whatley. 1970. Oxidative phospho-rylation coupled to oxygen uptake and nitrate reductionin Micrococcus denitrificans. Biochim. Biophys. Acta216:342-352.

9. King, T. E. 1966. Glucose dehydrogenase-particulate. II.

DENITRIFYING P. AERUGINOSA 263

Acetobacter suboxydans. Methods Enzymol. 9:98-103.10. Koike, I., and A. Hattori. 1975. Growth yield of a deni-

trifying bacterium, Pseudomonas denitrificans, underaerobic 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 ofobligatelyanaerobic Veillonella akalescens. J. Bacteriol.L22:245-249.

12. Konings, W. N., and H. R. Kaback. 1973. Anaerobictransport 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 andsome properties of a soluble nitrate reductase fromRhizobium japonicum. Biochim. Biophys. Acta85:377-389.

15. Mokrasch, L C., W. D. Davidson, and R. W. Mc-Gilvery. 1956. Purification properties of fructose-1,6-diphosphatase. 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. Transportand phosphorylation of glucose, fructose and mannitolby 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 complexformed by denitifying Pseudomonas aeruginosa. Bio-chem. 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 ofPseudo-monas. Arch. Microbiol. 112:49-55.

20. Strickland, J. D. H., and T. R. Parsons. 1968. A prac-tical handbook of seawater analysis. Bull. 167. FisheriesResearch Board of Canada, Ottawa.

21. van Hartingsveldt, J., and A. H. Stouthamer. 1973.Mapping and characterization of mutants of Pseudo-monas aeruginosa affected in nitrate respiration inaerobic 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 nitraterespiration in Aerobacter aerogenes. J. Bacteriol.96:1455-1464.

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