JOURNAL OF BACTERIOLOGY, Apr. 1984, p. 187-1940021-9193/84/040187-08$02.00/0Copyright © 1984, American Society for Microbiology

Vol. 158, No. 1

Role of the nifQ Gene Product in the Incorporation of Molybdenuminto Nitrogenase in Klebsiella pneumoniae

JUAN IMPERIAL, RODOLFO A. UGALDE, VINOD K. SHAH, AND WINSTON J. BRILL*Department of Bacteriology and Center for Studies of Nitrogen Fixation, University of Wisconsin, Madison, Wisconsin

53706

Received 6 September 1983/Accepted 3 January 1984

NifQ- mutants of Klebsiella pneumoniae are defective in nitrogen fixation due to an elevated requirementfor molybdenum. When millimolar concentrations of molybdate were added to the medium, the effects ofthe nifQ mutations were suppressed. NifQ- mutants were not impaired in the uptake of molybdate, butmolybdate accumulation was defective in these mutants. All of the nif-coded proteins were present in NifQ-cells derepressed in the absence of molybdenum. Molybdenum-activatable nitrogenase component I wasfound at the same level observed in the wild type. Molybdenum, thus, does not play a role in nifexpressionor in the short-term stability of nif-coded proteins. The defect in NifQ- mutants was in the incorporation ofmolybdenum into nitrogenase component I. The nifQ gene product acts together with the products of nifB,nifNV, and nifE in the biosynthesis of the iron-molybdenum cofactor of nitrogenase.

Nitrogen fixation in Klebsiella pneumoniae requires thecoordinate expression of at least 15 genes arranged in sevenoperons that constitute the nifcluster (reviewed in reference25). The protein products of 13 of these genes have beenidentified (24, 26). The functions of the products of most ofthe nif genes have been identified as structural proteins,regulatory proteins, electron transport proteins, or enzymesfor processing nitrogenase (7, 11, 19, 26, 31).The essential role of molybdenum in nitrogen fixation,

proposed as early as 1930 (1), was shown to be related to itspresence in component I of the nitrogenase system (3).Molybdenum in nitrogenase component I is present in acofactor (iron-molybdenum cofactor [FeMo-co]) containingFe, Mo, and S (28). This cofactor has been proposed as theactive site for the reduction of N2, since it restores theactivity of certain Nif mutants (26, 28), gives nitrogenasecomponent I its characteristic electron paramagnetic reso-nance spectrum which changes during enzyme turnover (23),and can catalyze acetylene reduction to ethylene (29).Very little is known about the pathway of biosynthesis of

FeMo-co and its insertion into nitrogenase component I.Three nif genes, nifB, nifN, and nifE, have been proposedas part of this process, since inactive component I in cellextracts of mutants with lesions in these genes can beactivated by the addition of pure FeMo-co (26).The gene nifQ was defined by MacNeil et al. (16) as the nif

complementation group closest to his, forming an operonwith nifB. All point mutations, Mu insertions, and deletionmutations in nifQ yield a very leaky phenotype, making thegenetic analysis difficult. This difficulty led Merrick et al.(17) to question the existence of nifQ as an independentcistron and led MacNeil et al. (16) to propose a nonessentialrole for the nifQ gene product in nitrogen fixation expres-sion. No protein product has yet been assigned to the nifQgene.

In this paper we characterize the physiology of NifQ-mutants and assign an essential role to the nifQ product inthe processing of molybdenum into nitrogenase.

* Corresponding author.

MATERIALS AND METHODSMedia and chemicals. K medium was used as an N-free

minimal medium (15). KN medium is K medium containing25 mM ammonium acetate. When molybdenum-free mediumwas required, Na2MoO4 was omitted, ultrapure chemicalswere used, and all glassware was treated with 4 N HCl andwashed with double-distilled water. Some batches of thisMo-free medium were further depleted of traces of molybde-num by a biological method. Azotobacter vinelandii cellswere grown in modified Burk medium (35), supplementedwith 25 mM ammonium acetate and lacking Na2MoO4, untilthey reached the early stationary phase, washed twice in thesame medium, and suspended to one-third of the originalculture density in the K medium to be treated. The suspen-sion was incubated for 15 min at 30°C and centrifuged for 30min at 20,000 x g and 4°C. The supernatant solutions weresterilized by filtration (0.2-,um pore size) and stored at 4°C.Carrier-free Na299MoO4 was purchased from Centichem,Inc., Tuxedo, N.Y.

Bacterial strains. Wild-type K. pneumoniae UN is strainM5al (2). Mutant strains of K. pneumoniae are described inTable 1. A. vinelandii OP (4) was used.Growth and derepression. To derepress for nitrogenase

activity, cells were grown overnight in Mo-free KN mediumunless otherwise stated. The cultures were washed with Mo-free K medium, and the absorbancy at 660 nm was adjustedto 1.2. One-milliliter aliquots were derepressed under an Aratmosphere; after 1.5 h of incubation, L-serine (50 pug/ml)was added; assays were performed after 5 h. The washedcells were used as the inoculum (1:40) for growth experi-ments with N2. The maximum cell yield was determined byabsorbancy at 660 nm.

Einzyme assays. Acetylene reduction activity was assayedin whole cells (2). ,3-Galactosidase activity assays followedpublished methods (18).

Polyacrylamide gel electrophoresis. Two-dimensional gelswere run with extracts obtained from nif-derepressed,[355]methionine-labeled cultures (24). Gels were treated withEn3Hance (New England Nuclear Corp., Boston, Mass.),dried, and autoradiographed (24). Anaerobic native electro-phoresis of 99Mo-labeled cell extracts was performed withslab gels (15 by 16.5 cm by 0.75 mm (27).

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188 IMPERIAL ET AL.

Strain

UNUN563UN1089UN1655UN1676UN1688UN2139UN2454UN2458UN4484UN4487UN4489UN4499UN4504UN4515UN4523UN4533UN4847UN4903

TABLE 1. Bacterial strains

Relevant genotype

Wild typeArfb gnd his nifnifD4409::MunifB4691nifE4712nifN4724nifQ4970nifQ5027::MunifQ503::MunifK5924::Mu dl(lac Ap9nifE5927::Mu dl(lac Ap)nijM5929::Mu dl(lac Ap)nifM5939::Mu dl(lac Ap)nifJ5944::Mu dl(lac Ap9nifDS955::Mu dl(lac Apr)nifF5963::Mu dl(lac Ap9niJB5973::Mu dl(lac ApDnifA6135::Mu dl(lac Ap)nijS6J91::Mu dl(lac Apr)

Reference

2161616161616161614141414141414141414

9Mo labeling. To study Mo accumulation, cultures werederepressed in the presence of 20 ,uCi of carrier-free Na-259MoO4 per ml. After 5 h, 50- -l aliquots were filteredthrough nitrocellulose filters and washed with 4 ml of Kmedium containing 10 ,uM Na2MoO4, and the filters werecounted in Bray scintillation fluid (New England NuclearCorp.) with the 32p setting of a Packard liquid scintillationcounter. The exchangeability of the incorporated label wasstudied by the incubation of anaerobic samples in thepresence of 400 ,uM Na2MoO4 and the filtration of the 50-,lIaliquots after 10 min.To obtain 9Mo-labeled extracts, the radioactive cultures

(20 ml) were washed in K medium containing 10 ,uMNa2MoO4, and extracts were prepared by osmotic shock (2)with sorbitol instead of sucrose. The final volume (0.5 ml)was frozen and thawed three times and centrifuged for 20min at 5,000 x g , and the supernatant solution wasrecovered and stored at -200C. All the manipulations wereperformed anaerobically under argon.

Serological assay. Quantitation of nitrogenase component Icross-reacting niaterial was achieved by immunoelectropho-resis (30).

RESULTSMolybdenum requirement for nitrogenase activity. The

requirement of molybdenum for nitrogenase activity wasstUdied by the use of acetylene reduction to follow nitroge-nase derepression under argon and by the growth of cultureswith N2 in molybdenum-free K medium with different con-centrations of Na2MoO4. Figure 1 shows the dependence ofacetylene reduction activity on the concentration of molyb-date added to derepressing cultures. The basal activity of thewild type in the absence of added molybdate varied, depend-ing on the batch of medium, and was as high as 50% of theactivity obtained with optimal molybdate. Treatment of themedium with A. vinelandii cells as described above reducedthis basal activity to 3 to 10% with 1-ml cultures. The use oflarger culture volumes (100 ml) further reduced this basalactivity (<1%). Maximum activity was reached at concen-trations above 10 nM molybdate. The NifQ- mutant had alower activity in the absence of added molybdate andrequired 104 times more molybdate to reach the maximumactivity, which was even higher than that obtained with the

wild type. Therefore, the addition of appropriate levels ofmolybdate suppressed the effect of the mutation. The sameresults (data not shown) were obtained with mutant strainsUN2458 (nifQ5031::Mu) and UN2139 (NifQ-). The leaki-ness reported for NifQ- mutants (16, 17) results from theamount of molybdate present in the media routinely used forgenetic analysis. Mutants with lesions in nifB, nifN, or nifE,three genes involved in the biosynthesis of FeMo-co, did notrespond to the concentration of molybdate.Maximum growth of the wild type and NifQ mutants on

N2 showed the same dependence on the molybdate added(Fig. 2), but the requirement for the maximum growth wasslightly higher.

Millimolar concentrations of molybdate inhibited bothacetylene reduction activity and growth on N2. NifQ- mu-tants showed a higher tolerance (Fig. 1). This toxicity wasnot due to salinity, since equivalent concentrations of sodi-um sulfate had no effect.

Transport and accumulation of 99Mo. The affinity of theMo transport systenm was studied in wild-type and NifQ-cells at short incubation times, and the accumulation ofnonexchangeable 99Mo label was measured at the end ofnitrogenase derepression.

(i) Mo uptake. Figure 3 shows double-reciprocal plots ofmolybdenum uptake after 30 s at different molybdate con-centrations. The system was biphasic, showing a high-affinity component at low concentrations and a secondcomponent with lower affinity at high concentrations of thesubstrate. No major differences were found between the wildtype and the NifQ mutants in either component. Similarresults were obtained for 30-mnii assays (data not shown).These results rule out the implication of a role for the nifQgene product in the transport of molybdate. These experi-ments cannot exclude the possibility that the high-affinity

300

.--

L..Uf)w-J0

C0

aw

0LLI

wzw-izw

250

200

150

100

50

0 101 102 103 104 105 106 lo7 lol

MoO ADDED (nM)FIG. 1. Effect of the molybdate concentration on nitrogenase

activity of K. pneumoniae. Symbols: 0, wild type; A, UN2458(nifQ-:Mu); O, UN1655 (NifB-), UN1676 (NifE-), and UN1688(NifN).

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MOLYBDENUM METABOLISM AND THE nifQ GENE 189

0.8 x E

ll~~~~ ~ ~~~~~~~~~~c aI.4 eersino irgns ciiyadmlbeu

w 75-1i5

O~~~~~~~~~~~~~10101314 I« I 0 cuuaini wl-yeK nuoie oacmlto:O

0.7 ( 0

c0.6o w(0 ~~ ~~~~~~~~~~~~~~~~~~50--J1

(0

z W C.4 0.4-Z4FIG. 2. Effect of the molybdate concentrationon25 0o K(c) 03 I-0 ~~~~~~~~~~~w

0.2

0 100 200 300 4000.1

TIME (min)

I ~~~~~~~~~~~FIG.4. Derepression of nitrogenase activity and molybdenum0 lo, 102 10 10 1 10* le lo,, accumulation in wild-type K. pneumoniae. Mo accumulation: 0,

-NH4+; A, +NH4+. Acetylene reduction; 0, -NH + ; A +NH4+.Mo0?- ADDED (nM) The assays were performed in 1-ml aliquots. 'MoO4- (20 I.Ci) was

FIG. 2. Effect of the molybdate concentration on growth of wild-thonysucofM inheexprmt.

type K. pneumoniae (0) and UN2458 (nifQ503J ::Mu) (A) on N2.

component is the result of adsorption on the surface of thecells.

(ii) "Mo accumulation. In K. pneumoniae, intracellularmolybdenum accumulation was dependent on the level ofderepression of nitrogenase (Fig. 4) and very low accumula-tion was observed when cells were grown on ammonia.Table 2 shows intracellular accumulation of molybdenum innonexchangeable pools after derepression and its relation-ship to acetylene reduction activity. When Na299MoO4 wasthe only source of Mo added, both UN and UN2454(nifQ::Mu) showed an increase in acetylene reduction activi-ty corresponding to a concentration of molybdate higherthan that calculated from the specific radioactivity (0.16nM). This discrepancy varied among batches of Na2 MoO4and is probably due to contamination by traces of nonradio-

0)U)0r 20a

(f)w-J0

-50 -40 -30 -10 0

active Mo in the preparation of the carrier-free isotope.However, the differences in activity between UN and NifQ-mutants when Na299MoO4 was used were still clear. Whenstrain UN was supplemented with enough molybdate toproduce maximum activity, the accumulation of nonex-changeable molybdenum increased. Similar levels of accu-mulation and activity were observed for the NifQ- mutantstrain in the same conditions. At low concentrations ofmolybdate, however, the mutant showed accumulation lev-els higher than those expected from the acetylene reductionactivity. No accumulation was detected when nifexpressionwas repressed by ammonia or by total deletion of the nifcluster (Table 2).

Effect of molybdenum starvation and the nifQ mutation onnitrogen fixation expression. nif-lac fusions in all the nifoperons were used to study the effect of Mo deprivation on

'/S (,M MoO2)FIG. 3. Double-reciprocal plot of the rate of molybdenum uptake versus molybdenum concentration in wild-type K. pneumoniae (0) and

UN2458 (NifQ-) (0).

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190 IMPERIAL ET AL.

TABLE 2. Molybdenum accumulation and acetylene reductionactivity

Na2MoO4 99Mo ac- C2H2 re-Strain NH4 add- added cumula- duction

ed (mM) (ILM) ted' activity

UN 0 0 100 51.4UN 25 0 2.2 <1.0UN2458 (nifQ5031::Mu) 0 0 44.9b 12.5cUN2458 25 0 1.5 <1.0UN1089 (nifD4409::Mu) 0 0 53.7 <1.0UN1089 25 0 2.9 <1.0UN563 (Anif) 0 0 7.6 <1.0UN 0 200 lOOb loodUN2458 (nifQ5031::Mu) 0 200 219 147.7

a Nonexchangeable counts per minute as described in the text.100% represents 20.0 x 106 cpm/ml of culture.

b 100%o represents 1.1 x 105 cpmlml of culture.c This value is an average of several experiments.d 100o represents 63.3 nmol/min per unit of absorbancy at 660

nm.

expression (Table 3). Molybdate had no effect on the expres-sion of the nifBQ operon or any of the other nif operons.

All the identified nif-coded proteins were detected by two-dimensional gel electrophoresis in a NifQ- mutant dere-pressed under molybdenum deprivation (Fig. 5), and thedistribution pattern was identical to that in gels of wild-typeextracts (data not shown; 24, 26).

Nitrogenase component I was quantitated by immunoelec-trophoresis in extracts of the wild type and NifQ- mutantderepressed under Mo-deficient or Mo-sufficient conditions(Table 4). Molybdenum had no effect on the amount ofnitrogenase component I cross-reacting material, despite the47-fold difference in activity between Mo-starved and Mo-sufficient cultures of the NifQ- mutant.Was the absence of molybdenum in nitrogenase compo-

nent I the only reason why normal levels of nitrogenasecomponent I produced very low activity in the NifQ-mutant? To answer this question we tried to activate culturesderepressed under Mo limitation by adding an excess ofmolybdate in the absence of protein synthesis (Table 5).Both UN and the NifQ- mutant recovered activity to thesame extent, indicating that there is no functional differencebetween nitrogenase component I from the wild type andthat from the NifQ- strain when the molybdenum concentra-tion is nonlimiting. However, only 30 to 40% of the maxi-mum activity was recovered. These data do not support afunction of molybdenum in the regulation of transcriptionand translation of nif genes or a function contributing to thestability of nitrogenase.

Nitrogenase activity and molybdenum in nitrogenase com-ponent I. As mentioned above, nitrogen fixation derepres-sion was required for the accumulation of molybdenum.However, the accumulation levels in NifQ- culturesderepressed under Mo deprivation and in UN1089(nifD4409::Mu), a mutant lacking both subunits of nitroge-nase component I (Table 2), indicated that nitrogenasecomponent I, although an important Mo reservoir in the cell,was not the only pool of Mo.

Nitrogenase component I could easily be identified as themajor radioactive band by polyacrylamide gel electrophore-sis of extracts from cells labeled with Na299MoO4 duringderepression (Fig. 6). No other radioactive bands wereobserved in its vicinity. This allowed us to quantitate the

accumulation of 99Mo in nitrogenase component I in wild-type and NifQ- strains derepressed in the presence of afixed amount of Na299MoO4 and different levels of nonradio-active molybdate and to correlate the accumulation withacetylene reduction activity of the cultures (Table 6). Eventhough the nitrogenase activities of NifQ- cultures werevery different from the wild-type activities at different mo-lybdenum concentrations, the ratio between acetylene-re-ducing activity and 99Mo concentration in nitrogenase com-ponent I was the same in both strains. Therefore, undermolybdenum-limiting conditions, the catalytic activity ofnitrogenase depends on the amount of molybdenum incorpo-rated into nitrogenase component I, and NifQ- mutants aredefective in this incorporation process.

DISCUSSIONMolybdenum is required for nitrogen fixation in K. pneu-

moniae. When no molybdenum was added to the medium,wild-type K. pneumoniae exhibited reduced nitrogenaseactivity. The basal activity was due to traces of molybdenumpresent in the medium which were difficult to eliminate andthat could be efficiently scavenged by K. pneumoniae. Whenthe medium was treated to reduce the amount of traceelements, this basal activity was reduced accordingly. Wehave been able to do this by a simple biological method. A.vinelandii cells exhibit a very high affinity for molybdenum(unpublished data) and have a high storage capacity due tothe presence of a constitutively produced molybdenum-binding protein (21). Short incubation of Mo-starved A.vinelandii cells caused the specific depletion of molybdenumtraces present in the medium. This incubation did not affectthe iron content of the medium and did not result in theexcretion of detectable levels of molybdenum-binding sider-ophores (as measured by previously described methods [20];unpublished data). Normal growth rate, growth yield, andnitrogenase activity were observed in K. pneumoniae cellsgrown in this medium supplemented with only ultrapuremolybdate.Among the nif genes described in K. pneumoniae, nifL

and nifQ have been characterized as nonessential for nitro-gen fixation (16, 17). The nifL product plays a role in therepression of nitrogen fixation (11) and in the destabilizationof nif mRNA (J. Collins and W. J. Brill, submitted for

TABLE 3. P-Galactosidase activity of nif-lac fusionsUnits of p-galactosidasea

Strain Repressed Derepressed cellscells 2c2(+MoO42-c)b +MoO42C -MoO42

UN4533 (nijB-lacZ) 0.4 4.7 6.3UN4847 (nifA-lacZ) 1.1 9.6 9.2UN4523 (nifF-1acZ) 0.2 5.6 5.8UN4489 (nifM-1acZ) 0.4 2.4 2.4UN4903 (nifS-IacZ) 0.9 17.6 18.3UN4499 (nifN-lacZ) 0.9 16.2 17.6UN4487 (nifE-lacZ) 1.1 17.2 18.4UN4484 (nifK-lacZ) 1.1 27.2 29.2UN4515 (nifJD-acZ) 1.1 22.1 25.7UN4504 (nifJ-1acZ) 0.4 17.0 21.9

a Optical density at 420 nm of nitrophenol released from o-nitrophenol galactoside per minute per optical density at 660 nm cellturbidity (18).

b Repressed by growth with 25 mM NH4+.c Na2MoO4 present at 400 ,uM during derepression.

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MOLYBDENUM METABOLISM AND THE nifQ GENE 191

I

4

I

IOJ

AON

O E

D

ov

K CL.

ilF

CM

F

FIG. 5. Two-dimensional polyacrylamide gel electrophoresis of an extract from 35S-labeled K. pneumoniae UN2458 (nifQS031::Mu)derepressed for nitrogenase in Mo-free medium. Protein spots were identified by comparison with UN gels by the method of Roberts and Brill(24). The letters refer to the nifgenes codifying the respective proteins. Spots for peptides M and L were poorly labeled but identifiable in theoriginal autoradiography.

publication). The nifQ product has not been identified, andno role has been ascribed to it due to a leakiness of NifQ-mutants (16, 17). The results presented here prove that nifQplays a role in nitrogen fixation and that the essentiality of itsfunction depends on the molybdenum concentration. Molyb-date concentrations of as low as 10 nM cause maximumnitrogenase activity in wild-type K. pneumoniae, whereasNifQ- mutants show only 4%, thus behaving like tight Nifmutants. The distinctive phenotype of NifQ- mutations isthe high requirement of molybdenum for nitrogenase activi-

TABLE 4. Nitrogenase component I antigenically cross-reactingmaterial

Acetylene-Strain MoO42- added reducing Cross-reactingStrain (.LM) activity material (%)O

(%)UN 0 3.8 97

400 ioob 100

UN2458 (nifQS031::Mu) 0 2.6 127400 122 105

a Antigenically cross-reacting material calculated from the peakheights.

b 100%o represents 77.7 nmol/min per unit of absorbancy at 660nm.

ty. High concentrations of Mo added to NifQ- mutantsduring nitrogenase derepression totally suppress the effectsof the mutation, both in point and Mu insertion mutants.This result was obtained with all NifQ- mutants tested. Ourlaboratory generally supplements media for K. pneumoniaewith 1 ,uM molybdate. At this concentration, NifQ- mutantshave 30% nitrogenase activity compared with the wild-typeactivity. Other groups (17) use higher concentrations ofmolybdate, which suppress the phenotype of the mutationeven more, thus explaining the nonconclusive genetic dataon the nifQ gene.

TABLE 5. Reactivation of K. pneumoniae nitrogenase activity byMoO42- after derepression in Mo-free medium

Acetylene-reducing activityStrain MoO42 added (%)

(ILM) -Tetracycline +TetracyclineaUN 0 NDb 39UN 200 ND 100CUN2458 (nifQS031::Mu) 0 17 9UN2458 (nifQS031::Mu) 200 73 86

a Fifty micrograms of tetracycline per milliliter was added toinhibit protein synthesis.

b ND, Not determined.c 100%o represents 22 nmol/min per unit of absorbancy at 660 nm.

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192 IMPERIAL ET AL.

A B CD EF

FIG. 6. Radioautography of 'Mo-labeled proteins from K. pneu-

moniae cells derepressed for nitrogenase. Extracts were subjectedto anaerobic polyacrylamide gel electrophoresis under nondena-

turing conditions. Lanes A (wild-type) and B [UN2458

(nifQS3J:Mu), no nonradioactive MoO42- added; lanes C

[UN2458 (nifQ503J::Mu)] and D (wild type), 20 nM nonradioactive

MoO42 added; lanes E (wild type) and F [UN2458 (nifQ5031::Mu)],1.2 liM nonradioactive MoO42 added. The arrow shows the

position of nitrogenase component The nature of the spots at the

origin and at the front is not known.

The availability of NifQ- mutants with high molybdenum

requirements allowed us to study the role of molybdenum in

the regulation of nif expression. Our results indicate that, to

the extent that it is possible to eliminate molybdenum traces

from the medium to give 4% of the maximum nitrogenase

activity, molybdenum does not play a role in the regulationof nifgene expression. The data on expression in strains with

nif-lac fusions under Mo deprivation agree with those report-

ed previously (6). In addition, neither the synthesis of nif-coded proteins nor the amount of nitrogenase component I

cross-reacting material is affected by molybdenum starva-

tion. This nitrogenase component I, inactive because of the

absence of molybdenum, can be activated by the addition of

molybdate to cells in the absence of protein synthesis. This

activation did not restore full activity. Other authors (12)

have reported a partial activation under similar conditions.

However, they found unexpectedly low levels of nitrogenase

cross-reacting material in Mo-starved cells, which could not

explain the levels of activity recovered. We found the

opposite results with respect to the cross-reacting material

found in Mo-deprived cells. Since no difference in the

amount of nitrogenase component I cross-reacting materialwas found between the wild type and the NifQ- mutant, weconclude that the only defect in NifQ- mutants is in themetabolism of molybdate.

Mutations in the chlD gene of Escherichia coli cause adeficiency in the biosynthesis of the molybdenum cofactorcommon to most molybdoenzymes and thus produce apleiotropic phenotype (5, 10, 34). The effects of the mutationcan be suppressed by the addition of high levels of molyb-date to the medium (10). Several authors (13, 33) have shownthat these mutations affect nitrogen fixation in the same wayin an E. coli strain having the nifgenes on a plasmid. Thus,the chlD-coded protein activity is an early step common toboth the molybdenum cofactor and FeMo-co, whereas nifQaffects only the biosynthesis of FeMo-co, but both mutationscan be phenotypically "cured" by molybdate. More recently(5, 34) it has been shown that chiG mutations can also bepartially cured by increasing the molybdate concentration inthe medium. Thus, phenotypic reversion by molybdateappears to be common to mutations in different steps of thebiosynthesis of molybdenum cofactors and is probably areflection of the possibility that some of these reactions canoccur nonenzymatically.

In most natural environments it is highly unlikely thatbacteria are going to encounter high levels of molybdenum.The fact that the abundance of molybdenum in the earth'scrust is just 0.015 mmol/kg (22), most of which is in aninsoluble form (MoS2), justifies this conclusion and thedevelopment of specific, high-affinity systems for the proc-essing of molybdenum. The possibility that some of thereactions can occur nonenzymatically, however, can beinterpreted as indirect evidence that cells do not tightlyregulate the size of internal pools of molybdenum. Thiswould be the reason for our finding that high concentrationsof molybdate are inhibitory for K. pneumoniae growing onN2. Since this effect is specific for nitrogen fixation, NifQ-mutants are more resistant than the wild type, and molyb-date is less toxic to cells nonderepressed for nitrogen fixa-tion (data not shown), this toxicity is probably due to theaccumulation of an intermediate in the metabolism of molyb-date.The affinity of the uptake system for molybdate is the

same for the wild type as for a NifQ- mutant; thus, the highmolybdenum requirement of NifQ- mutants is not due to adeficiency in molybdate transport. These data support theidea that nifQ mutations affect a later step than that of chlDfunction, which has been shown not to be transport (10).Little is known about molybdenum transport in bacteria (32).The only published study on transport of molybdate bynitrogen-fixing bacteria is that of Elliott and Mortenson (8, 9)with Clostridium pasteurianum, which seems to be very

TABLE 6. Enzymatic activity and accumulation of 9Mo in nitrogenase component I

MoO42 Acetylene-reducing activity (%) 9Mo in component I (%) Ratio (activity/99Mo)added UN2458 UN2458 UN2458(aeM) UN (nifQ503 ::Mu) UN (n 24Q5803:Mu) UN (nifQ503J :Mu)

0 35 10 looa 30 0.3 0.30.02 58 14 51 12 1.1 1.21.2 loob 53 5.6 2.7 17.9 19.6

200 69 102 NDC ND ND NDa 100% represents 37 x 103 cpm in nitrogenase component I, determined as discussed in the text.b 100% represents 58.5 nmol/min per unit of absorbancy at 660 nm.c ND, Not determined.

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MOLYBDENUM METABOLISM AND THE nifQ GENE 193

different from K. pneumoniae with respect to affinity, kinet-ics, and specificity (unpublished data).

In K. pneumoniae, molybdenum accumulation occursonly upon nif derepression, unlike A. vinelandii, in whichaccumulation is constitutively expressed due to the presenceof a storage protein (21). When K. pneumoniae is dere-pressed, accumulation increases with the external concen-tration of molybdate. When enough molybdate to cu-re theNifQ- mutants is given, they accumulate as much molybde-num as the wild type does; however, at low concentrationsof molybdate, NifQ- mutants show defective accumulation.The internal pool of molybdenum in these conditions ishigher than that of nonderepressed wild-type cells, thussuggesting the presence of intermediates in the metabolismof molybdate into FeMo-co which are formed before thenifQ-coded step. This point is further emphasized by thehigh molybdenumn accumulation shown by a mutant lackingboth subunits of nitrogenase component I.The lack of proportionality between molybdenum accu-

mulation and nitrogenase activity in NifQ- mutants at a lowmolybdenum concentration might be explained by the incor-poration of molybdenum into nitrogenase component I as aninactive form. To rule this out we quantitated the molybde-num content in nitrogenase component I and reiated it to theenzymatic activity exhibited by the cultures. The fact thatthe ratio between activity and molybdenum content innitrogenase component I is the same for both strains at thedifferent concenltrations of molybdate added to culturesproves that the defect in NifQ- mutants is in the incorpo-ration of all molybdenum into nitrogenase component I.

ACKNOWLEDGMENTSThis research was supported by the College of Agricultural and

Life Sciences, University of Wisconsin, and by Public HealthService grant GM22130 from the National Institutes of Health. J.I.was the recipient of a Postdoctoral Fellowship for the Ministerio deEducacion y Universidades of Spain. R.A.U. is a Fellow of theConsejo Nacional de Investigaciones Cientificas y Tecnicas, Repub-lica Argentina.

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