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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.000 DOI: 10.1128/AEM.67.10.46944700.2001

Oct. 2001, p. 46944700 Vol. 67, No. 10

Copyright 2001, American Society for Microbiology. All Rights Reserved.

Response of the Endophytic Diazotroph Gluconacetobacterdiazotrophicus on Solid Media to Changes in Atmospheric

Partial O2 PressureBO PAN AND J. KEVIN VESSEY*Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada

Received 5 June 2001/Accepted 31 July 2001

Gluconacetobacter diazotrophicus is an N2

-fixing endophyte isolated from sugarcane. G. diazotrophicus wasgrown on solid medium at atmospheric partial O

2pressures (pO

2) of 10, 20, and 30 kPa for 5 to 6 days. Using

a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a rangeof atmospheric pO

2(5 to 60 kPa). Nitrogenase activity was measured by H

2evolution in N

2-O

2and in Ar-O

2,

and respiration rate was measured by CO2

evolution in N2

-O2

. To validate the use of H2

production as an assayfor nitrogenase activity, a non-N

2-fixing (Nif) mutant of G. diazotrophicus was tested and found to have a low

rate of uptake hydrogenase (Hup) activity (0.016 0.009 mol of H2

1010 cells1 h1) when incubated in anatmosphere enriched in H

2. However, Hup activity was not detectable under the normal assay conditions used

in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO2 tested. However, when the assayatmospheric pO

2was below the level at which the colonies had been grown, nitrogenase activity was decreased.

Optimal atmospheric pO2

for nitrogenase activity was 0 to 20 kPa above the pO2

at which the bacteria had beengrown. As atmospheric pO

2was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase

activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO2

wasincreased, respiration rate increased marginally. A large single-step increase in atmospheric pO

2from 20 to

60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O2

, 80% ofnitrogenase activity was recovered within 10 min, indicating a switch-off/switch-on O

2protection mechanism

of nitrogenase activity. Our study demonstrates that colonies ofG. diazotrophicus can fix N2

at a wide range ofatmospheric pO

2and can adapt to maintain nitrogenase activity in response to both long-term and short-term

changes in atmospheric pO2

.

Gluconacetobacter diazotrophicus (47) (previously known asAcetobacter diazotrophicus [15]) is a strict aerobe and an N2-

fixing endophyte originally isolated from sugarcane roots andstems (6). It has been estimated that G. diazotrophicus can fixup to 150 kg of N ha1 year1 in sugarcane (2). Such highlevels of N2 fixation have not been reported in any other systemoutside legume-rhizobium symbioses. The bacterium has sub-sequently been isolated from sweet potato (38), coffee (23),pineapple (44), sorghum (22), finger millet (31), and severalother tropical grass species (24).

Aerobic endophytic diazotrophs require a high flux of O2 totheir respiratory systems to enable an adequate supply of re-ductant and ATP to support N2 fixation (e.g., see reference13), yet paradoxically, an excessive flux of O

2to the bacterium

can result in an inhibition of nitrogenase activity (14, 21, 26).

The inhibition of nitrogenase activity by O2 in aerobic dia-zotrophs can be reversible or irreversible, depending on theorganism and the nature (i.e., duration and severity) of theincrease in O2 flux (33, 37, 39). Reversible inhibition of nitro-genase activity (i.e., a temporary switch-off of the nitroge-nase activity while O2 flux is excessive) can be due to a con-formational change in nitrogenase, as seen in Azotobacter(11,32), to an ADP-ribosylation of dinitrogenase reductase, as seen

in the purple nonsulfur bacteria (46) and Azospirillum (49), orto a diversion of electrons from nitrogenase to other reduction

pathways, as proposed for Azotobacter(16, 29).G. diazotrophicus has the ability to fix N2 at ambient atmo-

spheric partial O2 pressures (pO2) (i.e., approximately 20 kPaof O2) in semisolid medium (6) and as colonies on solid me-dium (10). The ability to fix N

2in colonies on solid medium is

especially interesting, as there is evidence that G. diazotrophi-cus exists in situ in the intercellular spaces of sugarcane vas-cular tissue as mucoid microcolonies (9). Dong (8) also re-ported that colony morphology on solid medium and therelative distribution of the bacteria within these highly muci-laginous colony changed with changes in the partial pressure ofO2 surrounding the colonies.

Reis and Dobereiner (40) measured nitrogenase activity in

liquid cultures of G. diazotrophicus by acetylene reduction inclosed batch assays and found that activity was maximal whenthe culture was at equilibrium with 0.2 kPa of O2 in the gasphase. However, nitrogenase activity of G. diazotrophicusgrown in colonies on solid medium in response to changes inatmospheric pO

2has not yet been well characterized. Given

that G. diazotrophicus exists in situ as microcolonies adheringto plant cell walls (9), characterization of the response of thebacterium on solid medium to changes in atmospheric pO2 isparticularly relevant.

The objective of our study was to characterize the effect ofatmospheric pO2 on nitrogenase activity of G. diazotrophicusgrown on solid medium using flowthrough gas exchange mea-

* Corresponding author. Mailing address: Department of Plant Sci-ence, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.Phone: (204) 474-8251. Fax: (204) 474-7528. E-mail: [email protected].

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surements. Treatments included long-term growth of the bac-terium on a range of atmospheric pO2 (10 to 30 kPa) andsubsequent rapid changes in atmospheric pO

2in small (5- to

10-kPa) and large (40-kPa) steps. We found that nitrogenaseactivity by G. diazotrophicus is adaptive to both short-term andlong-term changes in atmospheric pO2 and that the bacterium

has a switch-off/switch-on mechanism for protection of nitro-genase from rapid changes in atmospheric pO2.

MATERIALS AND METHODS

Organism and culture. G. diazotrophicus PAL-5 (ATCC 49037; obtained from

the American Type Culture Collection, Manassas, Va.) was cultured for 2 days

at 30C, shaken at 150 rpm in LGI-P liquid medium (M. McCulley [Carleton

University], personal communication), a modified version of LGI medium (6).

LGI-P medium differs from the original LGI medium in containing 0.02 g of

Na2MoO4 2H2O liter1, 0.1 mg of biotin liter1, 0.2 mg of pyridaxol HCl

liter1, and 5 ml of sugarcane juice (pressed from fresh sugarcane stem) liter1,

and the final pH was adjusted to 5.5 using 1% acetic acid. Diluted cells were

spread on solid LGI-P agar medium (15 g of agar liter1 plus 50 mg of yeast

extract liter1). Before serial dilution, 5 ml of culture was vortexed with glass

beads to prevent clumping of the colonies and to obtain an even distribution of

individual separate colonies on the petri plates.G. diazotrophicus was grown on solid LGI-P medium in petri plates for 5 or 6

days at 30C prior to gas exchange measurements. Cultures were grown under

ambient atmospheric pO2

(approximately 20 kPa) or in a gas exchange chamber

(see below) with 10 or 30 kPa of O2 flowing through the chamber at approxi-

mately 65 ml per min. For cultures grown in the chamber, input air was bubbled

through a flask of water prior to being fed into the chamber to avoid desiccation

of the cultures.

Cell enumeration. The numbers of viable cells per colony of all petri plate

cultures used in gas exchange measurements were determined by plate counting.

Five colonies per plate were cut out of the agar, and as much agar subtending the

colonies as possible was removed without disturbing the integrity of the colonies.

These five colonies were then vortexed together in a small test tube containing 5

ml of 0.85% NaCl solution and glass beads. This suspension was then serially

diluted in 0.85% NaCl solution and plated. Cell number per plate was calculated

by multiplying the colony number per plate by the cell number per colony. On

average, colonies contained 107

to 108

viable cells each and 8.5-cm-diameter petriplates contained 100 to 150 colonies each. No differences were noted in these

growth parameters for cultures grown at 10, 20, or 30 kPa of O2.

Gas exchange measurements of respiration rate and nitrogenase and hydro-

genase activities. A flowthrough gas exchange system (45) was used to measure

the effects of changing atmospheric pO2 on respiration rate and nitrogenase

activity of G. diazotrophicus colonies. The system includes computer-controlled

mass flow controllers (MKS Instruments Inc., Nepean, Canada) for gas mixing

and delivery, an infrared gas CO2 analyzer (ADC-225MKS; Analytical Develop-

ment Co. Ltd., Hoddesdon, United Kingdom) for measurement of respiration

rate, and an H2 analyzer (27) for measurement of nitrogenase activity. A cham-

ber with inner dimensions of 50 by 20 by 5 cm with four shelves for holding up

to 40 petri plates was constructed from 0.9-cm-thick acrylic sheeting. The void

volume when the chamber was fully loaded with cultures was 1.86 liters. Gas was

introduced at one end of the chamber, flowed horizontally across the petri plates,

and exited at the opposite end of the chamber.

Measurements of nitrogenase activity and respiration rate by G. diazotrophicus

colonies were taken at various atmospheric pO2. Gas mixtures fed into the assay

chamber were composed of various partial pressures of O2 in N2 (N2-O2) or in

Ar (Ar-O2). Respiration rate was measured as the rate of CO2 evolved from the

colonies with N2-O

2as the input gas. Nitrogenase activity was measured as H

2

evolution in N2-O2 and in Ar-O2 (21, 26).

Gas exchange measurements were made in the following manner. Twenty to

40 petri plates containing 5- or 6-day-old cultures of G. diazotrophicus on solid

LGI-P medium were sealed in the chamber and then connected to the gas

exchange system. Gas mixtures were passed through the chamber at a rate of 500

ml min1. For testing the response of G. diazotrophicus to small (5- or 10-kPa)

changes in atmospheric pO2, gas exchange measurements were initiated at the

pO2 under which the cultures had been grown (i.e., 10, 20, or 30 kPa of O2).

Initially, H2 and CO2 evolution was quantified at this pO2 in a gas mixture of

N2-O2. Once these readings had been made, the input gas was switched to Ar-O2at the same pO2. Normally, 30 min to 1 h was required before the H 2 evolution

came to steady state, and the H2 evolution rate was always measured 1 h after the

switch from N2-O2 to Ar-O2. After the H2 evolution rate had been measured in

Ar-O2, the input stream was change back to N2-O2, but at a new atmospheric

pO2 (5 or 10 kPa from the previous reading). Upon returning to an atmosphere

of N2-O

2, 10 to 30 min was required for H

2and CO

2evolution rates to come to

steady state. This cycle was then repeated until nitrogenase activity and respira-

tion rate had been measured at all atmospheric pO2 (stepping down from initial

level of atmospheric pO2 to the lowest levels tested and then stepping up to the

highest levels tested). To observe the temporal response of G. diazotrophicus to

large (40-kPa), single-step increases and decreases in atmospheric pO2, gasexchange measurements were initiated at the atmospheric pO2 under which the

bacteria had been grown (approximately 20 kPa), and then atmospheric pO2 was

increased in a single step to 60 kPa, where it remained for approximately 15 min

before being returned to 20 kPa in a single step. All gas exchange measurements

were made at room temperature (22 1C). Preliminary experiments showed

that once steady-state rates of H2 and CO2 evolution were reached, they re-

mained steady for many hours (i.e., up to 12 h).

Production of H2 is an obligate reaction of the nitrogenase enzyme complex

during the fixation of N2 (4). The rate of H2 evolution in N2-O2 is a measure of

partial or apparent nitrogenase activity (i.e., proton reduction to H2

by nitro-

genase in the presence of N2 fixation) (21, 26). The rate of H2 evolution in Ar-O2is a measure of total nitrogenase activity (i.e., in the absence of N

2as a substrate,

total electron flow through nitrogenase is used to reduce protons to H 2) (20, 21,

26). In N2-O2, the proportion of total electron flow through nitrogenase being

directed to N2 fixation is known as the electron allocation coefficient (EAC) (12)

and is calculated as 1 (H2 evolution in N2-O2H2 evolution in Ar-O2). EACcan be viewed as a measure of an aspect of nitrogenase efficiency (i.e., the

higher the EAC, the greater the proportion of electrons going to fix N2 and the

lower the proportion of electrons going to the wasteful process of proton

reduction).

The accuracy of measuring nitrogenase activity by H2 evolution is dependent

upon a lack of hydrogenase activity leading to either H2

production or consump-

tion by the test organism under the assay conditions. Experiments with a non-

N2-fixing (Nif) mutant of G. diazotrophicus (strain MAD3A) (42) were per-

formed to determine if H2 evolution from G. diazotrophicus was associated only

with nitrogenase activity and if the bacterium had hydrogenase uptake (Hup)

activity. The Nif mutant was designed by insertional mutagenesis of the nifD

gene of wild-type G. diazotrophicus. The resulting mutant strain (MAD3A) was

generously donated by C. Kennedy, University of Arizona. G. diazotrophicus

MAD3A was grown and handled as described above for wild-type G. diazotro-

phicus PAL5 except that 200 g of kanamycin ml1 was added to the medium.

The growth rate of the Nif

mutant was not significantly different from that ofwild-type G. diazotrophicus for the first 5 days of culture.

Three replicates of 40 plates each ofG. diazotrophicus MAD3A were tested for

H2 production in air and Ar-O2 (80:20) in preliminary experiments in our gas

exchange system. MAD3A did not produce H2 production under any conditions

(data not shown).

Hydrogenase uptake activity by G. diazotrophicus was assessed in flowthrough

and closed-assay systems. For the flowthrough assay, MAD3A was grown for 4

days at ambient pO2

(approximately 20 kPa) as described above. Three replicates

of 20 petri plate cultures were then placed in the gas exchange chamber and

flushed continuously with air containing 2 ppm (vol/vol) of H2 at a flow rate of

20 ml min1 for approximately 24 h. This concentration of H2

was used because

it is the typical level of H2 evolution from wild-type G. diazotrophicus in air under

our normal assay conditions. After exposure to 2 ppm of H2 for 24 h, the gas flow

rate was increased to 500 ml min1 (the normal flow rate for our assays) and the

concentration of H2 exiting the chamber was measured. For the closed assay,

wild-type and Nif mutant strains of G. diazotrophicus were grown on solid

medium for 4 days. On the fifth day, 10 petri plate cultures were placed in the gas

exchange chamber and the chamber was flushed with 50 ppm of H2 in air at flow

rate of 20 ml min1. After 24 h at this flow rate, the chamber was sealed, and

evolution (wild-type strain) and consumption (Nif strain) were monitored im-

mediately after sealing and then every 30 to 60 min for the next 6 to 8 h. Gas

samples (1 ml) were taken from the chamber and injected into an air stream

entering the H2 analyzer at a flow rate of 300 ml min1 for analysis as described

by Layzell et al. (27). Hydrogen consumption and evolution rates were calculated

by linear regression. The tests were replicated four times each for the wild-type

and Nif strains of G. diazotrophicus.

The aerobic, facultative chemoautotroph Ralstonia eutropha (ATCC 17699)

was used as a positive control in the assessment of Hup activity. Early-log-phase

cells grown in Difco 0003 liquid medium (Becton Dickinson, Franklin Lakes,

N.J.) were plated onto Difco 0001 solid medium and grown for 4 days before

being assayed. H2 consumption by these colonies was assay as described above

for G. diazotrophicus MAD3A (i.e., closed assays at 50 ppm of H2 for 8 h).

VOL. 67, 2001 G. DIAZOTROPHICUS RESPONSE TO ATMOSPHERIC pO2 CHANGES 4695

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Four experiments were conducted to test the response of G. diazotrophicus to

changes in atmospheric pO2. The experiments consisted of (i) testing responses

of G. diazotrophicus grown at 20 kPa of O2 to small (5- or 10-kPa) stepped

changes in atmospheric pO2; (ii) testing responses of G. diazotrophicus grown at

20 kPa of O2 to a large (40-kPa) stepped change in atmospheric pO2; (iii) testing

responses of G. diazotrophicus grown at 10 kPa of O2 to small (5- or 10-kPa)

stepped changes in atmospheric pO2; and (iv) testing responses of G. diazotro-

phicus grown at 30 kPa of O2 to small (10-kPa) stepped changes in atmospheric

pO2. Gas exchange measurements in a single chamber containing 20 to 40 petri

plates of G. diazotrophicus cultures was considered a single replicate. For each

experiment, gas exchange measurements were replicated four times. All data

were normalized by calculating gas evolution per cell (cell number was deter-

mined for each replication of each experiment; see enumeration method above).Data were analyzed using the general linear model of the SAS statistical package

(SAS Institute, Cary, N.C.), assuming a completely randomized design, and mean

separation was tested using the least-significant-difference procedure (P 0.95).

RESULTS

Effects of small stepped changes in atmospheric pO2

on G.

diazotrophicus grown at 20 kPa of O2. For G. diazotrophicusgrown at 20 kPa of O

2, 10-kPa stepped increases in atmo-spheric pO2 above 30 kPa of O2 resulted in a decrease in totalnitrogenase activity (H2 evolution in Ar-O2) (Fig. 1). However,nitrogenase was still active even at atmospheric pO2 of 60 kPa(29% of the rate at 20 kPa of O2). Stepped decreases in

atmospheric pO2 from 20 to 10 to 5 kPa also resulted indecreases in total nitrogenase activity. The optimal atmo-spheric pO

2for G. diazotrophicus grown at 20 kPa of O2 were

20 and 30 kPa of O2.Stepped increases of 10 kPa of O

2above 20 kPa had no

significant effect on the EAC of nitrogenase activity (Fig. 2).However, as atmospheric pO

2was lowered from 20 kPa to10

and 5 kPa of O2, the EAC decreased.As atmospheric pO

2was increased from 20 to 60 kPa of O

2

in 10-kPa steps, the respiration rate of G. diazotrophicus cellsincreased marginally (Fig. 3). For example, the threefold in-crease in atmospheric pO2 from 20 to 60 kPa resulted in an11% increase in CO

2evolution per cell. In contrast, decreasing

atmospheric pO2 from 20 to 10 kPa and then 5 kPa resulted in

severe decreases in respiration rates of 39 and 51%, respec-tively.

Effects of a large (40-kPa) stepped change in atmospheric

pO2

on G. diazotrophicus grown at 20 kPa of O2. When G.diazotrophicus colonies grown at atmospheric pO2 of 20 kPawere exposed to a 40-kPa single-step increase in atmosphericpO

2, nitrogenase activity decreased rapidly and severely (Fig.

4). After this decrease, nitrogenase activity at 60 kPa of O2 wassteady at approximately 26% of the activity at 20 kPa of O

2.

After 15 min at 60 kPa of O2, oxygen concentration was then

switched back to 20 kPa, and nitrogenase activity increasedalmost immediately. Within 10 min of returning to 20 kPa ofO

2, nitrogenase activity by G. diazotrophicus had recovered toapproximately 80% of the original activity. Changes in nitro-genase activity (Fig. 4) and respiration rate (data not shown) in

FIG. 1. Effect of atmospheric pO2 on total nitrogenase activity (H2evolution in Ar-O2) of G. diazotrophicus colonies grown at 20 kPa of

O2. Data are means plus standard errors (n 4). Results with differentletters are significantly different at a P value of0.05.

FIG. 2. Effect of atmospheric pO2 on EACs of nitrogenase of G.diazotrophicus grown at 20 kPa of O2. Data are means plus standarderrors (n 4). Data with different letters are significantly different at

a P value of0.05.

FIG. 3. Effect of atmospheric pO2 on respiration rate (CO2 evolu-tion in N2-O2) of G. diazotrophicus colonies grown at 20 kPa of O2.Data are means plus standard errors (n 4). Data with differentletters are significantly different at a P value of0.05.

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response to the single-step change from 20 to 60 kPa of O2were similar in magnitude (i.e., a 74% decrease in nitrogenaseactivity and an approximate 10% increase for respiration) tothose observed when the increase in from 20 and 60 kPa of O

2

took place in several 10-kPa steps (Fig. 1 and 3).Effects of small stepped changes in atmospheric pO

2on G.

diazotrophicus grown at 10 or 30 kPa of O2. G. diazotrophicuscolonies were grown under 10 or 30 kPa of atmospheric O

2for

5 to 6 days and then assayed for total nitrogenase activity at arange of atmospheric pO

2(5 to 60 kPa) (Fig. 5 and 6). In both

cases, maximal nitrogenase activity occurred at 10 to 20 kPa ofO

2above the atmospheric pO

2at which the colonies had been

grown. For colonies grown under 10 kPa of O2, nitrogenaseactivity was maximized at 20 and 30 kPa of O

2(Fig. 5). For

colonies grown under 30 kPa of O2, nitrogenase activity wasmaximized at 40 kPa of O

2(Fig. 6).

Hydrogenase uptake activity of G. diazotrophicus. There wasno detectable H

2consumption by the Nif mutant of G. dia-

zotrophicus (MAD3A) under typical conditions experienced bythe wild-type strain when nitrogenase activity was assayed (2

ppm of H2 in air in a flowthrough system at ambient atmo-spheric pO2). However, when the strain was supplied with 50

ppm of H2 in air in a closed-assay system, we detected a H2consumption rates of 0.016 0.009 mol of H2 10

10 cells1

h1. This rate is low, as the H2 evolution rate by wild-type G.diazotrophicus assayed under the same conditions was 0.362 0.027 mol of H2 10

10 cells1 h1 and H2 consumption rate bythe Hup aerobe R. eutropha was 0.080 0.003 mol of H21010 cells1 h1.

DISCUSSION

The concentration of O2 at the sites of nitrogenase activity in

aerobic and microaerophilic diazotrophs is the result of theinterplay among (i) the concentration of O2 in the surroundingatmosphere, (ii) the diffusion rate of O

2from the surrounding

atmosphere to the sites of nitrogenase activity, (iii) the con-sumption rate of O

2in the vicinity of nitrogenase (predomi-

nantly via oxidative phosphorylation), and (iv) the role of car-riers of O

2which facilitate diffusion in some systems (such as

leghemoglobin in legume nodules) (21). The present studyinvestigated responses in nitrogenase activity to changes inatmospheric pO2 around colonies in a flowthrough system;previous studies (5, 40) injected enough pure O

2into a previ-

ously anaerobic, closed liquid system to achieve target pO2. Allthese studies enable observation of changes in nitrogenaseactivity in response to relative changes in O2 flux to the bac-

FIG. 4. Time course of the response of nitrogenase activity (H2evolution in Ar-O2) of G. diazotrophicus colonies to large, sudden

changes in O2 concentration. The gas composition (top axis) waschanged from 20 to 60 kPa of O2, maintained for 15 min, then changedback to 20 kPa of O2. The timeline of nitrogenase activity is typical ofthe response to the changes in pO2. The vertical bars represent thestandard errors for actual rates of nitrogenase activity at steady state(n 4).

FIG. 5. Effect of atmospheric pO2 on nitrogenase activity (H2 evo-lution in Ar-O2) of G. diazotrophicus colonies grown at 10 kPa of O2.Data are means plus standard errors (n 4). Data with differentletters are significantly different at a P value of0.05.

FIG. 6. Effect of atmospheric pO2 on nitrogenase activity (H2 evo-lution in Ar-O2) of G. diazotrophicus colonies grown at 30 kPa of O2.Data are means plus standard errors (n 4). Data with different

letters are significantly different at a P value of0.05.


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teria; however, neither the actual flux of O2

to the diazotrophsor the actual concentration of O2 at the sites of nitrogenaseactivity was determined.

In our study, nitrogenase activity by G. diazotrophicus wastolerant of atmospheric pO

2as high as 60 kPa (Fig. 1). These

findings are not in conflict with previous studies (5, 40) that

found that nitrogenase activity by G. diazotrophicus in liquidculture was totally inhibited when the culture was at equilib-rium with 6 kPa of O2 in the gas phase. These findings simplyreflect that in our study, atmospheric pO2 surrounding thecolonies was changed, and in the previous studies, the partialpressure of dissolved oxygen in liquid cultures was changed.However, comparison of these studies indicates that G. dia-zotrophicus can use the milieu of a colony as an effective re-sistance to O2 diffusion, resulting in an O2 concentration andO

2flux within the colony which enable the bacteria to fix N

2in

a broad range of atmospheric pO2 surrounding the colony.Using H

2production as a measure of nitrogenase activity in

closed-assay systems, Dong et al. (8, 10) showed that G. dia-zotrophicus could fix N2 in colonies with 2 and 20 kPa of O2 in

the surrounding atmosphere and suggested that colony struc-ture and location of bacteria within the colony played a role inthe protection of nitrogenase from excessive O2 flux. Bacterialmucilage is known to decrease the rate of oxygen diffusion tocells (3). The presence of extracellular polysaccharide sur-rounding Beijerinckia derxii cells is necessary to maintain ni-trogenase in this organism (1). Derxia gummosa forms smallnonfixing colonies if grown at 20 kPa of O

2; however, if grown

at 5 kPa of O2, the bacterium forms large, highly mucilaginouscolonies which fix N

2(17, 18). The motile diazotroph Azospi-

rillum brasilense (50) is known to display aerotaxis within sus-pensions to achieve the appropriate O

2environment for N

2

fixation.

For colonies grown at 20 kPa of O2 and assayed at the sameatmospheric pO2, total nitrogenase activity was approximately0.5 mol of H2 10

10 cells1 h1 (Fig. 1). Is this a relatively lowor high rate of nitrogenase activity? We have compared thelevel of nitrogenase activity in G. diazotrophicus to that ofBradyrhizobium japonicum in a typical soybean (Glycine max[L.] Merr.) nodule. Based on measurements of nodules on5-week-old soybean plants, Lin et al. (28) found that nodulescontained approximately 109B. japonicum bacteroids each andthat total nitrogenase activity was in the range of 2.0 to 4.0mol of H2 10

10 cells1 h1. Nitrogenase activity for G. dia-zotrophicus colonies at ambient atmospheric pO2 in our studywas approximately 12 to 25% of the rates calculated for B.

japonicum in soybean nodules. We consider such a level ofnitrogenase activity by G. diazotrophicus in colonies to be re-markably high considering that a soybean nodule is a highlysophisticated organ designed to provide a highly conducivemilieu (in terms of O

2flux, carbon supply, assimilation offixed

N, etc.) for bacteroids to fix N2.Our study is the first measure of EACs for G. diazotrophicus.

We found that the EAC of G. diazotrophicus at 20 kPa of O2was approximately 0.6 (Fig. 2), meaning that in air, approxi-mately 60% of electron flow through nitrogenase would beallocated to reduction of dinitrogen and 40% would be allo-cated to proton reduction. Again, the EAC of G. diazotrophi-cus can be put into context by comparing it to that of rhizobiain legume nodules. The theoretical maximum for EAC is 0.75

(i.e., at least one H2

produced for every N2 fixed by nitroge-

nase) (43). The EACs of legume symbioses are commonlybetween 0.59 and 0.70 (21). The reason for the variability in theEAC is not clearly understood (19, 26). Our measurements ofthe EAC for nitrogenase in G. diazotrophicus grown on solidmedium at ambient atmospheric pO2 is in the same range as

EACs commonly observed in legume nodules.The accuracy of measurements of nitrogenase activity by H2evolution is dependent upon the lack of hydrogenase uptakeactivity (21, 26). Although the Nif mutant of G. diazotrophi-cus was seen to have a low level of Hup activity when suppliedwith relatively high levels of H2 (50 ppm) in a closed-assaysystem, under the standard conditions in our flowthrough assaysystem (i.e., 2 ppm of H

2), Hup activity was not detectable.

Small (5- to 10-kPa) decreases in atmospheric pO2 resultedin declines in nitrogenase activity and respiration rate in G.diazotrophicus grown at 20 kPa of O2 (Fig. 1). This is clearlyrepresentative of an O

2limitation of cellular metabolism and

has been seen in other aerobically functional N2-fixing systems,such as Azotobacter(48) and soybean nodules (25). However,

small stepwise increases in atmospheric pO2 above 20 kPa alsoresulted in declines in nitrogenase activity (Fig. 1). The de-clines in nitrogenase activity with small (10-kPa) increases inatmospheric pO

2could occur for one of two reasons: (i) an

irreversible O2-induced denaturation of the nitrogenase en-zyme or (ii) a reversible controlled down-regulation of nitro-genase activity (14). The time course of nitrogenase activity inresponse to single-step, 40-kPa changes in atmospheric pO

2

(Fig. 4) indicates that the latter and not the former mechanismis at work in G. diazotrophicus. The rapid decrease in nitroge-nase activity when atmospheric pO2 was switched from 20 to 60kPa, and the subsequent rapid recovery when the bacteriareturned to 20 kPa, indicate that G. diazotrophicus has a

switch-off/switch-on protection mechanism in response tochanges in atmospheric pO2.

Reversible inhibition of nitrogenase activity has been seen ina number of diazotrophs in response to increases in pO2 and tothe addition of ammonium. Three underlying physiologicalmechanisms have been associated with switch-off/switch-on ki-netics of nitrogenase in diazotrophs. The switch-off/switch-onmechanism can be the result of an O2-induced conformationalchange in nitrogenase as seen in the Mo-dependent nitroge-nase of Azotobacter (11, 32, 35, 36, 41). Switch-off/switch-onmechanisms can also be facilitated by an ADP-ribosylation ofdinitrogenase reductase which halts nitrogenase activity. Thismechanism is coded for by the draT and draG genes and has

been observed in a number of diazotrophs, includingRhodobacter capsulata (46), Rhodospirillum rubrum, Azospiril-lum brasilense, and Azospirillum lipoferum (34, 49). Finally, thenitrogenase switch-off/switch-on mechanism in a number ofdiazotrophs may involve diversion of electrons from nitroge-nase to other (unidentified) electron acceptors (16) and/or an

ATP limitation of nitrogenase activity, possibly due to a switchto uncoupled respiratory chain as proposed for Azotobactervinelandii (29).

Which if any of the above identified switch-off/switch-onmechanisms are at work in G. diazotrophicus was not investi-gated in our study. However, it is highly unlikely that themechanism involves the ADP-ribosylation of dinitrogenase re-ductase. Although Burris et al. (5) found that G. diazotrophicus


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had a rather sluggish response to ammonium addition andrequired 10 M NH4

to switch off nitrogenase, they found noevidence of ADP-ribosylation of dinitrogenase reductase or ofthe draT-draG gene complex in G. diazotrophicus. Recently, S.Norlund (personal communication) also found evidence of aswitch-off/switch-on phenomenon in G. diazotrophicus in re-

sponse to changes in pO2, possibly involving a conformationalchange in nitrogenase medium by a Shethna-like protein (32).In this study, long-term adaptation of G. diazotrophicus to

different atmospheric pO2 was tested by growing the bacteriumfor 5 or 6 days at 10, 20, or 30 kPa of O2 before nitrogenaseactivity was measured. Although culture conditions were notexactly the same for all the cultures (see Material and Meth-ods), some trends are consistent in all three treatments. Duringassays of nitrogenase activity, when atmospheric pO2 was de-creased below the concentration at which the bacteria werecultured, nitrogenase activity was always lower (Fig. 1, 5, and6). This appears to be due to a generalized O

2limitation of

cellular metabolism (Fig. 3) (25, 48). G. diazotrophicus cultures which were grown under different atmospheric pO

2also

showed different optimal atmospheric pO2 for nitrogenase ac-tivity. The optimal atmospheric pO

2for cultures grown at 10

and 20 kPa of O2 was 20 to 30 kPa of O2 (Fig. 1 and 5); theoptimal atmospheric pO

2for nitrogenase activity for cultures

grown at 30 kPa of O2 was 40 kPa of O2 (Fig. 6) The fact thatthe cultures grown at the highest atmospheric pO

2showed a

higher optimal pO2 for nitrogenase activity indicates a long-term adaptation ofG. diazotrophicus colonies to different pO2.Other aerobically functional N2-fixing systems such as A. vine-landii (30) and the B. japonicum-soybean symbiosis (7) areknown to make long-term adaptations of nitrogenase activityto nonambient pO

2. Dong (8) noted differences in colony mor-

phology of G. diazotrophicus between cultures grown long-

term on 2 and 20 kPa of atmospheric pO2. We are currentlyinvestigating whether morphologic and structural characteris-tics of the colonies contribute to these long-term adaptations.

ACKNOWLEDGMENTS

We thank B. Luit and J. Foidart for their technical assistance, C.Kennedy for donating the Nif strain G. diazotrophicus MAD3A, andP. Hallenbeck, Universite de Montreal, for useful discussions on thispaper.

This study was funded by grants from Cargill Inc. and the AAFC/NSERC (Canada) Research Partnership Program.

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