APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1986, p. 143-149 Vol. 51, No. 10099-2240/86/010143-07$02.00/0Copyright © 1986, American Society for Microbiology

Evidence for NH4' Switch-Off Regulation of Nitrogenase Activityby Bacteria in Salt Marsh Sediments and Roots of the Grass

Spartina alternifloraDUANE C. YOCH* AND GARY J. WHITING

Department of Biology, University of South Carolina, Columbia, South Carolina 29208

Received 25 June 1985/Accepted 30 September 1985

The regulatory effect of NH4' on nitrogen fixation in a Sparlina alterniflora salt marsh was examined.Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4' in both the light anddark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, andrhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobicC2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significantrates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could bedistinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4'. Ammoniaalso inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appearedto be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kineticcharacteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine areconsistent with the N14' switch-off-switch-on mechanism of nitrogenase regulation.

The inhibitory effect of NH4' on nitrogen fixation in saltmarsh sediments and on bacteria associated with the roots ofthe marsh grass, Spartina alterniflora, has been observednumerous times (3, 4, 6, 10, 27, 28, 35). Because theseexposures to NH4+ were usually for periods of many hours(or even days), repression of nitrogenase synthesis has beenoffered as an explanation (6, 27). In preliminary studies onwashed Spartina roots, we also observed that NH4' inhib-ited nitrogenase activity as measured by acetylene reductionactivity (ARA), but the rate of inhibition was too rapid to beexplained by repression. This suggested that N2 fixation maybe regulated not only by a repression-derepression mecha-nism, but also by a system that affects the activity of theexisting enzyme. Such a system, called NH4' switch-off byYates (40), has been documented in both photosyntheticbacteria (9, 16, 24) and a closely related (34)chemoheterotroph, Azospirillum sp. (16, 38). Here, afterbeing converted to glutamine by glutamine synthetase (1, 14,41, 42), NH4' initiates the inactivation of nitrogenase (22,33) by covalent attachment of ADP-ribose (29) to the Feprotein component of nitrogenase (9, 24, 29, 31).

In this study, we present field and laboratory results of theNH4' effect on N2 fixation (ARA) in three microenviron-ments of the marsh sediment: the surface, sediment parti-cles, and Spartina roots. The different response of themicrobes in each habitat to NH4+ is discussed in terms of itsimplications for short-term regulation of nitrogen-fixing ac-tivity in a salt marsh environment.

MATERIALS AND METHODSARA in situ. ARA in the S. alterniflora (short form) zone

in the North Inlet salt marsh, South Carolina, was measuredin situ by using chambers and techniques developed by G. J.Whiting (Ph.D. thesis, Universtiy of South Carolina,Columbia, 1985). Chambers consisted of glass cylinders (30by 8.5 cm) fitted with rubber stoppers as tops through whichwere mounted a thermometer, a copper cooling coil (6-mm

* Corresponding author.

outer diameter), and two copper tubes (3-mm outer diameter)with attached three-way valves (Medex, Hillard, Ohio) (Fig.1). Chambers were placed between the Spartina stems onbare sediment surface. Chamber sides penetrated the marshsurface to a depth of 6 cm, forming a gastight seal. Placementof chambers into the sediment was facilitated by the cuttingof the sediment and rootmat with a sharpened steel core tube.ARA experiments were initiated by introducing 10% C2H2

(vol/vol) into the headspace through the sample valve (Fig.lb). The headspace was mixed before each sampling byusing the 50-ml syringe attached to the sampling valve. Thissyringe also allowed for pressure equalization inside thechamber by replacing the volume of headspace as sampleswere withdrawn. Gas samples (5 ml) were taken every 10 to15 min in 10-ml GasPak syringes (Becton Dickinson and Co.,Rutherford, N.J.) fitted with three-way Medex valves. Abase-line rate of C2H2 reduction was established before theintroduction of either seawater (20 ml) or NH4+-amendedseawater to the sediment surface via a tube (Fig. le). Gassamples were analysed on a Varian 3700 gas chromatographequipped with a flame ionization detector, a 1.8-m PorapakQ column, and a 1-ml sample loop-valve assembly (VarianAssociates, Palo Alto, Calif.). Peak areas were integrated ona HP 3390 integrator. Rates of activity were obtained by alinear regression analysis calculated with the SAS GLMprocedure (13).ARA in sediment slurries. Cores of salt marsh sediment

were collected from the short Spartina zone with an alumi-num tube (5.5-cm diameter), immediately sealed with rubberstoppers, and transported back to the laboratory. Sediment(free of roots) was obtained by passing the particulate corematerial (0- to 10-cm depth) through a 2-mnm sieve. Sediment(1 g [wet weight]) was added to 7-ml Fernbach flaskscontaining 1.0 ml of a seawater substitute (SWS). The SWScontained the following (in milligrams per liter of distilledwater): NaHCO3, 200; CaCl2 - 2H20, 400; MgC92, 1,000; andNaCl, 30,000. The solution was buffered with 10 mM potas-sium phosphate buffer adjusted to pH 7.8 with NaOH. Insome experiments, the medium was amended with either

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1-

4-

0910~

FIG. 1. The chamber used for in situ measurements of ARA onthe salt marsh. The components of this chamber are headspacethermometer (a), head gas mixing syringe with attached samplingvalve (b), solution addition syringe and valve (c), copper cooling coilfor pumping 00 water through the chamber to maintain ambienttemperature in the light (d), and solution addition tube (e). Darkconditions were created by placing aluminum foil around thechamber.

mannose or sodium lactate (1% [wt/vol]). The bottles con-taining sediment were sealed with serum bottle stoppers andevacuated and refilled four times with argon (which had beenpassed over a catalyst to remove any traces of 02). Acety-lene (0.2 ml) was added to give a final concentration ofapproximately 4%, and the vessels were incubated at 32°C.Ethylene was analyzed by taking 10-lI gas samples at thetimes indicated on the various figures. Additions of NH4Clwere made by adding 10 ,ul of a 300 mM anaerobic stocksolution.ARA of belowground plant material. Root and rhizome

material from the 0- to 10-cm depth interval of cores wasobtained by washing away the sediment on a 2-mm-meshsieve with tap water. Cores were all obtained from the shortSpartina zone. When the plant material was completely freeof sediment, it was separated into live roots, live rhizomes,and dead root material. Live roots were distinguished fromdead roots by visual observation as described by Valiela etal. (36). Living roots (and rhizomes) were creamy or pearlwhite and were rigid or turgid, whereas dead roots andrhizomes were brown or dull grey and flaccid. Live rootswere surface sterilized as described by McClung et al. (19).In this procedure, roots were immersed in 20% Clorox inseawater (final concentration, 1.05% sodium hypochlorite[NaOCl]) for 15 min. Surface-sterilized roots were immersedand rinsed for 5 min with three changes of sterilized seawa-ter. Surface sterility was verified by placing one of the rootson an agar plate of yeast extract-enriched SWS-malatemedium and observing no growth after 3 days. Acetylenereduction by the roots was determined by putting approxi-mately 200 mg (wet weight) of roots into 7-ml Fernbachflasks containing 1.5 ml of the SWS-mannose medium. The

100 B

80 _

L..1-

Q)

C)

0

60

40 _

20 _

1 2 1 2 3

FIG. 2. Percent weight contribution of sediment andbelowground plant material. (A) Dry weight distribution betweensediment particles (1) and belowground plant material (2). (B) Dryweight distribution between dead plant material (1), live rhizomes(2), and live roots (3).

roots (and rhizomes) were assayed anaerobically by theprocedure described above for sediments. Nitrogen amend-ments were also made in an identical manner.

Quantitation of ARA associated with sediment andbelowground plant material. The relative proportions of plantroots and sediments were determined in cores (10-cm depth,5.3-cm diameter) sieved on a 2-mm-mesh screen positionedover a large beaker; sediments were washed away from thebelowground plant material into the beaker with tap water.The plant material was further separated into live roots, deadroots, and live rhizomes. Both the sediment and the plantmaterial were dried at 60°C, and weights were expressed asa percentage of the total dry weight of the sediment core

TABLE 1. Distribution of ARA between sediment andbelowground plant material in the salt marsh

% of total C,H, reduction rate"Expt Fraction No carbon Plus

added mannose

1 Sediment <0.1 95.4Belowground 100 4.6

plant material

2 Live roots 36.5" 14.7Dead roots 48.2 82.7Live rhizomes 15.3 2.6

Weight-specific rate of C2H2 reduction. Data represent the mean of threereplications. Experiments were carried out in May 1985.bFrom June through October the contribution of live roots increased to

approximately 70% of the total C2H, reduction rate.

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N2 FIXATION (ARA) IN SALT MARSH MICROBES 145

(Fig. 2A). The plant material which had been sorted into liveroots, live rhizomes, and dead plant material was expressedseparately as a percentage of total root material (Fig. 2B).The relative amounts of ARA associated with sedimentparticles and with live and dead roots (the percentage of totalsediment C2H2 reduction rate) was calculated for eachbelowground component by multiplying the percent weightcontribution of each fraction by its rate of ARA (Table 1).

RESULTSEffect of NH4' on ARA in situ. To gain some insight into

the short-term effect of inorganic (NH4+) nitrogen on nitro-genase activity in the marsh, ARA at the marsh surface wasmeasured by using chambers (Fig. 1) covering an area of 227cm2 which were placed between the Spartina plants. A.

A120 Sw

/NH100 4

80 -

60-

40X

a. 20 a addtlonls

> 30 BSW

220/14Nf

0 60 120 180

MINUTESFIG. 3. Effect of ammonia on ARA measured in situ. C2H2

reduction was measured in chambers in the short Spartina zone ofthe salt marsh between August and September 1984. Ambienttemperatures ranged from 29 to 38°C. The initial base-line rates ofeach chamber were normalized to the mean rate, and then thenormalization constant was applied to the data acquired after theadditions. Symbols: 0, seawater (20 ml); 0, seawater containing 10mM NH4Cl (A) or 3 mM NH4Cl (B). Rates were calculated in light(A) and dark (B) conditions. Seawater was added to the chambers atthe times indicated by the arrows. The data are the means of tworeplications and are representative of three experiments carried outin late summer.

700 _live roots

80-8

dead roots

/ / /~~~~Ive rhizome

__C,

0 10 20 30 40HOURS

FIG. 4. Anaerobic ARA of sediment and belowground plantroots and rhizomes. (A) Marsh sediment components assayed forARA in the absence of an added carbon source. (B) ARA measuredin SWS solutions containing 1% mannose. These results, based ondry weights, represent activities measured in May 1985. Data are themean of three replications.

base-line rate was established for each chamber before theaddition of either seawater (the control) or seawater contain-ing NH4Cl (Fig. 3). The stimulatory effect of added seawaterwhich has been seen by others (3) was also evident here.Through the summer and fall we saw that ARA in the lightwas 8 to 12 times higher than ARA in the dark. Ammoniainhibited ARA in both the light and dark, relative to theseawater control, but inhibition was not complete, and therates remained linear for at least 2 h. The interstitial concen-tration of NH4' during the assay period was unknownbecause of the absorptive effect of the sediment and uptake

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.b

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'I)

'b

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0

HOURSFIG. 5. NH4' switch-off of ARA associated with excised roots (A) and roots of intact plants (B) in the absence of carbon amendments.

Excised roots came from cores taken from the short Spartina zone of the marsh 24 h earlier. Intact plants were grown in the greenhouse inpots of sand set in tubs of seawater. Plant roots (average dry weight, 0.2 g) were washed free of sand, and the plant was placed into atwo-chamber apparatus. The root chamber contained 290 ml of seawater and a 100-ml headspace to which C2H2 was added to achieve a finalconcentration of 10%o. The stem and leaves were fitted through an opening in a glass plate placed on top of the root chamber and sealed witha rubber stopper and silicon vacuum grease. The plant was illuminated with approximately 600 microeinsteins of light.

by plant roots. The NH4' added was estimated to be 450 and1,500 ,uM (for dark and light, respectively) in the top 5 cm ofthe sediment, which represents a sevenfold dilution of theoriginal additions based on water content measurements inthe sediment. These values, however, do not account foruptake by roots. Since both the ARA and NH4+ inhibition insitu represent the sum of all the microbes present, it wasdifficult to gain additional information from this approach.Therefore, the N2 fixers (C2H2 reducers) associated with theroots were separated from those in the rhizosphere sedimentto study the NH4' effect on each component separately.

Distribution of ARA between sediment and belowgroundplant material. To gain a quantitative understanding of theARA associated with sediment and belowground plant ma-terial, the weight ratio of each environment was determined.On a dry weight basis, sediment particles represented over90% of a 10-cm-deep core (Fig. 2A). When the belowgroundbiomass was separated, dead roots represented approxi-mately 70% of the dry weight of that material, with live rootsand live and dead rhizomes each representing about 10% ofthe remainder (Fig. 2B).The activity (on a dry weight basis) associated with bulk

sediment and the belowground plant components is shown inFig. 4. Because only low ARA could be observed in deadroot material, and none could be observed in sedimentslurries without an added carbon source (Fig. 4A), measure-ments were also made after a mannose amendment (Fig. 4B).Mannose stimulated live and dead root ARAs 5- and 30-fold,respectively.

When the percentage of total C2H2 reduction rate for themarsh components was calculated from in vitro data (seeMaterials and Methods), the unamended sediment madevirtually no contribution when compared to total plant rootmaterial (Table 1, experiment 1). However, if a carbonsource (such as mannose) was added, the potential for C2H2reduction by the sediment was great and represented about95% of the total activity in the top 10 cm of the sedimentcolumn. When a similar comparison was made of only thebelowground plant material, dead roots made up most (70%)of the dry weight (Fig. 2B) but made up only 48% of the totalC2H2 reduction rate (Table 1, experiment 2). As with sedi-ment particles, dead roots had a great potential for C2H2reduction if a carbon source was available, since the weight-specific rate increased from 48% to approximately 83% whenmannose was added. The weight-specific ARAs contributedby live roots and rhizomes were much lower: approximately15 and 3%, respectively.NH4' inhibition of ARA in vitro. Sediment particles, live

roots, live rhizomes, and dead roots all harbored N2-fixing(C2H2 reducing) populations of bacteria, but as the dataabove indicate, only on live roots was there enough carbonto support appreciable rates of ARA. Therefore, to elicitARA activity from all three bulk sediment microenviron-ments so that the NH4' effect on this activity could beexamined, all were assayed with carbon amendments. Thevalidity of this approach was indicated by the fact that NH4'also inhibited ARA in both excised roots and roots fromintact plants (Fig. 5).

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N2 FIXATION (ARA) IN SALT MARSH MICROBES 147

In sediments which were made anaerobic, we found thatacetylene-reducing populations of N2 fixers enriched bydifferent carbon substrates responded differently to NH4+.Although these populations were not strictly defined, NH4'inhibition of mannose-supported ARA was slow, taking over12 h to reach completion (Fig. 6A), while NH4+ inhibition ofARA supported by lactate was complete in 3 h (Fig. 6B).Root ARA (supported by mannose) was also rapidly(2 h) inhibited by NH4' (Fig. 6C). In this experiment, totalroot material was used, which contained over 90% deadroots.Ammonia inhibition of ARA in root-associated bacteria

Sediment no addition3 i(mannose)

20

1

Lj+ A4

CD 0 10 20' 30Z Sediment

3 (lactate) no addition

3

2

J +

E° ° 4

1C , / C

E ~ 1 o 20 30 40

HOURSFIG. 6. Effect of NH4+ on the ARA of salt marsh sediment- and

root-associated bacteria. Sediment (1 g [wet weight]) or plant rootmaterial (0.2 g [wet weight]) was suspended anaerobically in 1.5 mlof seawater. Sediment slurries were supplemented with eithermannose (A) or sodium lactate (1%) (B), and the root suspensionwas amended with 1%f mannose (C). At the time indicated by thearrow, 2 mM NH4Cl was added to each vessel. The rates areexpressed on the basis of dry weight (of sediment or total plant rootmaterial) and are the mean of three replications.

3

:1:3

(..)

0

2

A

2

0 20 30

B *

I

1.5[

1.01-

0.51

0 20 30 0 30 40

HOURS

FIG. 7. NH4+ switch-off of ARA in bacteria associated with S.alterniflora roots. (A) Dead roots; (B) live roots; (C) surface-sterilized roots. All root fractions were assayed anaerobically inSWS medium (described in the text) containing 1% mannose. Dryroot weights averaged 0.0216 g per vessel. Symbols: 0, Controls (noaddition); 0, 2 mM NH4Cl added anaerobically at the times indi-cated by the arrows.

was examined in more detail by separating the root materialinto dead roots, live roots, and surface-sterilized live roots.ARA was rapidly inhibited by NH4' in all three rootenvironments (Fig. 7) in a manner consistent with the NH4'switch-off phenomenon seen up to this time only in purecultures. Because methionine sulfoximine (MSX) is knownto inhibit NH4' switch-off of ARA, the effect of this drugwas tested on the S. alterniflora root-associated microbes.MSX at a concentration of only 25 ,uM could prevent ARAinhibition in dead roots by NH4' concentrations as high as25 mM (Fig. 8). MSX alone had no effect on root ARAactivity.

DISCUSSION

Nitrogen is considered to be a major limiting nutrient incoastal salt marshes, and increasing the nitrogen supplydramatically increases productivity and the standing crop ofmarsh plants (36). One of the sources of nitrogen in the saltmarsh is from nitrogen-fixing bacteria associated with thesediment and underground plant parts (4, 6, 10, 15, 20, 35).This input of nitrogen can be affected by many environmen-tal variables such as light, temperature, pH, oxygen, andmineral nutrients (2). Of the nutrients, both NH4' and NO3may regulate nitrogen fixation. When interstitial nitrogenlevels of the marsh were artificially increased by the appli-cation of sewage sludge (11, 39) or NH4' (4, 6, 11, 35), ARAwas decreased. Nitrogenase activity in excised Spartinaroots also varies inversely with the amount of NH4' in thewater used to cover the roots (27).One of the problems in assessing the regulatory effect of

interstitial NH4' on nitrogenase activity in the salt marsh isthat concentrations can range from 3 to 300 p.M (3, 6, 7, 10,21, 28, 35), with the higher concentrations apparently occur-ring early in the growing season (3). Since all in situ studiesin salt marshes have shown substantial rates of nitrogenaseactivity (26, 27), microenvironments probably exist wherelevels of NH4' are low enough to permit N2 fixation tooccur. The regulatory effects of interstitial nitrogen com-

pounds in situ remain a matter of speculation, but Dicker andSmith (6) have proposed by analogy with known free-livingheterotrophs that when inhibition by NH4' does occur, it is

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2F

'23

(Z)04

1 /VH4

MSX

MXIMSX

FIG. 8. Effect of MSX on NH4' switch-off by microbes associ-ated with dead roots of S. alterniflora. Symbols: 0, no addition; 0,

25 ,uM MSX added at the time indicated by the arrow; A, 25 mMNH4' added at the time indicated by the arrow; A, 25 ,uM MSX and25 mM NH4' added at the times indicated by the arrows. All rootmaterial was suspended in SWS containing 1% mannose. Dry rootweights averaged 0.0162 g per vessel, and the assays were carriedout under argon.

probably due to repression of nitrogenase synthesis. TheNH4' switch-off mechanism (inhibition of nitrogenase activ-ity) was not considered by them. Evidence presented heresuggests that some bacteria in the bulk sediment have thepotential of using this process, but it was most evident inbacteria associated with S. alterniflora root material.To examine the effect of NH4' in an undisturbed setting,

chambers (Fig. 1) were placed on the marsh surface, allow-ing us to measure in situ ARA in both surface fixers(presumably cyanobacteria and aerobes such as Azotobacterspp.) and belowground heterotrophs. The lack of significantNH4' inhibition in the light (Fig. 3A) was consistent with thepresence of cyanobacteria, since NH4' inhibition in theseorganisms is minimal (25, 41). Below the marsh surface, inthe anaerobic sediment, Clostridium and Desulfovibrio spp.

(5, 11, 15, 28) are the predominant N2 fixers. However, inanaerobic sediment slurries, little ARA was seen without theaddition of a carbon source, an observation made previouslyby others (10, 12, 15, 35, 37). To stimulate fixation byfermenters such as Bacillus and Clostridium spp., slurrieswere supplemented with mannose, and C2H2 reduction was

inhibited slowly (over 12 to 15 h) by NH4' (Fig. 6A). Gordonet al. (8 and references therein) have shown that NH4' doesnot inhibit clostridial ARA in the short term. Assuming thatthe in vitro sediment slurry activity (Fig. 6A) is caused by

that group of microbes, our NH4' inhibition data are con-sistent with that finding. In sediments amended with lactate,a good substrate for desulfovibrio spp. (but not for clostrid-ium spp.), ARA was inhibited by NH4+ within several hours(Fig. 6B). In support of this finding is the recent report thatNH4' rapidly switches off acetylene reduction in pure cul-tures of Desulfovibrio gigas (30).Except for high ARA in the presence of mannose, the

characteristics and identity of the organisms located on theroot material remain unknown. We suggest, however, thatthe organisms associated with the roots may be differentfrom those mannose fermenters on the sediment particles,because only the former show a rapid NH4' switch-off.Because of the necessity of enrichment in demonstratingARA in sediments and dead roots, our examination couldonly explore the NH4' effect of those fixers that couldrespond to the carbon substrate provided. Like the rice,sugarcane, and millet rhizospheres, it is probable that nu-merous species of N2 fixers will also be isolated from theSpartina root environment. An N2-fixing Campylobacter sp.has recently been isolated from surface-sterilized Spartinaroots (17, 18). This isolate is an obligate microaerophile thatuses organic acids, not carbohydrates such as mannose andglucose, as a source of energy, suggesting that it is not theorganism responsible for the anaerobic mannose-supportedARA observed here in surface-sterilized roots.Concerning the mechanism ofNH4+ inhibition, it is known

that this molecule can inhibit N2 fixation by several pro-cesses; therefore, a more specific analysis of the inhibitorymechanism was undertaken. MSX, an inhibitor of glutaminesynthetase activity (32) was added to mannose-amended rootsuspensions. Glutamine, the product of NH4' assimilation,appears to catalyze the switch-off inactivation process, andinhibition of glutamine synthetase (by MSX) relieves thisinhibition (1, 14, 41, 42). Consistent with the NH4' switch-off mechanism, MSX effectively prevented ARA inhibitionby NH4' in root cultures (Fig. 8).

In summary, we have shown that NH4' could inhibitnitrogenase activity in bacteria both located on sedimentparticles and in association with Spartina roots, indicatingthat regulation was possible. The mechanism of inhibitionappeared to differ between the subcomponents of the marsh(i.e., sediment particles and roots). Depending on whichmicrobial population was enriched by the added carbonsource, either repression or switch-off of ARA could beobserved in sediment particles. This was in contrast to rootARA, which rapidly responded to NH4' independently ofcarbon additions. The rate of inhibition by NH4' and itssensitivity to MSX in carbohydrate-amended dead rootsamples are both consistent with the switch-off-switch-onmechanism of nitrogenase regulation.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantGM 32183from the National Institute of General Medical Studies (to D.C.Y.)and National Science Foundation grant DEB-8119752 (to F. JohnVernberg, Director of the Baruch Institute, University of SouthCarolina, who supported G.J.W. during his Ph.D. work).We thank Edwin Gandy and Jiudi Li for their technical assistance,

and Gary King for his suggestions and criticisms of the manuscript.

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22. Neilson, A. H., and S. Nordlund. 1975. Regulation of nitrogenasesynthesis in intact cells of Rhodospirillum rubrum: inactivationof nitrogen fixation by ammonia, L-glutamine and L-asparagine.J. Gen. Microbiol. 91:53-62.

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