APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1983, p. 1247-12530099-2240/83/041247-07$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 45, No. 4

Comparison of Denitrification by Pseudomonas stutzeri,Pseudomonas aeruginosa, and Paracoccus denitrificans

CURTIS A. CARLSON AND JOHN L. INGRAHAM*Bacteriology Department, University of California, Davis, California 95616

Received 29 July 1982/Accepted 29 December 1982

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudo-monas aeruginosa, and Paracoccus denitrjjicans. Although all three organismsreduced nitrate to dinitrogen gas, they did so at different rates and accumulateddifferent kinds and amounts of intermediates. Their rates of anaerobic growth onnitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold.Cell yields from nitrate varied threefold. Rates of gas production by resting cellsincubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold,respectively, among the three species. The composition of the gas produced alsovaried markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonasaeruginosa and Paracoccus denitrificans produced nitrous oxide as well; andunder certain conditions Pseudomonas aeruginosa produced even more nitrousoxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidlyreduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobicallywhen any of these nitrogen oxides were present in the medium. Pseudomonasaeruginosa reduced these oxides slowly and was unable to grow anaerobically atthe expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction byPseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite.Thus, although it has been well studied physiologically and genetically, Pseudo-M"onas aeruginosa may not be the best species for studying the later steps of thedenitrification pathway.

Denitrification is the anaerobic reduction of afixed nitrogen oxide, coupled with respiratorygeneration of ATP and release of a gas. Theprocess has been the topic of several recentreviews (6, 15, 24, 25, 27, 38). Complete denitri-fication is thought to involve the sequentialreduction of nitrate, nitrite, nitric oxide, andnitrous oxide to dinitrogen gas. A variety ofincomplete denitrification pathways also exist:some denitrifying bacteria reduce both nitrateand nitrite; others reduce only nitrite. Someproduce only dinitrogen; some produce a mix-ture of dinitrogen and nitrous oxide; othersproduce only nitrous oxide. Even within a singlespecies, Pseudomonasfluorescens, the biotypesdiffer in the end product of the pathway (11). In aspecies of Vibrio, only nitrate and nitrous oxideare reduced (41). Such diversity, in a sense anatural set of mutant phenotypes, has contribut-ed to our understanding of the intermediates ofdenitrification and the significance of each re-ductive step. However, it is becoming increas-ingly evident that significant differences exist inthe rate and regulation of each step even amongorganisms that possess the complete pathway(i.e., reduction of nitrate to dinitrogen). Knowl-edge about such differences may help in choos-

ing the appropriate organism for particular deni-trification studies. Pseudomonas aeruginosa hasbeen extensively studied genetically and there-fore is often considered a favorable organism foruse in studies on denitrification that involve theanalysis of mutant strains. However, it may notbe completely desirable. We undertook thisstudy to compare several aspects of denitrifica-tion in Pseudomonas aeruginosa with two otherorganisms capable of complete denitrification,Paracoccus denitrificans and Pseudomonasstutzeri. The latter appeared to be better suitedfor our physiological genetic studies concentrat-ing on nitrous oxide reduction.

MATERIALS AND METHODSMaterials. Nitrous oxide and a mixture of 10%

methane-909o helium were products of Matheson Sci-entific, Inc., Rutherford, N.J. Nitric oxide, helium,and dinitrogen were obtained from Liquid Carbonic,Chicago, Ill. Cleve's acid (a-naphthylamnine-7-sulfonicacid) was from Polysciences, Inc., Warrington, Pa.

Media. The medium used routinely was modifiedLuria-Bertani (LB) broth (5), consisting of (per liter):10 g of tryptone, 5.0 g of yeast extract, 5.0 g of NaCl,1.0 g of glucose, 10 ml of 1 M Tris-hydrochloride (pH7.5), 1.0 ml of 1 M MgSO4 - 7H20, and water. For

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TIME OF INCUBATION (hr)FIG. 1. Cell growth and denitrification of nitrate. The increase in absorbance (optical density at 650 nm) (0,

left-hand scale) and the total accumulation of the intermediate and final products of denitrification (inmicromoles, right-hand scale) were measured in cultures incubated anaerobically at 37°C with 23 mM sodiumnitrate. (A) Pseudomonas stutzeri. (B) Pseudomonas aeruginosa. (C) Paracoccus denitrificans. Symbols: X,N02-, A, N20; 0, N2-

solid medium, 20 g of Bacto-Agar (Difco Laboratories,Detroit, Mich.) was added.Measurement of denitrification intermediates. Nitrite

was measured by a modification of the method ofNicholas and Nason (23). Samples were mixed vigor-ously into 4 ml of reagent containing 0.25% sulfanilicacid, 0.005% Cleve's acid, and 15% acetic acid, andthe absorbance at 540 nm was measured 30 min later.Gas samples were analyzed with a Perkin-Elmer Sig-ma 4 gas chromatograph equipped with a thermalconductivity detector. Samples were injected onto acolumn (3/16 in. by 18 ft [ca. 0.476 by 550 cm]) ofPorapak Q at 40°C and eluted with helium at a flowrate of 32 ml/min. Retention times (in seconds) of thegases of interest were N2 (173), 02 (183), NO (196),CH4 (280), CO2 (530), N20 (700), and C2H2 (958).Quantitation was aided by a Perkin Elmer M-2 calcu-lating integrator standardized with known gas mix-tures.

Organisms. Pseudomonas stutzeri JM300 was a nat-ural soil isolate, obtained from B. A. Bryan and C. C.Delwiche of this university, characterized as previous-ly described (4a). Pseudomonas aeruginosa PAO1 wasprovided by T. C. Hollocher, Brandeis University.Paracoccus denitrificans ATCC 19367 was obtainedfrom M. K. Firestone, University of California,Berkeley.Growth of cultures. The stock cultures were main-

tained aerobically on LB agar. Cells were routinelygrown anaerobically at 37°C in 70-ml stationary serumvials completely filled with LB broth supplementedwith sodium nitrate. Anaerobic cultures to be assayed

for gas production as well as growth rate were incubat-ed in vials with a 20-ml head space, filled with 90%helium-10%o methane; the latter was used as an inter-nal standard to normalize measurements of gas vol-ume. Samples of culture or gas were removed bysyringe. Alternatively, growth rates alone were mea-sured on anaerobic cultures incubated in half-filledErlenmeyer flasks modified by the addition of a nephe-lometer tube side arm and fitted for gas sparging. Theflasks were made anaerobic by being sparged withhelium or with nitrous oxide when appropriate. Tur-bidity was measured in a Klett-Summerson photoelec-tric colorimeter equipped with a red filter. Dry cellmass was determined by standard methods (10).Assay of denitrification. Cells grown anaerobically to

stationary phase on limiting nitrate were harvested bycentrifugation at 20°C for 10 min at 5,000 x g, washedin fresh LB broth, recentrifuged, and resuspended inLB broth to a cell concentration of about 1011 ml-1.Then 0.3 ml of the suspension was transferred to LBbroth supplemented with 40 mM sodium succinate in a9-ml serum vial, sealed with a rubber septum. Theheadspace was evacuated and regassed with 10%omethane in helium, and a solution of electron acceptorin LB broth was injected to initiate the reaction. Thefinal volume of the cell suspension was 3 ml. Nitrousoxide, as well as nitrate and nitrite, was added as asolution (i.e., nitrous oxide-saturated LB broth) toavoid a lag. The vials were agitated at 150 rpm on arotary shaker at 24°C. Gas samples (100 p.l) werewithdrawn from the headspace with a Hamilton gas-tight syringe; samples for analysis of nitrite were

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TABLE 1. Anaerobic growth of denitrifiers on nitrate

Doubling Gas production' Cell yield' DNR activity'Organism time (h) (nmol of N min- (mg [dry wtl/ (U/mg of protein)mg [dry wtf1') ~ mol of nitrate)

Pseudomonas stutzeri 1.9 85.6 0.034 183Pseudomonas aeruginosa 1.3 287.8 0.028 33.3Paracoccus denitrificans 1.5 33.7 0.078 202

a The sum of volatile nitrogen oxides (i.e. NO, N20, N2) produced during early stationary growth phase. Cellmass was estimated from the optical density converted to dry weight by a standard curve prepared for eachorganism.

b Yield was determined from a series of limiting nitrate concentrations (6 to 45 mM) in LB. The value given isthe average of at least eight determinations.

c Dissimilatory nitrate reductase activity; the in vitro production of nitrite measured in extracts of cellsharvested in exponential growth phase.

withdrawn with a Hamilton liquid syringe. The rate ofdenitrification was proportional to the concentrationof cells in suspension. Each vial was sampled at leastfive times over a period of 2 to 4 h.

Nitrate reductase activity was measured in vitrowith crude extracts prepared by harvesting cells bycentrifugation, suspending them in 50 mM Tris-hydro-chloride buffer (pH 7.5) containing 1 mM EDTA, 5mM 2-mercaptoethanol, 5 mM MgSO4, and 1 mg ofDNase per ml, and breaking them in a French pressurecell, all at 4°C. The production of nitrite was estimatedin the presence of dithionite-reduced benzylviologenas electron donor by a modification of the method ofLowe and Evans (18). The reaction mixture contained,in a final volume of 2 ml, 80 ,umol of potassiumphosphate buffer (pH 7.4), 40 ,umol of NaNO3, 30,umol of benzyl viologen, and cell extract. After sever-al minutes of equilibration at 30°C, the reaction wasinitiated by adding 0.2 ml of a freshly prepared solu-tion of 46 mM sodium dithionite in 0.1 M sodiumbicarbonate. Samples were removed at various timesfor the measurement of nitrite. Reaction rates wereproportional to time and protein concentration, thelatter measured by the method of Lowry et al. (19).One unit of activity was defined as the amount ofenzyme that catalyzed the production of 1 nmol ofN02- min t.

RESULTSDenitrification of nitrate. Measurements were

made during anaerobic growth on nitrate tocompare the denitrification properties of Pseu-domonas stutzeri, Pseudomonas aeruginosa,and Paracoccus denitrificans. Cell yield reflect-ed the net energy gain, and the amount andcomposition of the gases evolved reflected therelative rates of intermediate steps of the path-way. The results (Fig. 1 and Table 1) indicatedthat there were slight differences in growth rateamong the three organisms but dramatic differ-ences in gas production and cell yield fromlimiting nitrate. In all cases N2 was producedduring the growth phase and continued to beevolved as the cultures entered stationary phase(Fig. 1). The pattern of accumulation of nitrogenoxide intermediates varied markedly among the

three species. Nitrite accumulated transiently ingrowing cultures of all three species; it washighest with Pseudomonas stutzeri (Fig. 1), butit disappeared from the medium late in thegrowth phase. A small amount of nitrous oxideappeared as Paracoccus cells entered stationaryphase (Fig. 1C). Larger amounts were producedby Pseudomonas aeruginosa at about the samegrowth phase (Fig. 1B). Pseudomonas stutzerinever produced detectable extracellular N20.The rates of gas production by early-station-

ary-phase cells are shown in Table 1. Pseudomo-nas aeruginosa was by far the most active, but alarge proportion of the gas produced was nitrousoxide (Fig. 1). Pseudomonas stutzeri and Para-coccus denitrificans produced N2 exclusively,but the former produced it at a significantlyhigher rate.The two pseudomonads produced similar cell

yields from denitrification of nitrate (Table 1).However, the yield of Pseudomonas stutzericells per mole of nitrate was proportional to theconcentration of nitrate up to 40 mM, whereasthe yield of Pseudomonas aeruginosa cells wasproportional only up to about 25 mM NaNO3(data not shown). The cell yield of Paracoccusdenitrificans was two to three times that of theother cultures and was proportional to nitrateconcentration up to 40 mM (data not shown).The assay for dissimilatory nitrate reductase

activity provided an in vitro index of the abilityof the cells to reduce nitrate. Since an artificialelectron donor was employed, the assay mightnot necessarily be expected to reflect in vivorates, but in fact it did. The specific activities ofnitrate reductase in cell extracts of Pseudomo-nas stutzeri and Paracoccus denitrificans wereboth higher than in extracts of Pseudomonasaeruginosa cells (Table 1), and growing cells ofthe latter species excreted less nitrite (Fig. 1).Growth on nitrous oxide. Unlike Pseudomo-

nas aeruginosa, Pseudomonas stutzeri andParacoccus denitrificans grew vigorously withnitrous oxide as the sole electron acceptor. The

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TABLE 2. Rates of denitrification by anaerobic resting-cell suspensionsGas production (nmol of N min-' mg [dry wt]-l)a from electron acceptor at

indicated concn (mM):Organism Nitrate Nitrite Nitrous oxide

33 100 36 2.5 17

Pseudomonas stutzeri 90.2b 96.4b 58.0b 77.6b 232.8bPseudomonas aeruginosa 45.2b 44.0b 9.4c 7.6b 15.2bParacoccus denitrificans 34.2b 64.4b 58.8d 92.4b 166.8b

a The sum of volatile nitrogen oxides. Values are the average of measurements made during incubation for twoor more samples.

b Entirely N2.c Predominantly NO, with 9% N20 and 11% N2 after 5 h.d Predominantly N2, with 6 to 17% N20. There was no NO.

doubling time (90 min) of Pseudomonas stutzericells in LB broth continuously sparged withnitrous oxide was even shorter than its doublingtime in the same medium with nitrate (114 min),but longer than in the same medium spargedwith air (55 min).

Production of gases by resting cell suspensions.We also compared the rates of gas evolutionfrom various nitrogen oxides by concentratedsuspensions of washed cells grown on nitrate.The rate of gas production from nitrate byPseudomonas stutzeri and Paracoccus denitrifi-cans cells (Table 2) was similar to that bygrowing cells (Table 1). N2 was the only gasreleased from either 33 or 100 mM nitrate.Neither nitrite, nitric oxide, nor nitrous oxideaccumulated during nitrate reduction. The rateof gas production from nitrate by resting cells ofPseudomonas aeruginosa (Table 2) was muchlower than by growing cells (Table 1). Unlikegrowing cells that produced mainly nitrous ox-ide, resting cells produced only N2.The three species metabolized nitrite quite

differently. Both pseudomonads produced gasmuch more slowly from nitrite than from nitrate(Table 2). However, Paracoccus denitrificansproduced gas from both electron acceptors atabout the same rate. Like growing cultures,resting cells of both Pseudomonas stutzeri andParacoccus denitrificans produced primarilyN2, with a trace of nitrous oxide accumulatingfrom the latter. With 100 mM sodium nitrite, gas

production was further inhibited, and nitrousoxide constituted the major portion (70%) of theproduct from both species (data not shown).Pseudomonas aeruginosa differed dramatically:the predominant product with 36 mM nitrite wasnitric oxide, and only traces of N2 and nitrousoxide were present (Table 2). Since the reduc-tion of nitric oxide appeared to be almost com-

pletely inhibited by 36 mM nitrite, the effect ofhigher concentrations was not examined. Lowerconcentrations differentially inhibited the reduc-tion of NO and N20 (Fig. 2). Cells supplied with

6 mM nitrite produced nitric oxide transiently inaddition to nitrous oxide and N2. Total gasproduction was 35 nmol of N min-1 mg (drywt)-1. At a nitrite concentration of 12 mM, totalgas production decreased, the accumulation ofnitric oxide was higher and persisted longer, andproduction of both N2 and nitrous oxide wasdepressed (Fig. 2B). These trends continued inthe presence of 18 mM nitrite (data not shown),and with 36 mM nitrite only nitric oxide waspresent even after several hours of incubation(Fig. 2C). The evolution of gas by Pseudomonasstutzeri cells incubated with 36 mM nitrite isshown (Fig. 2D) for comparison, and that byParacoccus denitrificans cells was similar, asmentioned above.Pseudomonas stutzeri and Paracoccus deni-

trificans both vigorously reduced nitrous oxideto N2 (Table 2), but the rate of reduction byPseudomonas aeruginosa was an order of mag-nitude slower. Furthermore, gas production bythe other two species from nitrous oxide wasmarkedly faster than it was from either nitrate ornitrite; this was not true for Pseudomonas aeru-ginosa. Exogenous nitrous oxide was not toxicto this organism, because its growth on nitratewas not inhibited by 1.0 atm of nitrous oxide(data not shown). Rather, the slow rate of reduc-tion of N20 and the failure of cells growing onnitrate to reduce it completely to N2 (Fig. 1)must reflect a defect in the ability of this speciesto metabolize nitrous oxide.

DISCUSSIONBacteria that carry out complete denitrifica-

tion differ fundamentally in a number of ways.For the three species in this study, the ability todenitrify serves the same function, allowing an-aerobic growth of these facultative aerobes, butthe growth rates, cell yields, ability to utilizeexogenous intermediates, and relative rates ofvarious steps of the pathway varied greatly.Pseudomonas aeruginosa grew most rapidly(doubling time, 1.3 h) and released the largest

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Go- ~ ~ / 60

40 I40 - --

20 ,.. //20 p'

:I-~~~ ~ ~ ~ ~ ~ ~ I

20 -Y )-(-100

TIME OF INCUBATION (hr)

FIG. 2. Production of gases from nitrite by restingcells. Cell suspensions of Pseudomonas aeruginosa(A, B, and C) or Pseudomronas stutzeri (D) wereincubated with 6 (A), 12 (B), or 36 mM (C andD) sodium nitrite. Volatile products were nitricoxide (V --V), nitrous oxide (A A), or dini-trogen (A ).

amount of gas; however, it had the lowest cellyields (28 g/mol of nitrate) and produced thehighest proportion of nitrous oxide. In contrast,Pseudomonas stutzeri and Paracoccus denitrifi-cans grew more slowly (doubling times, 1.9 and1.5 h, respectively) and released gas more slow-ly, but the gas produced was nearly all N2 andthe cell yields were markedly higher (34 and 78g/mol of nitrate, respectively). These speciesapparently realized a significantly greater netATP yield from denitrification; Paracoccus den-itrificans cells derived more than twice as muchATP per electron transported to a nitrogen oxideacceptor as did cells of Pseudomonas aerugino-sa.The patterns of accumulation of intermediates

also differed. Presumably these intermediatesare excreted into the medium when a subsequentstep in the pathway is rate limiting, as suggestedrecently by the kinetic study of Betlach andTiedje (3). We found that growing cells of Pseu-domonas stutzeri and Paracoccus denitrificansexcreted nitrite; their high nitrate reductase ac-tivity in vitro suggested that nitrite reductionwas rate limiting. In contrast, only a third asmuch nitrite accumulated in the medium of thePseudomonas aeruginosa culture, and its nitratereductase activity was fivefold lower.The relative rates of various steps of denitrifi-

cation in the three representative denitrifierswere also compared by measuring gas evolutionin resting cells incubated with nitrate or interme-diate electron acceptors; again the three species

had different patterns (Table 2). Pseudomonasstutzeri reduced nitrate, nitrite, and nitrous ox-ide completely to N2 at the highest rates. Para-coccus denitrificans behaved similarly, althoughsome nitrous oxide was excreted when cellswere incubated with 36 mM nitrite. Pseudomo-nas aeruginosa was strikingly different; it re-duced each of the nitrogen oxides much moreslowly. The reduction rates for nitrite and ni-trous oxide were one-fourth and one-fifteenththose of the other species, respectively. Surpris-ingly, nitric oxide accumulated as the majorproduct of nitrite reduction. Only with low con-centrations of nitrite were nitrous oxide and N2produced. Possibly the reductases constitutingthe latter part of the denitrification pathway inPseudomonas aeruginosa are differentially in-hibited by nitrite. Payne (26) discussed earlierstudies that also concluded that nitrite plays adominant role in determining the composition ofthe gaseous products of denitrification. Wefound that concentrations of nitrite greater than15 mM markedly inhibited anaerobic growth ofPseudomonas aeruginosa (data not shown),consistent with reports that 10 mM nitrite inhib-ited respiration, active transport, and oxidativephosphorylation in this organism (40). This con-centration of nitrite was also reported to inhibitthe growth of Paracoccus denitrificans (39);however, we found that the resting cells of thisorganism were much less sensitive to nitrite thanwere those of Pseudomonas aeruginosa.

Recently, a group of denitrifiers was com-pared for their apparent affinity for the uptake ofnitrate (R. M. Edwards and J. M. Tiedje, Abstr.Annu. Meet. Am. Soc. Microbiol. 1981, N48, p.181, and personal communication). In that re-port Pseudomonas stutzeri JM300 comparedvery favorably with the others. Its apparent Kmfor nitrate was 5.7 + 2.4 ,uM, and its Vma, was1.56 ,uM min-1; the comparable values for Pseu-domonas fluorescens, for example, were 6.0 +1.8 ,uM and 1.60 p.M min-1, respectively. Suchdata suggest that Pseudomonas stutzeri maycompete effectively in low-nitrate natural envi-ronments.A number of denitrifiers have been shown to

grow on nitrous oxide (Table 3). Indeed, thiscapability is probably limited to denitrifyingbacteria, but t is not common to all of them.Several species otherwise capable of denitrifica-tion cannot grow on nitrous oxide. Most of theseproduce only nitrous oxide from nitrate or nitritedenitrification and thus apparently lack the laststep in the pathway. However, Pseudomonasaeruginosa is unique. It can produce N2 andreduce exogenous nitrous oxide, but it is unableto grow with nitrous oxide as the sole oxidant (4,37). The biochemical basis of this phenomenonis particularly obscure because, as Bryan and

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TABLE 3. Classification of denitrifying bacteria bytheir metabolism of nitrous oxide

Species Reference

Nitrous oxide utilizing'Pseudomonas stutzeri...........1Pseudomonas perfectomarinus. .. 24Pseudomonas denitrificansb ..... 20Pseudomonas lemoingeii ........ 32Pseudomonas fluorescens ....... 11Pseudomonas chlororaphisB561cd...................... 11

Pseudomonas pickettii .......... 9Paracoccus denitrificans ........ 31Paracoccus halodenitrificans .... M. R. Betlach

andL. Hochsteine

Agrobacterium tumefaciens ..... 30Agrobacterium radiobacter...... 30Bacillus azotoformans .......... 28Bacillus stearothermophilus .....8Flavobacterium sp.............. 29Alcaligenes faecalis ............ 33Vibrio succinogenesf............ 41Azospirillum brasilenseg......... 21, 22Thiobacillus denitrificans........ 14

Nitrous oxide nonutilizing'Pseudomonas fluorescens

PJ188 ...................... 11Pseudomonas chlororaphisd ..... 11Pseudomonas aureofaciens'......7Azospirillum lipoferumg ......... 21, 22Azospirillum itersonii ........... E. J. HanseniCorynebacterium nephridiik...... 12, 34Pseudomonas aeruginosa ....... 4, 37

a Can grow with nitrous oxide as sole electronacceptor and produce dinitrogen gas from nitrate ornitrite.

b Reclassified as an Alcaligenes (2).C N2 produced from N03-; N20 growth not estab-

lished.d Now assigned to Pseudomonas fluorescens bio-

type D (36).e Personal communication.f An exception: N2 is produced from N20 but not

from N03 or N02.8 Formerly Spirillum lipoferum (17).h Cannot grow on nitrous oxide. All of these species

produce only nitrous oxide from nitrate or nitriteexcept Pseudomonas aeruginosa, which also pro-duces dinitrogen gas.'Now assigned to Pseudomonas fluorescens bio-

type E (36).i M.Sc. thesis, University of Iowa, Iowa City, 1972.k Now provisionally classified as Achromobacter

(27).

Delwiche showed, acetylene inhibition of ni-trous oxide reduction decreased the cell yieldfrom nitrate of Pseudomonas aeruginosa andPseudomonas stutzeri to the same extent (B. A.Bryan, Ph.D. thesis, University of California,Davis, 1980).

We initiated this comparative study as an aidin choosing an organism suitable for use inphysiological genetic investigations on denitrifi-cation. Our aim was to select an organism thatwas typical of those carrying out complete deni-trification, easily manipulated in culture, andcapable of genetic exchange. Even from thecomparison of only three organisms, it was clearthat the level of variation among their patterns ofdenitrification was too great for any of them tobe considered typical. Pseudomonas aeruginosamight still be considered desirable because it hasa well-characterized genetic system (13, 35), butits inability to grow anaerobically with exoge-nous N20 argues persuasively against it. Mu-tants blocked in the last step of the pathwaywould be difficult to select or score. We chosePseudomonas stutzeri for our further studies. Itdenitrifies actively, is easy to manipulate inculture for physiological and genetic experi-ments, and, as we found recently, has an effec-tive natural transformation system for the ex-change of genetic information between cells(4a).

ACKNOWLEDGMENTSThis work was supported in part by National Science

Foundation grant AER77-07301 and by U.S. Department ofAgriculture grant 59-2063-1-1627-0.We thank JoAnne J. Rosen for able technical assistance.

LITERATURE CITED

1. Allen, M. B., and C. B. van Niel. 1952. Experiments onbacterial denitrification. J. Bacteriol. 64:397-412.

2. Ambler, R. P. 1973. The amino acid sequence of cyto-chrome c' from Alcaligenes sp. N.C.1.B. 11015. Biochem.J. 135:751-758.

3. Betlach, M. R., and J. R. Tiedje. 1981. Kinetic explana-tion for accumulation of nitrite, nitric oxide, and nitrousoxide during bacterial denitrification. Appl. Environ. Mi-crobiol. 42:1074-1084.

4. Carlson, C. A., and J. L. Ingraham. 1981. The physiologi-cal genetics of denitrification in Pseudomonas, p. 429-444. In J. M. Lyons, R. C. Valentine, D. A. Phillips,D. W. Rains, and R. C. Huffaker (ed.), Genetic engineer-ing of symbiotic nitrogen fixation and conservation of soilnitrogen. Plenum Publishing Corp., New York.

4a.Carlson, C. A., L. S. Pierson, J. J. Rosen, and J. L.Ingraham. 1983. Pseudomonas stutzeri and related spe-cies undergo natural transformation. J. Bacteriol. 153:93-99.

5. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advancedbacterial genetics, p. 201. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y.

6. Firestone, M. K. 1981. Biological denitrification, p. 289-326. In F. J. Stevenson (ed.), Nitrogen in agriculturalsoils. Agronomy monograph no. 22. American Society forAgronomy, Madison, Wis.

7. Firestone, M. K., R. B. Firestone, and J. M. Tiedje. 1979.Nitric oxide as an intermediate in denitrification: evidencefrom nitrogen-13 isotope exchange. Biochem. Biophys.Res. Commun. 91:10-16.

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10. Gerhardt, P. 1981. Diluents and biomass measurement, p.504-507. In P. Gerhardt, R. G. E. Murray, R. N. Costi-low, E. W. Nester, W. A. Wood, N. R. Kreig, and G. B.Phillips (ed.), Manual of methods for general bacteriolo-gy. American Society for Microbiology, Washington,D.C.

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12. Hart, L. T., P. D. Larson, and C. McCleskey. 1965. Deni-trification by Corynebacterium nephridii. J. Bacteriol.89:1104-1108.

13. Holloway, B. W., V. Krishnapillai, and A. F. Morgan.1979. Chromosomal genetics of Pseudomonas. Microbiol.Rev. 43:73-102.

14. Ishaque, M., and M. I. H. Aleem. 1973. Intermediates ofdenitrification in the chemoautotroph Thiobacillus denitri-ficans. Arch. Mikrobiol. 94:269-282.

15. Jeter, R. M., and J. L. Ingraham. 1981. The denitrifyingprokaryotes, p. 913-925. In M. P. Starr, H. Stolp, H. G.Truper, A. Balows, and H. G. Schlegel (ed.), The pro-karyotes: a handbook on habitats, isolation and identifica-tion of bacteria. Springer-Verlag, New York.

17. Krieg, N. R. 1977. Taxonomic studies of Spirillum lipo-ferum, p. 463-472. In A. Hollander (ed.), Genetic engi-neering for nitrogen fixation. Plenum Publishing Corp.Ltd., London.

18. Lowe, R. H., and H. J. Evans. 1964. Preparation andsome properties of a soluble nitrate reductase from Rhizo-biumjaponicum. Biochim. Biophys. Acta 85:377-384.

19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

20. Matsubara, T. 1971. Studies on denitrification. XII. Someproperties of the N20-anaerobically grown cell. J. Bio-chem. 69:991-1001.

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