JouuNAL op BAcTEiOLOOY, Sept. 1983, p. 1138-1146 0021-9193/83/091138-09$02.00/0 Copyright C 1983, American Society for Microbiology Vol. 155, No. 3 Nitrate Uptake in Aspergillus nidulans and Involvement of the Third Gene of the Nitrate Assimilation Gene Cluster ALAN G. BROWNLEE AND HERBERT N. ARST, JR.* Department of Genetics, Ridley Building, The University, Newcastle upon Tyne, NE] 7RU, England Received 20 December 1982/Accepted 23 June 1983 In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 FM was estimated. In uptake assays, the crnAI mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnAI reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction. One of the most thoroughly studied metabolic processes in eucaryotic microorganisms is ni- trate assimilation in the ascomycete Aspergillus nidulans (reviewed by Cove [10]). One aspect of this process, nitrate uptake, has, however, re- ceived scant attention apart from a few prelimi- nary experiments reported briefly in a review (20). Tightly clustered but discrete genes specify functionally related products in nitrate assimila- tion by A. nidulans. Three clustered genes occur in the order (27) crnA niiA niaD in linkage group VIII. niaD and niiA are the structural genes for nitrate and nitrite reductases, respectively (10), whereas crnA is a heretofore uncharacterized gene in which loss of function confers resistance to the toxic analogs chlorate and bromate with- out any obvious nutritional impairment (27). The phenotypes of deletion mutations covering this region give no reason to suppose that the cluster contains any additional genes (27). Two posi- tive-acting regulatory genes, both recombining freely with the cluster, control expression of niaD and nUiA: nirA mediates induction by ni- trate and nitrite, and nirA- mutations (loss of function) lead to non-inducibility of nitrate and nitrite reductases and inability to utilize nitrate or nitrite as a nitrogen source (10). areA medi- ates nitrogen metabolite repression, and areAr mutations (loss of function) lead to extremely low levels of many activities involved in nitro- gen nutrition, including nitrate and nitrite reduc- tases, and inability to utilize nitrogen sources other than ammonium (2, 10, 26). The require- ment for the areA product for expression of niaD and niiA is partially alleviated by nirAcld alleles, containing two separate mutations in nirA, resulting in constitutivity and nitrogen metabolite derepression, respectively (26). D. J. Cove (personal communication) orig- inally proposed that the crnA gene product might be involved in nitrate uptake in A. nidu- lans, but early attempts to demonstrate defec- tive nitrate uptake by mycelia of crnA- strains were unsuccessful (A. B. Tomsett, Ph.D. thesis, University of Cambridge, Cambridge, England, 1977). We were encouraged to pursue this possi- bility by the finding that crnA- strains can be distinguished from crnA+ strains in two kinds of growth tests on nitrate-containing solid media (unpublished data). First, although crnA- strains utilize even very limiting levels of nitrate normally, they are hypersensitive to a number of growth inhibitors such as Cs' and methylam- monium when nitrate serves as the nitrogen source, but not on other nitrogen sources, in- cluding nitrite. Such inhibitor hypersensitivity characteristically results from mutations reduc- ing the rate of nitrogen source utilization (15, 22). Second, various mutations in A. nidulans result in toxicity of nitrate and nitrite or nitrite alone (10, 22, 26), and in double mutants, crnA mutations protect against nitrate, but not nitrite, toxicity. Here we examine some characteristics of net nitrate uptake in A. nidulans, demonstrating that a typical crnA mutation impairs nitrate uptake in 1138 on March 5, 2019 by guest http://jb.asm.org/ Downloaded from
Transcript
JouuNAL op BAcTEiOLOOY, Sept. 1983, p. 1138-11460021-9193/83/091138-09$02.00/0Copyright C 1983, American Society for Microbiology
Vol. 155, No. 3
Nitrate Uptake in Aspergillus nidulans and Involvement of theThird Gene of the Nitrate Assimilation Gene Cluster
ALAN G. BROWNLEE AND HERBERT N. ARST, JR.*Department of Genetics, Ridley Building, The University, Newcastle upon Tyne, NE] 7RU, England
Received 20 December 1982/Accepted 23 June 1983
In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a processseparate and distinct from, but dependent upon, the nitrate reductase reaction.Uptake was inhibited by uncouplers, indicating that a proton gradient across theplasma membrane is required. Cyanide, azide, and N-ethylmaleimide were alsopotent inhibitors of uptake, but these compounds also inhibited nitrate reductase.The net uptake kinetics were problematic, presumably due to the presence ofmore than one uptake system and the dependence on nitrate reduction, but anapparent Km of 200 FM was estimated. In uptake assays, the crnAI mutationreduced nitrate uptake severalfold in conidiospores and young mycelia but had noeffect in older mycelia. Several growth tests also indicate that crnAI reducesnitrate uptake. crnA expression was subject to control by the positive-actingregulatory gene areA, mediating nitrogen metabolite repression, but was notunder the control of the positive-acting regulatory gene nirA, mediating nitrateinduction.
One of the most thoroughly studied metabolicprocesses in eucaryotic microorganisms is ni-trate assimilation in the ascomycete Aspergillusnidulans (reviewed by Cove [10]). One aspect ofthis process, nitrate uptake, has, however, re-ceived scant attention apart from a few prelimi-nary experiments reported briefly in a review(20). Tightly clustered but discrete genes specifyfunctionally related products in nitrate assimila-tion by A. nidulans. Three clustered genes occurin the order (27) crnA niiA niaD in linkage groupVIII. niaD and niiA are the structural genes fornitrate and nitrite reductases, respectively (10),whereas crnA is a heretofore uncharacterizedgene in which loss offunction confers resistanceto the toxic analogs chlorate and bromate with-out any obvious nutritional impairment (27). Thephenotypes of deletion mutations covering thisregion give no reason to suppose that the clustercontains any additional genes (27). Two posi-tive-acting regulatory genes, both recombiningfreely with the cluster, control expression ofniaD and nUiA: nirA mediates induction by ni-trate and nitrite, and nirA- mutations (loss offunction) lead to non-inducibility of nitrate andnitrite reductases and inability to utilize nitrateor nitrite as a nitrogen source (10). areA medi-ates nitrogen metabolite repression, and areArmutations (loss of function) lead to extremelylow levels of many activities involved in nitro-gen nutrition, including nitrate and nitrite reduc-tases, and inability to utilize nitrogen sourcesother than ammonium (2, 10, 26). The require-
ment for the areA product for expression ofniaD and niiA is partially alleviated by nirAcldalleles, containing two separate mutations innirA, resulting in constitutivity and nitrogenmetabolite derepression, respectively (26).D. J. Cove (personal communication) orig-
inally proposed that the crnA gene productmight be involved in nitrate uptake in A. nidu-lans, but early attempts to demonstrate defec-tive nitrate uptake by mycelia of crnA- strainswere unsuccessful (A. B. Tomsett, Ph.D. thesis,University of Cambridge, Cambridge, England,1977). We were encouraged to pursue this possi-bility by the finding that crnA- strains can bedistinguished from crnA+ strains in two kinds ofgrowth tests on nitrate-containing solid media(unpublished data). First, although crnA-strains utilize even very limiting levels of nitratenormally, they are hypersensitive to a number ofgrowth inhibitors such as Cs' and methylam-monium when nitrate serves as the nitrogensource, but not on other nitrogen sources, in-cluding nitrite. Such inhibitor hypersensitivitycharacteristically results from mutations reduc-ing the rate of nitrogen source utilization (15,22). Second, various mutations in A. nidulansresult in toxicity of nitrate and nitrite or nitritealone (10, 22, 26), and in double mutants, crnAmutations protect against nitrate, but not nitrite,toxicity.Here we examine some characteristics of net
nitrate uptake in A. nidulans, demonstrating thata typical crnA mutation impairs nitrate uptake in
NITRATE UPTAKE MUTANT OF ASPERGILLUS NIDULANS 1139
conidiospores and young mycelia. We believethis to be the first report of a mutation affectingnitrate uptake in a microorganism, such muta-tions having been reported previously only inthe higher plant Arabidopsis thaliana (11).(A preliminary report of this work was pre-
sented to the Genetical Society of Great Britain[A. G. Brownlee and H. N. Arst, Jr., Heredity48:321, 1982].)
MATERIALS AND METHODS
Strains. All strains ofA. nidulans carried markers instandard use which have been described previously (6;A. J. Clutterbuck, Aspergillus Newsl. 15:58-75, 1981;Table 1).
Genetic techniques and growth tests. Genetic tech-niques followed procedures in standard use (6).Growth testing ofA. nidulans by stab inoculation ontosolid media at 37°C has been described previously (2).The minimal medium described by Cove (7) was usedthroughout. It contained 10 g of D-glucose per liter as acarbon source. Unless other concentrations are speci-fied, nitrogen sources were added at 10 mM (ammoni-um, nitrate, nitrite), 5 mM (L-alanine, L-arginine, L-proline), 720 FM (hypoxanthine), or 590 ,uM (uricacid). Nitrate and nitrite were added as the sodiumsalts, ammonium was added as the (+)-tartrate, and L-arginine was added as the hydrochloride. Bromate andchlorate were added as the potassium salts. Cesiumand methylammonium were added as the chlorides.
Chemicals. Analytical reagent grade chemicals wereused wherever commercially available. Carbonyl cya-nide m-chlorophenylhydrazone (CCCP), carbonyl cya-nide p-trifluoromethoxyphenylhydrazone (FCCP),2,4,-dinitrophenol, cycloheximide, valinomycin, andNADPH were obtained from Sigma Chemical Co.,Ltd., London. Miracloth was purchased from Calbio-chem-Behring Corp., San Diego, Calif.
Conidiospores and mycelia. Conidiospores were har-vested from petri dishes containing complete medium(7) into sterile distilled water containing 0.01% Tween80 and were used either directly for uptake studies orfor inoculation for growth of mycelia (at approximate-ly one-fifth plate per flask) in 1-liter polypropyleneErlenmeyer flasks containing 200 ml of liquid medium.The standard nitrogen-free liquid minimal medium ofCove (7) was used throughout. Unless otherwise indi-cated, it contained 10 g of D-glucose per liter as carbonsource. Growth requirements were supplemented atstandard levels (7). Nitrogen sources were present asindicated. Urea was used at a final concentration of 5
mM. Mycelia were grown at 37°C in a GallenkampOrbital Shaker (Gallenkamp and Co., Ltd., London)with a shaker speed of 200 rpm.
Net nitrate uptake. Nitrate uptake by conidiosporesand mycelia was estimated by following the disappear-ance of nitrate from the medium, a procedure whichwas successfully employed with both Penicillium chrysogenum (14) and Neurospora crassa (24). Nitrateconcentrations were determined from the absorbanceat 204 nm in 5% perchloric acid (5), using an SP 1800spectrophotometer (Pye-Unicam Instruments Ltd.,Cambridge, England). Mycelia were harvested on ny-lon cloth, washed with prewarmed (37°C), nitrogen-free minimal medium (7), and transferred to 50 ml of
TABLE 1. Strains used
Relevantgenotype Additional Reference or originRelevntgeotype markers' e
apabaAl, biAl, and pyroA4 are requirements forp-aminobenzoate, biotin, and pyridoxine, respectively.fwAl results in fawn conidial color. None of thesemarkers affects nitrogen metabolism. Growth require-ments were fully supplemented as described by Cove(7).
b The biAl strain was used for conidial uptakestudies, and the pabaAl strain was used for mycelialuptake studies and enzyme assays. This choice wasarbitrary, as the biAl and pabaAl mutations have noeffect on these activities.
nitrogen-free minimal medium (pH 6.5) containing D-glucose and supplemented with (per liter) 10 ,ug ofbiotin, 4 mg of p-aminobenzoic acid, and, wherenecessary, 5 mg of pyridoxine hydrochloride in 250-mlErlenmeyer flasks held at 37°C in a shaking waterbath. The mycelial density was usually 1 to 2 mg (dryweight) per ml. After the addition of 500 ,uM sodiumnitrate, nitrate uptake rates were estimated from thedecrease in absorbance at 204 nm of acidified samplesof media (suitably diluted). Rates were based upon atleast four and usually five rapidly filtered 3-ml samplestaken within 20 min. Controls from which nitrate wasomitted were periodically tested. Any possible contri-bution to the absorbance by nitrite produced fromnitrate was determined by using samples with andwithout 0.2% sulfamic acid (see reference 5). Applica-tion of these controls seldom necessitated significantcorrection. Flasks were shaken at 100 rpm. Uptakerates were linear over a wide range of nitrate concen-trations. For conidial uptake studies, this procedurewas slightly modified (see the legend to Fig. 1).CCCP, FCCP, and valinomycin were dissolved in
ethanQl at 100 times the final concentration, and, inthese cases, the controls also contained 1% ethanol.For inhibitor studies, an additional control was em-ployed which measured absorbance in the presence ofinhibitor and the absence of nitrate (in the event thatan inhibitor absorbed or caused efflux of a sub-stance[s] absorbing at 204 nm).
Nitrate reductase (NADPH:nitrate oxidoreductase)assay. Harvested mycelia were ground in a mortar onice with an equal weight of acid-washed sand in 10volumes of cold extraction buffer, half of which wasadded midway through grinding. The extraction buffercontained (final concentrations) 100 mM phosphate(sodium salts, pH 7.0), 170 mM NaCl, and 1 mMmercaptoethanol. The crude homogenate was centri-fuged at 32,000 x g for 30 min, and the supematantfluid was saved. Nitrate reductase (EC 1.6.6.3) was
assayed by following the oxidation of NADPH at 340nm spectrophotometrically in a 1-ml assay mixturecontaining 1 nmol of flavin adenine dinucleotide, 200nmol of NADPH, 10 ,umol of NaNO3, and 50 ,umol of(sodium) orthophosphate (pH 7.0) and, typically, 50 1d
of cell-free extract. Assays were conducted at 32°Cand were initiated by adding the nitrate after measur-ing (and correcting for) the nitrate-independent oxida-tion ofNADPH by the extract. This correction rangedfrom approximately 2% for the highest nitrate reduc-tase activities reported here to slightly under 15% forthe lowest activities.
Nitrite reductse (NADPH:nitrite oxidoreductase) as-say. Crude extracts of freshly harvested mycelia wereprepared as described for nitrate reductase, but in apH 8.6 buffer containing (final concentrations) 50 mM(sodium) pyrophosphate, 33% (vol/vol) glycerol, 10mM L-cysteine, and 1 mM flavin adenine dinucleotide.Nitrite reductase (EC 1.6.6.4) was assayed by follow-ing the oxidation of NADPH at 340 nm spectrophoto-metrically in a 1-ml assay mixture containing 2 pmol ofsodium nitrite, 250 nmol of NADPH, and 100 Ftmol of(sodium) phosphate (pH 7.0) at 25°C. The reaction wasinitiated by adding 50 ,ul of cell-free extract (containingthe flavin adenine dinucleotide essential for the reac-tion).
Protein determination. Soluble protein in extractswas determined (16) by using crystalline bovine serumalbumin as standard. Soluble protein accounts forapproximately 12% of mycelial dry weight (unpub-lished data). Total protein in conidiospore suspensions(see Fig. lc) was determined by the same method aftersolubilization of samples in 100 mM NaOH.
RESULTS
Nitrate and nitrite reductase levels in crnAlstrains. Under growth conditions resulting ini-tially in maximal induction, the crnAI mutationdid not lower levels of either enzyme (Table 2).The modest but consistent elevation in enzymeactivities shown by the crnAl strain in Table 2might result from reduced self-nitrogen metabo-lite repression if crnAI reduces nitrate uptake,limiting conversion of nitrate to the repressingmetabolite.
Nitrate uptake by conidiospores. Freshly har-vested conidiospores of A. nidulans wild type(whether or not suspended overnight in diluteTween 80 to enhance their wettability) exhibitednet uptake of nitrate only after a lag of up to 2 hat 37C (Fig. la). Acquisition of net nitrateuptake capacity was prevented by the proteinsynthesis inhibitor cycloheximide and did notoccur in conidiospores ofa niaD17 strain lackingnitrate reductase (Fig. la). Metabolism of nitratebeyond nitrite is not, however, required; coni-diospores of a niiA4 strain lacking nitrite reduc-tase exhibited nearly normal acquisition of ni-trate uptake capacity (data not shown).Appearance of conidial net nitrate uptake capac-ity required an exogenous carbon and energysource (Fig. lb). Comparison of Fig. la with lb
TABLE 2. Nitrate-assimilating enzymes in the wildtype and a crnAI strainaNitrate reductase Nitrite reductase
a Mycelia were grown in supplemented glucose min-imal medium, with 20 mM nitrate as the nitrogensource, at 37°C for 8 or 17 h, as indicated.
I Enzyme activities are given as nanomoles ofNADPH oxidized per minute per milligam of protein.Values are mean t standard deviation (SD) of at leastthree independent experiments.
shows that preincubation of conidiospores inglucose solution allowed the appearance of up-take capacity to commence immediately uponaddition of nitrate. This indicates that the lag isdistinct from nitrate induction. After the lagperiod, net nitrate uptake capacity appearedsooner in a nirA101cd strain than in the wild type(Fig. lc). Addition of an equimolar concentra-tion of ammonium reduced net uptake capacityin the wild type to ca. 5% of the level found onaddition of nitrate alone (Fig. lc). In identicalconditions, an nirA1O1Ocd strain had ca. 40% asmuch net uptake capacity when NH4NO3 re-placed NaNO3 (data not shown).crnAI reduced net nitrate uptake by conidio-
spores approximately threefold (Table 3). Coni-diospores were preincubated to avoid the prob-lem of some variability in length of the lagperiod, and nirA101dd strains were used to ex-ploit the high, linear initial rate of acquisition ofuptake capacity resulting from this allele (Fig.lc).
Nitrate uptake by mycelia. Net nitrate uptakecapacity was inducible by nitrate in wild-typemycelia (Table 4; Fig. 2). Nitrite (at 10 mM) isalso a good inducer of net nitrate uptake capaci-ty, but variability in the degree of induction,possibly because of the reactivity and slighttoxicity of nitrite, precluded its use. As in coni-diospores, functional nitrate reductase was es-sential for net nitrate uptake activity both inyoung (grown for 8 h at 37°C) and older (grownfor 16 h at 37°C) mycelia, because under induc-ing growth conditions (Table 4), neither aniaD17 nor a cnxG2 strain had any nitrate up-take activity whatsoever. niaD17 is a mutationin the structural gene for the nitrate reductaseapoprotein and leads to chlorate resistance andconstitutive syntheses of nitrite reductase andancillary activities associated with nitrate reduc-tase, whereas cnxG2 is a mutation in a geneinvolved in biosynthesis of a molybdenum-con-taining cofactor common to nitrate reductase
NITRATE UPTAKE MUTANT OF ASPERGILLUS NIDULANS 1141
0)
. '
0z-o0
(min)
q OU
co4Q 60 -
cn/0n 40 -
z-
e 20 -
01 -
L I
0 20 40 60*C 1.2 Time (min)
~ 0.8_ pZ 12 - //+0
Tim (min
E*0
0)
0.4
co0.0.2-
0z 0
0 15 30 45 60 75
Time (min)
FIG. 1. Net nitrate uptake by conidiospores. (a)Conidiospores were harvested into distilled water con-taining 0.01% Tween 80, filtered through Miracloth toremove mycelial debris, washed with distilled waterusing low-speed centrifugation for sedimentation, andsuspended in distilled water. Before assay, one sampleof wild-type conidiospores was left overnight in 0.01%Tween 80 at 4°C. Approximately 5 x 101 conidio-spores were suspended in 50 ml of supplementedglucose minimal medium with or without 10 gg ofcycloheximide, and 200 ,uM nitrate was added. Sam-ples (1.5 ml) were filtered rapidly over a vacuum.Nitrate uptake is expressed as a percentage of theinitial concentration (200 ,LM). Symbols: 0, freshlyharvested wild-type conidiospores; 0, wild-type coni-diospores left overnight in Tween 80; U, freshly har-vested wild-type conidiospores in the presence ofcycloheximide; O, niaD17 strain. (b) Wild-type coni-
and two purine hydroxylases and does not leadto chlorate resistance or constitutive synthesesof nitrite reductase and ancillary activities asso-ciated with nitrate reductase (8-10, 21). Patemanand Kinghom (20) have observed a lack ofaccumulation of 15NO3- by mycelia of a niaD17strain.
In young (7 to 9 h) mycelia, there was a three-to fourfold reduction in net nitrate uptake capac-ity in a crnAl strain as compared with the wildtype in inducing growth conditions (Table 4; Fig.2a). In older mycelia (16 h), there was little or nodifference between crnAl and wild-type strains(Table 4). Nitrate-induced net nitrate uptakeactivity varied with mycelial age in an oscilla-tory fashion (Fig. 2b).Data in Fig. 2a compare the kinetics of induc-
tion of nitrate reductase as well as of net nitrateuptake capacity in wild-type and crnAl strains.The kinetics of induction of nitrate reductase inwild type grown on urea as the nitrogen sourceat 37°C were very similar to those at 25°Cdescribed by Cove (8). Induction kinetics of netnitrate uptake capacity in the same myceliaappeared to differ, suggesting nonidentity ofnitrate reductase and the nitrate uptake sys-tem(s). More convincing evidence for this non-identity was provided by several experiments inwhich the wild type was grown on nitrate as thenitrogen source: under these conditions, netnitrate uptake activity was very low (less than 2nmol/min per mg [dry weight]), whereas nitratereductase activity was maximally induced. (Thediminished nitrate reductase levels and induc-tion lag in the crnAl strains seen in Fig. 2a aretypical of results obtained in suboptimal induc-tion conditions.)
Further evidence for the nonidentity of nitratereductase and the nitrate uptake system(s) iscontained in Table 5. Three nirAcld alleles, in-cluding the most extreme alleles available,nirA103cId and nirAl13c/d (26), led to significantdegrees of nitrogen metabolite derepression ofnitrate reductase, with no derepression of theuptake system(s) evident. (The low residual up-
diospores (1.8 x 107) were suspended in 25 ml of thevarious nitrogen-free preincubation media shown for4.5 h before the addition of 200 ,uM NaNO3 at timezero. For comparison with distilled water, no pHadjustment was made to minimal media in this experi-ment, so that its pH was approximately the same asthat of the distilled water (pH 5.0). Symbols: 0,supplemented minimal medium lacking a carbonsource; 0, supplemented minimal medium containing1% glucose; A, distilled water containing 1% glucose.(c) Conidiospores (2.5 x 101) were suspended in 50 mlof supplemented glucose minimal medium and prein-cubated for 150 min before the addition of 200 ,uMN03 (as the Na+ or NH4' salt) at time zero. Sym-bols: 0, wild type plus NH4NO3; 0, wild type plusNaN03; *, nirA01/d plus NaNO3.
a Nitrate reductase activity is expressed as nanomoles ofNADPH oxidized per minute per milligrarh of proteinand is the average of at least two experiments ± SD. Nitrate uptake rates are expressed as nanomoles of N03removed per minute per milligam (dry weight) and are averages of at least two experiments ± SD.
b Freshly harvested conidiospores were preincubated for 4 h in nitrogen-free supplemented glucose minimalmedium at 37°C, collected by centrifugation, and suspended in 20 ml of supplemented glucose minimal medium;50O M NaN03 was added. Nitrate uptake was followed as described in the text.
c Mycehia were grown in supplemented glucose minimal medium, with urea as the nitrogen source, at 37°C for8 or 16 h, as indicated. Nitrate uptake rates were deternined immediately by using half of the freshly harvestedmycelia, and the remainder was frozen at -15°C for subsequent (within a few days) assay of nitrate reductase.
I ND, Not determined.
take activities measured in the nirAlOldcd andnirAl13cId strains grown on ammonium probablyresult from rapid derepression during harvestingof the mycelia and the short incubation in uptakemedium.)Comparison of data in Tables 3, 4, and 5
showed that nirAcld alleles led to a high constitu-tive level of mycelial net nitrate uptake capacity.This provided an opportunity to investigate theeffect of crnAl under conditions in which induc-tion was unnecessary. The nirAJOlc/d crnAldouble mutant had less than half of the netnitrate uptake activity of the nirA1OlcId crnA+strain in young (8 h) mycelia, but this differencewas much less in older (16 h) mycelia (Table 3).Nitrate reductase levels in the two strains werecomparable.
Characterization of the mycellal nitrate uptakesystem(s). Net nitrate uptake was inhibited byvarious metabolic inhibitors including cyanide,azide, N-ethylmaleimide, and the protonophoresCCCP, FCCP, and 2,4-dinitrophenol (Table 6).2,4-Dinitrophenol (500 ,uM) inhibited nitrate re-ductase only by approximately 18% in crudeextracts, and 100 FzM CCCP and 10 ,uM FCCPhad no effect (A. G. Brownlee, unpublisheddata). That CCCP and FCCP did not affectnitrate reductase activity at 10 times a concen-tration at which they strongly inhibited nitrateuptake (Table 6) suggests that they act directlyon transport.The effect of pH on net nitrate uptake is
shown in Fig. 3. Strains carrying nirA101cd wereused to avoid the requirement for induction andto enhance uptake activities. In 8-h mycelia ofthe nirA101C crnA+ strain, net nitrate uptakewas high between pH 4.0 and 7.0 (Fig. 3a).Activity fell rapidly outside this range (althoughnitrate was ionized throughout the pH rangeexamined). The profile ofpH dependence in 8-hmycelia of the nirA101lOd crnAI double mutant
was somewhat different, lower between pH 4.0and 8.5, but most markedly at pH 4.0 (Fig. 3a).In 17-h (at 37°C) mycelia, the two strains showedalmost identical pH dependence (Fig. 3b). Func-tional nitrate reductase is a prerequisite for netnitrate uptake at any pH; no uptake was ob-served in a niaD17 strain at pH 4.2 with the useof 8- or 16-h (at 37°C) mycelia (in addition to thedependence on nitrate reductase under standardassay conditions at pH 6.5 noted above).
Chlorate was a potent and rapid inhibitor ofnitrate uptake by 16-h (at 37°C) mycelia in boththe wild type and a crnAI strain (Fig. 3c).Similar chlorate inhibition was observed in 8-hwild-type mycelia, in which 1 mM chlorate in-hibited uptake of 500 ,uM nitrate by 80%. Theextent of chlorate inhibition was apparently pHdependent, however. In a preliminary experi-ment (data not shown), chlorate was markedlymore inhibitory at pH 4.2 than at pH 7.0 in both8- and 17-h mycelia from a nirAlOlc/d strain.
TABLE 4. Nitrate uptake in the wild type and acrnAl strain
Age of mycelia Concn of Nitrate uptake mtec in:at harvesting nitrate added
aMycelia were grown in supplemented glucose min-imal medium, with urea as the nitrogen source, at3rC.
b NaNO3 (10 mM) was added 100 min before har-vesting.
c Rates are expressed as nanomoles of NO3 re-moved per minute per milligram (dry weight) and arethe means ± SD from at least three independentexperiments.
NITRATE UPTAKE MUTANT OF ASPERGILLUS NIDULANS 1143
o (a)_ 10 _
02
E_ 8 -
.C
E 6 -
EC
4 -
e
C2 -
_ . ,
0,q-
EE
.C
E .'
o 0
_
c
0 0Z Z
20 40 60 80 100Time (min) Age of mycelia (h)
FIG. 2. Induction of mycelial nitrate uptake capacity. (a) Mycelia were grown for 9 h in supplemented glucoseminimal medium, with urea as the nitrogen source, at 37°C. At time zero, NaNO3 (5 mM) was added to all flasks.Contents of separate flasks were harvested at each time point, and nitrate uptake activity and nitrate reductasewere determined. (b) Mycelia were grown for the times indicated. NaNO3 (10 mM) was added 100 min beforeharvesting. N03 uptake was determined in the standard assay. Each point was obtained by using a separateflask. Variation between flasks of one strain at any given time was less than 10%o (determined separately).Symbols: 0, 0, wild type; A, A, crnAI; 0, A, nitrate uptake activity; 0, A, nitrate reductase activity. In both(a) and (b), uptake rates are expressed per milligm (dry weight), whereas nitrate reductase activities are
expressed per milligram of soluble protein.
Chlorate is a weak inhibitor of nitrate reductase(17).Using the standard uptake assay with 8- and
17-h mycelia of a nirA10cl/d strain grown with 5
mM urea as the sole nitrogen source, nitrateuptake proceeded at the same rate in the pres-ence or absence of 10 mM ammonium. Thisexperiment was repeated, using 8- and 17-hmycelia grown on 10 mM L-glutamate or 17-hmycelia grown on 10 mM L-phenylalanine as thesole nitrogen source, with the same result. Ap-parently, therefore, ammonium ion does not actas an uncoupler of nitrate transport.
Analysis of the kinetics of net nitrate uptakeproved problematic, presumably as a result ofthe relative insensitivity of the assay, the depen-dence of uptake on functional nitrate reductase,and the possible presence of more than one
uptake system. Repeated attempts to determinethe affinity of the nitrate uptake system in oldermycelia have revealed complex kinetics andnonlinear double reciprocal plots. However, thesystem(s) did exhibit saturation above 1 mMnitrate (Fig. 3d). A Hofstee plot (Fig. 3d insert)indicated an apparent Km of approximately 200,uM, but the V,n,, value was found to be subjectto some variation. No metal cation requirementfor uptake was evident, nor was there anyapparent effect of low ionic strength. Uptakeproceeded equally well in glass-distilled waterplus D-glucose and in 20 mM phosphate (as thepotassium salts) buffer (pH 6.5) plus glucoseplus 0 to 50 mM sodium or lithium chloride as instandard minimal medium.
Regulation of crnA expression. Although ni-trate uptake was inducible (Table 4; Fig. la), it
TABLE 5. Effects of growth in the presence of ammonium on nitrate reductase and nitrate uptake levels innirAcld strains
a Mycelia were grown for 8 to 9 h at 37°C in supplemented glucose minimal medium with the nitrogen source(s)indicated. Other details were as described for mycelia in Table 3.
a Additions were made to the standard nitrate up-take assay described in the text.
b Control rates (no additions) were 8 h, 9.6 ± 1.1; 17h, 14.1 + 0.7 nmol of NO3 per min/mg (dry weight) ±SD. Rates shown in the presence of inhibitors are theaverages of duplicate determinations, none of whichvaried >7% about the mean.
c Mycelia of the nirAlOlcid strain were grown insupplemented glucose minimal medium, with urea asthe nitrogen source, for 8 or 17 h at 37C.dND, Not determined.
cannot as yet be determined whether synthesisof the crnA product itself is inducible or whetherthis apparent inducibility stems entirely from theinducibility of nitrate reductase. Nitrogen me-tabolite repression of the syntheses of geneproducts essential for nitrate uptake, includingpresumably crnA, is, however, distinct fromnitrogen metabolite repression of nitrate reduc-tase (Table 5). A comparison of the phenotypesof crnAI and crnA+ strains carrying in additionvarious regulatory mutations yielded further in-formation on the regulation of crnA expression.crnAl was additive in double mutants with
nirAl-. For example, on glucose minimal medi-um containing 5 mM L-arginine as the nitrogensource, crnAl nirAl- double mutants wereclearly more resistant to the toxicity of 20 mMchlorate than were single mutants carrying ei-ther mutation. As nirAl- is a nonleaky nirAmutation (9, 10) and therefore probably resultsin complete loss of nirA product function, theadditivity of crnAI and nirAI indicates thatexpression ofcrnA is largely, possibly complete-ly, independent from nirA control. This conclu-sion is supported by data in Table 5 indicatingthat, although they relieved nitrogen metaboliterepression of nitrate reductase synthesis,nirAlOldd, nirA103c/d and nirAl13"jd did notlead to nitrogen metabolite derepression of netnitrate uptake capacity.Double mutants carrying crnAI and an areAr
mutation were constructed by using two differ-ent areAr alleles. areA18r is a complete loss offunction allele resulting from a translocation
breakpoint (1, 22), whereas areAlr is thermosen-sitive for the utilization of certain nitrogensources (2). Three independent double mutantsof each genotype were definitively identified bytheir ability to yield crnAl areA+ progeny uponoutcrossing to a wild-type strain. These doublemutants were indistinguishable from corre-sponding areAr single mutants in a range ofgrowth tests at 37°C, testing nitrogen sourceutilization and resistance to chlorate and bro-mate. At 25°C, a permissive temperature, crnAlareAlr strains were readily distinguishable fromareAlr single mutants by their inability to utilizenitrate, although they retained the ability toutilize nitrite, hypoxanthine, uric acid, and L-proline. This phenotype is consistent with in-volvement of crnA in nitrate but not nitriteuptake. The epistasis of areAr mutations tocrnAI suggests that the areA gene product medi-ates nitrogen metabolite repression of synthesisof the crnA product.The location of crnA in a gene cluster with
niaD and niiA (27) is intriguing. One approachto consideration of whether crnA is expressedvia a polycistronic mRNA is to examine expres-sion of crnA in strains carrying nis-5, a niiApromoter/initiator mutation associated with aninsertional translocation (3, 22). In nis-5 strains,the relevant gene configuration is crnAniiA ... niaD, as compared with crnA niiA niaDin translocation-free (nis+) strains. The proximi-ty of crnA to one of the translocation break-points increased the difficulty of constructingcrnAI nis-5 double mutants. This was achievedbty selecting puA' niiA+ recombinants from across of relevant partial genotype puA2 crnAI xniiA203 nis-5 (consult reference 3 for methods ofgenetic manipulation of nis-S). In growth tests,nis-5 had no effect on chlorate and bromatetoxicities, and the differences in phenotype be-tween crnAI nis-5 and crnA+ nis-5 strains werethe same as those between crnAl nis+ andcrnA+ nis+ strains. Given that niiA is almostcertainly transcribed towards crnA (3), thisshows that crnA is not to any significant degreeexpressed via a niaD nijA crnA tricistronic mes-senger.
DISCUSSIONEvidence is presented that, although depen-
dent upon intracellular nitrate reduction, netnitrate uptake is a separate and distinct process.It is sensitive to specific inhibitors, saturable athigh concentrations of nitrate, and subject tometabolic and developmental regulation. It has acharacteristic pH dependence and is partiallydefective in strains carrying the crnAl mutation.The ability of crnAl strains to take up nitratesuggests the existence of more than one mode ofnitrate uptake. The broad pH dependency is
NITRATE UPTAKE MUTANT OF ASPERGILLUS NIDULANS 1145
20k
10-
II I I I I I
2 3 4 5 6PH
la
E
Ec
0
.X
'C.z0z
0-
E
I-C
E-.
2
0)
0.90 2 4 6 8 10 12 14 0 1.0 2.0
Time (min) NO' concn (mM)FIG. 3. Properties of the mycelial nitrate uptake system. Mycelia were grown in supplemented glucose
minimal medium, with urea as the nitrogen source, at 37rC. pH dependence of nitrate uptake by 8-h (a) or 17-h (b)mycelia was determined by using 500 F.M NaNO3 in 20 mM phosphate (as the potassium salts) buffer, at the pHindicated, in the presence of 1% glucose. Values are the means of at least three independent experiments; barsdenote 1 SD. Symbols: 0, nirA101d; A, nirA01d crnAL. (c) Mycelia of the wild-type (0, 0) and crnAl (A, A)strains were grown for 16 h. For the purpose of induction, 10mM NaNO3 was added 3 h before harvesting. Usingthe standard assay, uptake of 500 ,M nitrate was determined in the-presence (0, A) and absence (0, A) of 1 mMKCl03. (d) Concentration dependence of nitrate uptake was determined in 17-h mycelia of the nirA101d strain.The concentrations of NaNO3 indicated were used in the standard uptake assay. Points are the means of threetypical experiments. Bars denote 1 SD.
consistent with this notion, and the kinetics ofnitrate uptake suggest systems with differentaffinities for nitrate. Evidence for the existenceof two assimilatory nitrate transport systems inKlebsiella pneumoniae has been reported (25).The crnAI mutation reduces net nitrate up-
take by conidiospores and young mycelia, butnot by older mycelia, perhaps explaining whyTomsett (Ph.D. thesis) was unable to detect anyeffect of crnAI on nitrate uptake. Thus, nitrateuptake by A. nidulans might bear some resem-blance to sulfate uptake by N. crassa, in whichthere are two genetically and biochemically dis-tinct permeases, one operative in conidiosporesand another operative in mycelia (18). In addi-tion to this developmental regulation, crnA isapparently under the control of the areA regula-tory gene mediating nitrogen metabolite repres-
sion. The available evidence suggests, however,that crnA is not under the control of the nirAregulatory gene mediating nitrate and nitriteinduction. In vivo evidence suggests that crhA isnot expressed via a tricistronic transcript of thenitrate assimilation gene cluster.The electrogenic proton-translocating ATPase
of fungal plasma membranes probably functionsin the transport of both ions and unchargedcompounds into the cell against a large electricpotential or concentration gradient (reviewed inreference 13). The effect of uncouplers on netnitrate uptake in A. nidulans observed heremight implicate the proton gradient in supplyingenergy for the transport of the nitrate anionagainst a probably large membrane potential(which in N. crassa can reach more than -300mV, inside negative [23]). It is also pertinent that
N-ethylmaleimide inhibits the purified N. crassaplasma membrane proton-translocating ATPase(4). However, N-ethylmaleimide, cyanide, andazide are very potent inhibitors of nitrate reduc-tase, both purified and in crude extracts (12, 17,19; A. G. Brownlee, unpublished data). Thus,the mode(s) of action of these inhibitors inblocking nitrate transport remains equivocal: asecondary consequence of inhibition of nitratereductase, a more direct effect on transport, orboth. Depolarization studies have provided evi-dence for nitrate-proton contransport in Lemnagibba (28).The potential nitrate-reducing activity (mea-
sured in vitro at saturating nitrate and NADPHconcentrations) was in excess, often consider-able excess, of net nitrate uptake capacity of thesame cells in all strains and growth conditionsemployed here (e.g., Tables 3 and 5; Fig. 2a).The best available estimate of the Km for nitrateof purified nitrate reductase from A. nidulans is80 pM (17). This suggests that our estimate of200 PM for the Km for nitrate uptake is ameasure of the saturability of an uptake systemand not of nitrate reductase.
ACKNOWLEDGMENTSThis work was supported by a grant from the Science and
Engineering Research Council.We thank Shelagh Cousen for technical assistance and
Claudio Scazzocchio and Mark Caddick for comments on themanuscript.
LITERATURE CITED1. Arst, H. N., Jr. 1981. Aspects of the control of gene
expression in fungi. Symp. Soc. Gen. Microbiol. 31:131-160.
2. Ant, H. N., Jr., sad D. J. Cove. 1973. Nitrogen metabo-lite repression in Aspergillus nidulans. Mol. Gen. Genet.126:111-141.
3. Ant, H. N., Jr., K. N. Raad, and C. R. Bafley. 1979. Dothe tightly linked structural genes for nitrate and nitritereductases in Aspergillus nidulans form an operon? Evi-dence from an insertional translocation which separatesthem. Mol. Gen. Genet. 174:89-100.
4. Brooker, R. J., ad C. W. Slayn. 1982. Inhibition ofplasma membrane [H+l-ATPase of Neurospora crassa byN-ethylmaleimide. Protection by nucleotides. J. Biol.Chem. 257:12051-12055.
5. Cawn, P. A. 1967. The determination of nitrate in soilsolutions by ultra-violet spectrophotometry. Analyst(London) 92:311-315.
6. Cluttertuc, A. J. 1974. Aspergillus nidulans, p. 447-510.In R. C. King (ed.), Handbook of genetics, vol. 1. PlenumPublishing Corp., New York.
7. Cove, D. J. 1966. The induction and repression of nitratereductase in the fungus Aspergiflus nidulans. Biochim.Biophys. Acta 113:51-56.
8. Cove, D. J. 1967. Kinetic studies of the induction ofnitrate reductase and cytochrome c reductase in thefungus Aspergillus nidulans. Biochem. J. 104:1033-1039.
9. Cove, D. J. 1976. Chlorate toxicity in Aspergillus nidu-lans. Studies of mutants altered in nitrate assimilation.Mol. Gen. Genet. 146:147-159.
10. Cove, D. J. 1979. Genetic studies of nitrate assimilation inAspergillus nidulans. Biol. Rev. 54:291-327.
11. Dddema, H., J. J. Hobtra, and W. J. Feenstra. 1978.Uptake of nitrate by mutants of Arabidopsis thalianadisturbed in uptake or reduction of nitrate. I. Effect ofnitrogen source during growth on uptake of nitrate andchlorate. Physiol. Plant. 43:343-350.
12. Downey, R. J., and F. X. Steiner. 1979. Further character-ization of the reduced nicotinamide adenine dinucleotidephosphate:nitrate oxidoreductase in Aspergillus nidulans.J. Bacteriol. 137:105-114.
13. Gofeau, A., and C. W. Slayman. 1981. The proton-trans-locating ATPase of the fungal plasma membrane. Bio-chim. Biophys. Acta 639:197-223.
14. Gokkmth, J., J. P. LIvonI, C. L. Norberg, and I. H.Segel. 1973. Regulation of nitrate uptake in Penicilliumchrysogenum by ammonium ion. Plant Physiol. 52:362-367.
15. Jones, S. A., H. N. Arst, Jr., and D. W. MacDonald. 1981.Gene roles in the prn cluster of Aspergillus nidulans.Curr. Genet. 3:49-56.
16. Lowry, 0. H., N. J. Rosebrough, A. L. Fan, and R. J.Randfll. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.
17. M nld, D. W., and A. Coddlngton. 1974. Propertiesof the assimilatory nitrate reductase from Aspergillusnidulans. Eur. J. Biochem. 46:169-178.
18. Marauf, G. A. 1970. Genetic and metabolic controls forsulfate metabolism in Neurospora crassa: isolation andstudy of chromate-resistant and sulfate transport-negativemutants. J. Bacteriol. 192:716-721.
19. M napwa, N., and A. Yddlmoto. 1982. Purification andchaacterization of the assimilatory NADPH-nitrate re-ductase of Aspergillus nidulans. J. Biochem. 91:761-774.
20. Pateman, J. A., and J. R. Ki1ghorn. 1976. Nitrogen me-tabolism, p. 159-237. In J. E. Smith and D. R. Berry(ed.), The filamentous fungi, vol. 2, Biosynthesis andmetabolism. Edward Arnold, Ltd., London.
21. Pate_nan, J. A., B. M. Rever, and D. J. Cove. 1967. Ge-netic and biochemical studies of nitrate reduction inAspergilus nidulans. Biochem. J. 104:103-111.
22. R_d, K. N., and H. N. Ant, Jr. 1977. A mutation inAspergilus nidulans which affects the regulation of nitritereductase and is tightly linked to its structural gene. Mol.Gen. Genet. 155:67-75.
23. Sanders, D., U.-P. Hansen, and C. L. Slaybn. 1981. Roleof the plasma membrane proton pump in pH regulation innon-animal cells. Proc. Natl. Acad. Sci. U.S.A. 78:5903-5907.
24. Sc , R. H., and R. H. Garrett. 1974. Nitrate trans-port system in Neurospora crossa. J. Bacteriol. 118:259-269.
25. Tlyer, J. R., and R. C. Huffaker. 1982. Kinetic evalua-tion, using 13N, reveals two assimilatory nitrate transportsystems in Klebsiella pneumoniae. J. Bacteriol. 149:198-202.
26. Tolervey, D. W., and H. N. Art, Jr. 1981. Mutations toconstitutivity and derepression are separate and separablein a regulatory gene ofAspergillus nidulans. Curr. Genet.4:63-68.
27. Tonsett, A. B., and D. J. Cove. 1979. Deletion mapping ofthe nilA niaD gene region of Aspergillus nidulans. Genet.Rcs. 34:9-32.
28. UDrc, W. R., and A. Novacky. 1981. Nitrate-dependentmembrane potential changes and their induction in Lemnagibba Gl. Plant Sci. Lett. 22:211-217.
