0099-2240/82/100923-06$02.

00/0 Copyright 0 1982, American Society for Microbiology

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1982, p. 923-928

Vol. 44, No. 4

Nitrogen Redox Metabolism of a Heterotrophic, NitrifyingDenitrifying Alcaligenes sp. from Soil

DOMENIC CASTIGNE1TI AND THOMAS C. HOLLOCHER* Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254

Received 5 February 1982/Accepted 4 June 1982

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (Vmax) of 0.066 and 0.003 ,umol of N x min-' mg of protein-, respectively, at 22C. Km values were 15 and 42 ,uM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.
x

Heterotrophic nitrifiers oxidize reduced nitrogenous compounds and utilize organic carbon
as carbon and energy sources. They are thus distinct from autotrophic nitrifiers, which use energy gained solely from the oxidation of inorganic nitrogen compounds for growth and carbon dioxide fixation (10). Heterotrophic nitrifiers generally have low N oxidation activities and yield small amounts of nitrification products, compared with autotrophs (10). Recently, however, an Alcaligenes sp. isolated from soil by Castignetti and Gunner (5, 6) was observed to yield large amounts of nitrite when cultured with pyruvic oxime (PO) as the sole source of carbon and nitrogen. The bacterium can also oxidize hydroxylamine (NH2OH) to nitrite and can synthesize the entire complement of denitrifying enzymes under appropriate conditions (7). The possible ecological importance of oximes stems from their presence in and isolation from a variety of plant tissues, including the leaves of tobacco, mulberry, spinach, turnip, and onion plants, and even from animal tissues, such as the tissues of silkworms and the kidneys, hearts, and livers of oxen (27). Hydroxamic acids, which also contain nitrogen at the redox level of NH20H, are produced in significant quantities by a number of microbes, including aspergilli,

penicillia, mycobacteria, Ustilago sphaerogena, and Rhodotorula pilimanae (9). Soils oxidize perfused PO to nitrite at appreciable rates (20). This oxidation apparently is not mediated by autotrophic nitrifiers, such as Nitrosomonas spp., because classical inhibitors of such organisms are without effect. In the case of soils induced to oxidize ammonium and nitrite and thus to contain high levels of autotrophic nitrifiers, the PO oxidizing capacity is low but can be induced to high levels by a suitable period of incubation with PO. Nitrification occurs in soils which seem to contain few autotrophic nitrifiers (20, 25). Such observations have been taken as evidence for the existence in these soils of a population of active heterotrophic nitrifiers. We note that two of the most abundant bacterial genera in soils, Alcaligenes and Arthrobacter (1), include species which can carry out heterotrophic nitrification (6, 10) and that Arthrobacter spp. can release NH2OH upon oxidation of ammonium (10). In this study, we examined some of the oxidative and reductive capacities of the Alcaligenes sp. of Castignetti and Gunner (5, 6) as a function of growth conditions and provided evidence against the hypothesis that hydrolysis of PO occurs before oxidation of its nitrogen.

923

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CASTIGNETTI AND HOLLOCHER

MATERIALS AND METHODS

APPL. ENVIRON. MICROBIOL.

Materials. PO was synthesized by the method of Quastel et al. (21) and was used as described previously (5, 6). Sodium pyruvate, sodium succinate, potassium nitrate, sodium nitrite, sodium azide, and hydroxylamine hydrochloride were obtained from Fisher Scientific Co., Pittsburgh, Pa. Hydroxylamine hydrochloride was recrystallized from ethanol-water, and stock solutions were prepared and neutralized immediately before use. L-Alanine was obtained from Sigma Chemical Co., St. Louis, Mo., and yeast extract, peptone, and agar were obtained from Difco Laboratories, Detroit, Mich. Cultures. The Alcaligenes sp. studied was isolated from soil (5) and has been partly characterized (5-7). The bacterium was maintained on PO-mineral saltsagar plates (5). When it was grown as a nitrifier, colonies were initially grown to mid-log stage in 0.3% PO-mineral salts medium (5), and this culture was used to provide a 1% inoculum for 1 or 1.5% POmineral salts medium. Cultures (400 ml) were grown in 2-liter Erlenmeyer flasks at 30C with shaking (255 rpm on a New Brunswick Gyrotory shaker [8]) and were harvested during logarithmic growth at a cell density of about 0.1 mg of protein x ml-'. Oxygen electrode measurements (Oxygen Monitor; Yellow Springs Instrument Co., Yellow Springs, Ohio) indicated that the dissolved 02 concentration in the cultures just before harvest was 80 to 95% of that expected at equilibrium under air. When the bacterium was grown as a denitrifier, colonies were first inoculated into 115 ml of anaerobic yeast extract-peptone medium with 15 mM nitrate (7) in 125-ml Erlenmeyer flasks and grown statically at 30C to mid- to late-log stage. Aliquots (1 ml) of this culture were then used to inoculate identical flasks, and these cultures were grown to mid-log stage. When the bacterium was grown aerobically on organic carbon sources other than PO, the mineral salts medium (5) was supplemented with 0.4% sodium pyruvate, ethanol, sodium succinate, or L-alanine. Except for alanine medium, the media were also supplemented with 0.1% ammonium chloride. Otherwise, conditions of growth were as described above for PO-grown cells. Cells were harvested by centrifugation at 0 to 40C, washed twice with cold mineral salts medium lacking carbon and nitrogen sources, and finally resuspended in an appropriate volume of the same medium. Cells could be stored for at least several hours at 0C before use without changes in the activities surveyed. Procedures. Two procedures were employed for measurement of the PO or NH2OH oxidation activity of Alcaligenes sp. In the first procedure, a cell suspension was placed in a vessel the dimensions of which were sufficient to provide a high surface-to-volume ratio and stirred vigorously with a magnetic stirring bar. Oxidation was initiated by the addition of a neutralized PO or NH2OH stock solution to an initial concentration of 1 to 3 mM PO or '1 mM NH2OH. Immediately after the substrate was added and at appropriate intervals thereafter 0.5- to 1-ml portions were removed and centrifuged for 1.5 min in an Eppendorf Microfuge to sediment the cells. The supernatant was assayed for nitrite, ammonium, PO, and NH2OH, as appropriate. In the second procedure, 2 ml of a cell suspension was placed in a 9-ml serum bottle, the bottle was stoppered, and the air was

replaced with a gas mixture consisting of 135 ,mol of 02, 8.9 ,umol of argon, and helium at a final pressure of 1 atm (100 kPa). The aqueous phase was stirred vigorously with a small magnetic stirring bar, and the reaction was initiated by injection of PO or NH2OH. At appropriate intervals thereafter, the gas phase was sampled (11) and analyzed for nitrous oxide (N20) and dinitrogen (N2). Nitric oxide (NO), if formed, would react with oxygen (02) to form nitrate and nitrite (11, 24) and so would not be observed. Samples of the aqueous phase were removed and processed as described above. In certain cases, samples of reaction mixtures containing NH2OH were mixed with equal volumes of H2SO4 to stop the reaction and dissolve the cells. Subsequent NH20H analysis was not affected by H2SO4 because of sufficiently great dilution factors. For measurement of nitrate reductase activity, the cell suspension was placed in contact with a nitrate electrode assembly in a vessel purged with argon and was stirred with a magnetic stirring bar (14, 16). After anaerobic conditions had been established, the system was supplemented with 10 mM neutralized sodium pyruvate, and the reaction was initiated by the addition of an equal concentration of anaerobic KNO3. Nitrate disappearance was followed potentiometrically, and samples were taken at appropriate intervals for nitrite analysis. (The Enzyme Commission [EC] numbers for respiratory nitrate reductase, nitrite reductase, and nitric oxide reductase are 1.7.99.4, 1.7.99.3, and 1.7.99.2, respectively. The reaction depicted for nitric oxide reductase is reduction of NO to N2, but this reaction in denitrifiers is a two-step reaction with N20 as an intermediate and nitrous oxide reductase as the second enzyme (24). Nitrous oxide reductase, PO oxidase, and hydroxylamine dehydrogenase have not been assigned numbers. The number for hydroxylamine oxidase is 1.7.3.4 and that for hydroxylamine reductase is 1.7.99.1 or, if reduced by NADH, 1.6.6.11. Which number applies in this study is unknown.) For measurement of nitrite reductase, nitric oxide reductase, and nitrous oxide reductase activities, analysis of gaseous products was carried out by gas chromatography-mass spectrometry (11). A cell suspension (1 ml) was placed in a 9-ml serum bottle, the bottle was stoppered, and the air was replaced with a gas consisting of helium and 8.9 ,umol of argon at a pressure of 1 atm. The aqueous phase was stirred vigorously with a small magnetic stirring bar. The system was supplemented with 20 ,umol of anaerobic sodium pyruvate, and the reaction was initiated by the injection of 20 Fmol of nitrite, 13.4 Fmol of NO, or 13.4 Fmol of N20. At appropriate times thereafter, the gas phase was sampled and analyzed for the appearance of NO, N20, and N2. All reactions were run at 21 to 23C. Every experiment included a control containing a cell suspension which previously had been heated to 100C for S min. Analytical methods. PO and NH20H were measured colorimetrically as previously reported (5). Nitrite was determined colorimetrically (26), and nitrate was measured with an Orion nitrate electrode assembly (14). A Hewlett-Packard model 5992A gas chromatographmass spectrometer was used as described previously (11) to determine NO, N20, and N2. Ammonium was measured by the phenate method (2), and cell protein was determined by the method of Herbert et al. (12).

VOL. 44, 1982

ALCALIGENES SP.: NITROGEN REDOX METABOLISM

0.20

925

23
.9

E 0.16 E
0

0.12
0

0.08
0.

E
0

x
I
I

__ _

0.04
s

20

40 Time (Min.)

60

8(0

as a

FIG. 1. PO metabolism by Alcaligenes sp. grown nitrifier. Cell density, 1.2 mg of protein x ml-'.

,
0

.
20

.
40 Time (Min.) 60

so

Data on the uptake of PO, NH2OH, and nitrate were used to generate progress curves from which Km values were estimated. The Ki of sodium azide as a competitive inhibitor of nitrate reductase was calculated by equation 1 (23): apparent Km = [Km(I)/Ki] + Km (1)

sp. grown as a nitrifier.

X

FIG. 2. HydroxylaminemetabolismbyAlcaligenes Cell density, 2.4 mg of protein

ml-,.

RESULTS Figures 1 and 2 show representative kinetic results for the oxidations of PO and NH20H, respectively, to nitrite by Alcaligenes sp. grown as a nitrifier. The specific activities (V,t,) for the oxidations of PO and NH20H were 0.066 and 0.003 ,umol x min-' x mg of protein-', respectively-a difference of about 20-fold. Km values were estimated from progress curves of the type presented in Fig. 1 and 2 by determining the concentration of substrate at which the slope equaled one-half the maximum slope observed in the linear (Vmax) region of the curve. Km values thus estimated were about 15 and 42 p,M for oxidations of PO and NH2OH, respectively. Product inhibition by nitrite did not create a significant artifact in estimating Km values, as predicted from the remarkable tolerance of nitrite exhibited by Alcaligenes sp. (5, 6). When Alcaligenes sp. was grown on nitrate as a denitrifier and then challenged with 3.5 mM PO in mineral salts medium under aerobic conditions, production of nitrite was not detected and there was no disappearance of PO. When challenged with NH2OH under similar conditions, however, NH2OH uptake was significant, and a specific activity of 0.016 pumol x min-' x mg of protein-' was determined. Remarkably, nitrite production was not detected, but ammonium ion and N20 were produced from NH2OH at nearly equal rates: 0.0047 and 0.0045 ,umol x min-' x mg of protein-1, respectively. Because N20

contains two N atoms per molecule, the rate of N20 production was 0.009 pxg atom of N x min-' x mg of protein-1. The sum of the rates of appearance of these two products was therefore 0.014 ,ug atom of N x min-' x mg of protein-'. NH2OH was thus converted largely or entirely into ammonium and N20, and these products accumulated in nearly equimolar amounts. Production of N2 was not detected. NH2OH uptake and ammonium and N20 production were very much inhibited or abolished in controls that had been heated to 100C for S min before the addition of NH2OH (Table 1). The possibility that a small portion of NH2OH disappeared within cells or was converted to oxime or hydroxamate was tested by comparing the NH2OH contents of cell supematants with that of the H2SO4 lysate. No difference was observed. Alcaligenes sp. grown as a nitrifier was examined to determine whether denitrifying enzymes were present when the cells were harvested and placed in medium supplemented with sodium pyruvate and various nitrogen oxides under anaerobic conditions. Although nitrite reductase and nitrous oxide reductase activities were not detected under any circumstances, nitrate reductase and nitric oxide reductase activities were consistently detected. Nitrate reductase and nitric oxide reductase specific activities (Vm..) were typically 0.04 and 0.007 ,umol x min-' x mg of protein-1, respectively, and were constant throughout the course of a 40- to 60-min experiment. The uptake of nitrate mea-

926

CASTIGNETTI AND HOLLOCHER

2.0

APPL. ENVIRON. MICROBIOL.

TABLE 1. Metabolism of NH2OH by Alcaligenes sp. grown as a denitrifier on nitratea

System
Cells
Incubation

time (min) 0 30 60 120

Amt of Concn (mM) of: N20 NH20H NH4+ N02- (,umol)

1.6
I

4.9

0.07

0 0.22 0.37

1.2 1

2.2
5.3
5.1

0.86
0.61
0.76

0 0
0 0 0
0 a 0
._

Heat-killed cells

0 a Denitrifying cells were prepared as described in the text. The cell density was 1.4 mg of protein x ml-1. In the process of heat killing the cells, 0.6 to 0.7 mM ammonium was released. N20 production was measured in a gas-tight vial containing 1 ml of a cell suspension (1.4 mg of protein).

0 30 60 120

0.8 0

0.4

20

sured potentiometrically was in good agreement with the production of nitrite measured colorimetrically. Thus, nitrate appeared to be converted to nitrite stoichiometrically. Similarly, the sole product of NO reduction appeared to be N20. Heat-killed cells failed to reduce nitrate and NO. The Km for nitrate was estimated as described above from progress curves of the kind illustrated in Fig. 3 and was found to be about 250 ,uM. The Km increased to 710 ,uM, for example, in the presence of 9.7 ,uM azide. From such data, a competitive Ki of about 5 ,uM was calculated for azide. Nitrate reductase activity was inhibited >95% by 02 at partial pressures essentially equal to that in air. The inhibition was completely and immediately reversible. For comparison, the nitrate reductase activity of Alcaligenes sp. grown anaerobically on nitrate as a denitrifier was inhibited >90% by aerobic conditions and was equally sensitive to azide. Although nitrate reductase activity was observed when Alcaligenes sp. was grown both as a denitrifier (7) and as a nitrifier, this activity was not found in cells grown aerobically on organic carbon sources other than PO. Nitric oxide reductase, nitrite reductase, and nitrous oxide reductase activities were also not detected in these cells. On the other hand, PO oxidase activity (0.004 ,umol x min-1 x mg of protein-1) and NH2OH -* ammonium reducing activity (0.003 ,umol x min-' x mg of protein-') were detected. These results and previously published data (7) provided for comparison are summarized in Table 2.
DISCUSSION The Alcaligenes sp. used in this study is the most active heterotrophic nitrifier yet reported

40 Time (Min.)

60

80

as a

FIG. 3. Nitrate reduction by Alcaligenes sp. grown nitrifier and suspended anaerobically. Cell density, 1.6 mg of protein x ml-'. Symbols: 0, nitrate reduction; 0, nitrate reduction in the presence of 9.7 ,uM sodium azide.

and exhibited a rate of oxidation of PO to nitrite of 0.07 ,umol of N x min-' x mg of protein-' under nongrowth conditions. In addition, it can grow in the presence of 130 mM nitrite without being inhibited (6). For comparison, the second largest rate for a heterotrophic nitrifier, 0.04 ,umol of N x min-' x mg of protein-', was reported for the oxidation of acetohydroxamate to nitrite by a Pseudomonas aeruginosa isolate (22). Nitrosomonas spp. and Nitrobacter spp. can oxidize ammonium and nitrite, respectively, at rates 10 times greater than that of the oxidation of PO by Alcaligenes sp. (10); for example, Hollocher et al. (13) recently reported that the rate of oxidation of ammonium to nitrite by Nitrosomonas europaea is 0.5 p.mol of N x min-' x mg of protein-', and the rate of oxidation of nitrite to nitrate by Nitrobacter agilis is 0.8 ,umol of N x min-1 x mg of protein- under nongrowth conditions. Enzymatic makeup. The oxidation of PO by Alcaligenes sp. grown aerobically on an organic carbon source and ammonium was greatly reduced, and the N oxide reductases characteristic of the denitrification apparatus were lacking. These enzymes therefore appeared not to be constitutive in the bacterium. When grown as a denitrifier, the bacterium exhibited all four of the classical N oxide reductases at high levels (7) but lacked the ability to oxidize PO. Remarkably, such denitrification-

VOL. 44, 1982

ALCALIGENES SP.: NITROGEN REDOX METABOLISM

927

TABLE 2. Enzyme activities detected in Alcaligenes sp. adapted to different growth habits Rate (,umol x min-' x mg of protein-') of substrate disappearance (Km [>M] value for substrate) Reference Culture condition NOb N20b p0a Nitrateb Nitriteb NH20H0 0 This paper Organic carbon (other 0.004c 0 0 0.003d 0 than PO) and 02

Nitrifying on PO and 02

0.066c (15)

0.003c (42)

0.04 (250)c

0.007'

This paper

0.035 0.06 This paper, 6 Denitrifying on enriched 0 0.15 0.12 0.014f medium and nitrate a 02 was the terminal oxidant. b Anaerobic conditions. Sodium pyruvate provided the reducing equivalents, except in the case of denitrifying cells, which were suspended in yeast extract-peptone medium. c Chief product was nitrite. d Chief product was ammonium ion. I Product was N20. f Chief products were ammonium ion and N20 in a mole ratio of about 1.

adapted cells were able under aerobic conditions to catalyze the disappearance of NH20H with production of N20 and ammonium in nearly equimolar amounts. If this reaction were simply catalysis of a redox dismutation of NH20H (17), the expected product N20/ammonium mole ratio would have been 0.5. The simplest explanation for this production of N20 and ammonium by denitrifying cells (and also for production of ammonium from NH20H by cells grown on organic carbon sources other than PO) is the existence of two enzyme systems that can act on NH20H. One may be termed hydroxylamine dehydrogenase (equation 2); the second may be termed hydroxylamine reductase (equation 3). NH20H + X-- 1/2 N20 + XH2 + 1/2 H20 (2) NH20H + YH2 + H+ -- NH4+ + Y + H20 (3) It is unknown whether equation 2 is relevant in any way with the hydroxylamine oxidase (NH20H -+ N02) of nitrifying cells. It seems unlikely that the reaction shown in equation 2 could proceed by way of free NO because the

its reversible inhibition by 02 (18, 19). Whether this nitric oxide reductase was the same as that of denitrification-adapted cells was unclear because no nitric oxide reductase has been characterized yet. At least it was clear that the nitric oxide reductase of nitrification-adapted cells could not represent a second reaction of nitrite reductase, because the cells failed to demonstrate nitrite reductase activity. Nitrite, which was present during aerobic growth, has been shown to be an inducer of Alcaligenes nitrite reductase under anaerobic conditions (15). We thus assume that 02 prevented this action of nitrite during growth. The existence of enzymes of denitrification in nitrifying cells is novel and may be of ecological utility to a soil bacterium. Such cells could

NOH

02

H3C

Coo-

N02_

reaction of NO with 02 would produce nitrogen dioxide (NO2) and then nitrate and nitrite by hydration (11), but nitrite was not detected as a product. The simplest route for the reaction shown in equation 2 is via nitroxyl (NOH), which is known to undergo rapid dimerization and dehydration (3, 4). When grown as a nitrifier on PO, Alcaligenes sp. could oxidize PO and NH20H to nitrite (5, 6) and, remarkably, also exhibited nitrate reductase and nitric oxide reductase activities (Table 2). The former activity could be attributed to the respiratory (dissimilatory) nitrate reductase on the basis of its extreme sensitivity to azide and

H21Wn
NH2 OH
+

02

H3C

COO0

FIG. 4. N oxidation of PO by Alcaligenes sp. The data support the upper pathway, i.e., direct oxidation of PO.

928

CASTIGNETTI AND HOLLOCHER

APPL. ENVIRON. MICROBIOL.

Nature (London) 175:173-174. 9. Emery, T. 1974. Biosynthesis and mechanism of action of hydroxamate-type siderochromes, p. 107-123. In J. B. Neilands (ed.), Microbial metabolism, a comprehensive treatise. Academic Press, Inc., New York. 10. Focht, D. D., and W. Verstraete. 1977. Biochemical ecology of nitrification and denitrification. Adv. Microb. Ecol. 1:135-214. 11. Garber, E. A. E., and T. C. Hollocher. 1981. '5N tracer studies on the role of nitric oxide in denitrification. J Biol. Chem. 256:5459-5465. 12. Herbert, D., P. J. Phipps, and R. E. Strange. 1971. Chemical analyses of microbial cells, p. 242-252. In J. R. Norris and D. W. Ribbons (ed.), Methods in Microbiology, vol. SB. Academic Press, Inc., New York. 13. Hollocher, T. C., S. Kumar, and D. J. D. Nicholas. 1982. Respiration-dependent proton translocation in Nitrosomonas europaea and its apparent absence in Nitrobacter agilis during inorganic oxidations. J. Bacteriol. 149:10131020. 14. John, P. 1977. Aerobic and anaerobic bacterial respiration monitored by electrodes. J. Gen. Microbiol. 98:231-238. 15. Kakutani, T., B. Teruhiko, and K. Arima. 1981. Regulation of nitrite reductase in the denitrifying bacterium Alcaligenesfaecalis S-6. Agric. Biol. Chem. 45:23-28. 16. Kristjansson, J. K., and T. C. Hollocher. 1979. Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane. J. Bacteriol. 137:12271233. 17. Mason, K. G. 1967. Hydroxylamine, p. 115-157. In Supplement to Mellor's comprehensive treatise on inorganic and theoretical chemistry, vol. VIII, supplement II, Nitrogen (part II). Longmans, Green and Co. Ltd., ,London. 18. Pichinoty, F. 1963. L'effet oxygene et la biosyntheses des enzymes d'oxydoreduction bacteriens, p. 507-521. In Mecanismes de regulation des activites cellulaires chez les microorganismes. Editions du Centre National de la Recherche Scientifique, Paris. 19. Pichinoty, F. 1964. A propos des nitrate-reductases d'une bacterie denitrificante. Biochim. Biochem. Acta 89:378381. 20. Quastel, J. H., and P. G. Scholefield. 1949. Influence of organic nitrogen compounds on nitrification in soil. Nature (London) 164:1068-1072. 21. Quastel, J. H., P. G. Scholefield, and J. W. Stevenson. 1952. Oxidation of pyruvic acid oxime by soil organisms. Biochem. J. 51:278-284. 22. Ralt, D., R. F. Gomez, and S. R. Tannenbaum. 1981. Conversion of acetohydroxamate and hydroxylamine to nitrite by intestinal microorganisms. Eur. J. Appl. Microbiol. Biotechnol. 12:226-230. 23. Segel, I. H. 1976. Biochemical calculations: how to solve mathematical problems in general biochemistry, 2nd ed., p. 248-252. John Wiley & Sons, Inc., New York. 24. St. John, R. T., and T. C. Holiocher. 1977. Nitrogen-15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa. J. Biol. Chem. 252:212-218. 25. Strayer, R. F., C.-J. Lin, and M. Alexander. 1981. Effect of simulated acid rain on nitrification and nitrogen mineralization in forest soils. J. Environ. Qual. 10:547-551. 26. 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. 27. Yamafugi, K., H. Kondo, and H. Omura. 1950. Distribution of oxime in plant and animal tissues. Enzymologia 14:153-156.

conceivably switch quickly from heterotrophic nitrification (aerobic) to nitrate respiration (anaerobic) in response to changes in the partial pressure of 02. If anaerobic conditions persisted, the bacterium would synthesize the remainder of the denitrification apparatus and promote reduction of nitrite to N2. Pathway for the oxidation of PO and nitrite. Figure 4 shows two possible pathways for the oxidation of PO by Alcaligenes sp. The lower pathway involves hydrolysis of PO to NH2OH and subsequent oxidation of this inorganic species. The upper pathway represents oxidation of nitrogen before detachment from carbon. Kinetic results (Fig. 1 and 2) showed that the Vmax for oxidation of PO exceeded that for NH2OH oxidation by 20-fold. This result is inconsistent with the lower hydrolytic pathway and implies that oxidation of PO proceeds almost entirely by the upper pathway, in which PO per se is the specific substrate. In the lower pathway, the Vmax for PO could not exceed that for NH2OH unless a permeability barrier existed for NH20H. No such barrier is known in bacteria.
ACKNOWLEDGMENTS This work was supported in part by grant PCM79-12566 from the National Science Foundation. D.C. was the recipient of a Jessie Smith Noyes postdoctoral fellowship. We thank E. Garber for his technical assistance and the personnel of E. Grunwald's laboratory (Brandeis University, Waltham, Mass.) for assistance in the operation of the gas chromatograph-mass spectrometer.
LITERATURE CITED 1. Alexander, M. 1977. Introduction to soil microbiology. John Wiley & Sons, Inc., New York. 2. American Public Health Association. 1975. Standard methods for the examination of water and wastewater, 14th ed., p. 429-436. American Public Health Association, Washington, D.C. 3. Bonner, F. T., and M. J. Akhtar. 1981. Formation of nitrosyltricyanonickelate (NiNO(CN)32 ) in a direct NOdisplacement reaction. Inorg. Chem. 20:3155-3160. 4. Bonner, F. T., L. S. Dzelzkalns, and J. A. Bonucci. 1978. Properties of nitroxyl as intermediate in the nitric oxidehydroxylamine reaction and in trioxidinitrate decomposition. Inorg. Chem. 17:2487-2494. 5. Castignettl, D., and H. B. Gunner. 1980. Sequential nitrification by an Alcaligenes sp. and Nitrobacter agilis. Can. J. Microbiol. 26:1114-1119. 6. Castignettl, D., and H. B. Gunner. 1981. Nitrite and nitrate synthesis from pyruvic-oxime by an Alcaligenes sp. Curr. Microbiol. 5:379-384. 7. Castignetti, D., and T. C. Hollocher. 1981. Vigorous denitrification by a heterotrophic nitrifier of the genus Alcaligenes. Curr. Microbiol. 6:247-252. 8. Colfins, F. M. 1955. Effect of aeration on the formation of nitrate-reducing enzymes by Pseudomonas aeruginosa.