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Effect of various growth conditions on nitrate uptake and nitrate reductase activity in Aspergillus nidulans nitrate assimilation defective mutants.

Introduction

Nitrate is the preferred source of nitrogen for photosynthetic and a microorganism including fungi and it is the nutrient that most frequently limits their growth (Crawford and Glass, 1998; Daniel-Vedele, et al., 1998). Furthermore, the central importance of nitrate is highlighted by reports that it is involved in certain plant metabolic and morphogenic processes (Schieble, et al., 1997; Zhang and Forde, 1998). The nitrate assimilation pathway involves nitrate uptake from the external environment then its release into the cell by specific nitrate transporters. The influx of nitrate into cells is an active process since it occurs against a nitrate gradient (Brownlee and Arst, 1983; Vidmar, et al., 2000; Forde, 2002). This is followed by the reduction of nitrate to nitrite then the subsequent conversion of nitrite to ammonium, catalyzed by the enzymes nitrate reductase (NR) (NADPH: nitrate oxidoreductase E.C. 1.6.6.3) and nitrite reductase (NiR) (NADPH: nitrite oxidoreductase 1.6.6.4) respectively (Cove 1979; Scazzhacchio and Arst, 1989; Kinghorn and Unkles, 1994). There are at least two classes of nitrate transport systems, high and low affinity, that have been identified (Trueman, et al., 1996). The Aspergillus nidulans nrtA (formerly crnA) gene (Brownlee and Arst, 1983) encodes a membrane protein that belongs to a family of high affinity nitrate transporters (Forde, 2000; Unkles, et al., 1991, 2001). A collection of mutants in the nrtA gene of A. nidulans was isolated in order to predict the peptide motifs and amino acid residues which are important for the transport of nitrate across the membrane. Missense mutations were found to inactivate the A. nidulans nrtA gene (Kinghorn, et al., 2005). Since the publication of nrtA sequence, other eukaryotic high affinity transporters have been identified and characterized at the DNA and protein levels. In the green alga Chlamydomonas reinhardtii three different nitrate/nitrite transport systems (Nar2, Nrt2;1, Nrt2;2) have been identified as high affinity nitrate/nitrite transport genes (Quesada, et al., 1994; Galvan, et al., 1996). In higher plants, two transport systems are involved. The first, is a high affinity system which is inducible by nitrate, the other is a low affinity system expressed constitutively (Hole, et al., 1990; Trueman, et al., 1996; Quesada, et al., 1997; Glass, et al., 2000). A putative nitrate transporter cDNA clone has been isolated from the higher plant Arabidopsis thaliana (Nrt1) where it is proposed to encode a low affinity nitrate transporter (Doddema and Telkamp, 1978; Tsay, et al., 1993). This clone does not show significant homology with any other algal or fungal nitrate transporters (Tsay, et al., 1993; Quesada, et al., 1994). A gene from barley plant was cloned using primers based on the A. nidulans nrtA sequence (Trueman, et al., 1996). The Aspergillus nitrate uptake system was found to be different from other nitrate-inducible nitrate uptake systems (with km's around 250 liM) in other related fungi, such as Neurospora crassa (Schloemer and Garrett, 1974; Quesada, et al., 1994) or Penicillium chrysogenum (Goldsmith, et al., 1973). The Aspergillus nitrate uptake system requires a functional nitrate reductase activity in order to be active. Genetic studies have revealed that mutants in niaD (the structural gene for NR) gene and the nrtA gene (the structural gene for nitrate transporter) have shown high resistance to chlorate; the toxic analogue to nitrate, but not to bromate; the toxic analogue to nitrite (Tomsett and Cove, 1979; Brownlee and Arst, 1983; Kanan, 2002; Kanan, et al., 2002). It had been proposed previously that chlorate is not itself toxic, but became toxic after its conversion to chlorite as a result of nitrate reductase-catalyzed conversion of chlorate to chlorite (MacDonald and Coddington, 1974). The reduction of bromate as well as chlorate by denitrifying pseudomonas bacterial species under anaerobic conditions has been studied extensively (Malmqvist, et al., 1991 , Malmqvist and Welander, 1992). Chlorate is a substrate for nitrate reductase (Krul and Veeningen, 1977), and the same enzyme may be responsible for the reduction of bromate. Chlorate is an effective electron acceptor for bacteria (Malmqvist, et al., 1991) and it is likely that bromate is used in the same way. A. nidulans (Emericella nidulans) is the most commonly used filamentous fungus for genetic research studies (David et al., 2008; Wortman, et al., 2009). A. nidulans has a sophisticated sexual genetic system, where it can undergo sexual reproduction, in addition to the a sexual cycle (Pyrzak, et al., 2008; Bennett, 2010). Due to amenability of this fungal model to combined genetic and biochemical approaches and since nothing is known about NRT gene (s) exact function (s) we have used biochemical approaches to study the effects of various growth conditions (i.e. cell age; presence or absence of inducers and/or inhibitors) on net nitrate transport and NR activity in various nrtA; nrtB (chlorate resistant but bromate sensitive) and cbrn (nrtRR) (chlorate and bromate resistant) mutant strains generated after NTG-chemical mutagenesis and subjected to genetic recombination analysis (pair-wise sexual crosses). Such a strategy has yielded valuable information on the E. coli lacY permease (Bailey and Manoil, 1998).

Materials and Methods

2.1 Aspergillus nidulans strains and media.

Two wild-type strains (biAland y[A.sub.2]pyro[A.sub.4]) carrying different color markers were used for the isolation of mutant strains with the nrtA, nrtB and cbrn (nrtRR) phenotypes after NTG-chemical mutagenesis. The nrtAl (yA2biA1nrtA1) mutant strain was provided as a gift from Dr. J. R. Kinghorn, University of St-Andrews-U.K. Gene symbols are those in standard use (Clutterbuck, 1974). Routine Aspergillus growth media and handling techniques were as described before (Clutterbuck, 1974).

2.2 Isolation of mutant strains after NTG-chemical mutagenesis.

Mutagenesis with N-methyl-N-nitro-N- nitrosoguanidine (NTG) was performed according to the procedure described previously by Adelberg and co-workers (1965) as modified by Kanan (2002). Five ml conidiospores suspension (approximately 1x [10.sup.8] conidiospores per ml) were added to 20 ml of 0.1 M potassium orthophosphate buffer (pH 5.8) containing 20 mg/ml NTG. The treatment was performed for 30 min at 37 [degrees]C. Conidiospores were pelted (4233R refrigerated centrifuge) at 6000 rpm for 10 min, washed thrice with sterile distilled water and resuspended in sufficient volume of saline-Tween 80 solution. Conidiospores were plated on selective media and incubated at required selection temperature. Mutant strains were isolated on the basis of resistance to chlorate toxicity (200 mM) with arginine (10 mM) as the sole source of nitrogen (Cove, 1976a). Arginine was previously proposed to serve as the best N-source for the isolation of high yield nrt mutants (Cove, 1976a, b; Kanan, 1996; Kanan, 2002; Kanan et al., 2002). Sodium deoxycholate (0.08 %) was added to the medium to reduce colony size (Mackintosh and Pritchard, 1963). Confirmed mutant strains were further tested at both selection temperatures (25[degrees]C and 37[degrees]C) on chlorate and bromate (200 mM) with arginine, N[H.sub.4]Cl, or uric acid (10 mM). The nrt and cbrn mutants were selected (at 25[degrees]C and 37[degrees]C) from other chlorate resistant, bromate sensitive nitrate assimilation defective strains, by their ability to utilize nitrate, nitrite and hypoxanthine (10 mM) (Cove, 1976 b).

2.3 Genetic recombination analysis of nrt mutants: Pair--wise sexual crosses.

Meiotic recombination analysis based on previously described procedures by Yager (1992) as modified by Kanan (2002), were used for the genetic analysis of nrt (i.e. chlorate resistant, nitrate and nitrite utilizing) mutant strains. Therefore, sexual pair-wise crosses were performed between two different haploid strains carrying different color markers on minimal medium (without vitamin solution) and incubated at 30[degrees]C for 10- 12 days. One hundred Lil from each hybrid cleistothecium suspension (i.e. having both colour markers of parents) were streaked on solid complete media and incubated at 37[degrees]C for 3 days. Progeny from each color marker in the cross were replicated along with the wild-type and parental strains from the prepared master plates into different types of selective and non-selective media including: bromate (200 mM) with uric acid, N[H.sub.4]Cl, or arginine (10 mM); chlorate (200 mM) with the same nitrogen sources; nitrate (10 mM); nitrite (10 mM) and adenine (10 mM). Such growth tests were performed at 37[degrees]C to determine the phenotypes of recombination and required for the identification and isolation of recombinant strains. Pair-wise sexual crosses were performed between a designated nrtAl (formerly termed crnAl: yA2biA1nrtA1) and each of 10 nrt (isolated in this study) mutants. Further sexual crosses were performed between a designated nrtB strain (namely nrtB1- yA2 nrtB1: obtained from a cross with the yellow wild-type strain- y[A.sub.2]pyro[A.sub.4]) and the remaining non-nrtA strains (non-parental chlorate sensitive recombinants). Another interesting phenomenon was noted during observation of the genetic analysis of nrt mutants, that is, wherever bromate sensitive and chlorate resistant recombinant progeny observed in pair-wise crosses between nrt mutants mapped genetically in two different nrt Ioci, other recombinant progeny were observed. These latter recombinants have shown resistance to both bromate and chlorate and were observed in low frequency and designated- nrtRR (i.e.

cbrn: chlorate and bromate resistant, nitrate utilizing strains).

2.4 Mutant strains included in the biochemical studies

The wild-type biA1 and the yA2 biA1 nrtA1 mutant strain in addition to the following mutant strains which were generated in this study after NTG-chemical mutagenesis and genetic recombination analysis were included in the biochemical studies. The included mutant strains were: biA1nrtA2, biA1nrtA3, biA1nrtA4, y[A.sub.2]pyro[A.sub.4]nrtB1, biA1nrtB2, biA1nrtRR1 (cs: cryso-sensitive), biA1nrtRR2, yA2 pyroA4nrtRR3, yA2 pyroA4nrtRR4 and biA1nrtRR5 (cs: cryso-sensitive).

2.5 Net nitrate transport assays.

Strains included in the uptake studies were grown for 8 and 16 h at 37[degrees]C in liquid minimal medium with 5 mM urea as the sole source of nitrogen (Cove, 1966). The inducer of nrt expression, sodium nitrate (10 mM) was added 3 h prior to harvesting by filtration. Nitrate uptake by mycelia was estimated by following the disappearance of nitrate from the medium. Nitrate concentration was determined spectrophotometrically from the absorbance at 204 nm in 5% perchloric acid. Assays were carried out as described previously (Brownlee and Arst, 1983; Kanan, 1996; AL-Najjar, 2005; Kanan and Al-Najjar, 2010).

2.6 Nitrate reductase assay

A slightly modified procedure by Kanan (1996) from that described previously by Wray and Filner (1970) was used in this work. Nitrate reductase was assayed by following the oxidation of NADPH, were the intensity of the resultant pink color was proportional to the amount of nitrite present and was estimated by determining the absorbance of the assay mixture at 540 nm spectrophotometrically. Values were converted into amounts of nitrite by means of applying the absorbance value to a nitrite standard curve. The amount of soluble protein in the extract was estimated by using the Bio-Rad protein standard II (lypholized BSA). The protein content in the extract was estimated by measuring the absorbance at 595 nm spectrophotometrically. The units of enzyme activity obtained per ml extract were divided by the protein concentration in that sample in order to calculate the enzyme specific activity in terms of units per min per mg protein.

2.7 Disappearance of nitrate reductase activity at different time intervals

Mycelia of tested strains were grown for 8 and 16 h at 37[degrees]C under induced conditions, then mycelial samples of 50 ml were harvested at different time intervals (i.e. 0 time, 8, 16 and 24 min) and assayed for nitrate reductase activity as mentioned above.

2.8 Disappearance of enzyme activity under various growth conditions

Mycelia of tested strains were grown under induced conditions and transferred to various media types and incubated further for 30, 60 and 90 min. The various growth conditions include media with: 10 mM NaN[O.sub.3], absence of nitrogen source, 10 mM NaN[O.sub.3] + 10 mM KCl[O.sub.3], 10 mM KCl[O.sub.3], 10 mM NaN[O.sub.3] + N[H.sub.4]Cl and 10 mM N[H.sub.4]Cl. Mycelial samples of 50 ml were harvested at different time intervals as indicated above and assayed for nitrate reductase activity as mentioned before.

2.9 Statistical analyses

Values are means [+ or -] SD of at least three independent replicates. All experiments were repeated at least three times.

3. Results

3.1 Isolation of chemically induced nitrate and / or nitrite transport defective mutants.

Results presented in Table 1 indicate that mutant strains defective in nitrate and / or nitrite transport (nrt, brn and cbrn strains) were successfully isolated at both selection temperatures (25[degrees]C and 37[degrees]C) on the basis of resistance to chlorate and / or bromate toxicity (200 mM) with arginine (10 mM) (Fig. 1a). The selected mutant strains with nrt (nitrate transporter) phenotypes have shown wild-type growth on (10 mM) nitrate, nitrite and hypoxanthine, in addition of being highly resistant to (200 mM) chlorate (The toxic analogue of nitrate) and / or bromate (The toxic analogue of nitrite) (Fig 1 c,d). The proportion of chlorate and bromate resistant nrtRR (i.e. cbrn) mutant strains have ranged from 3% to 7% when selection was performed at 25[degrees]C and from 5% to 13% when selection was at 37[degrees]C (Fig. 1d5). The chlorate sensitive bromate resistant mutants have shown a percentage in the range of 5% to 11% and from 5% to 8% when selection was performed at 25[degrees]C and 37[degrees]C, respectively (Fig. 1d6). However, the percentage of chlorate resistant bromate sensitive (RS) mutant strains (Fig. 1c, d7) have ranged from 63% to 83% at both selection temperatures. Furthermore, some of the generated nrt mutants have shown high resistance to chlorate and bromate at 37[degrees]C (Fig. 1d5) and complete sensitivity to chlorate but not to bromate at 25[degrees]C and these were considered cryso-sensitive (cold-sensitive) mutants. These strains were included in the biochemical studies in order to understand how the assembly of the protein at the non-permissive temperature will affect the net nitrate uptake and nitrate reductase activity.

3.2 Genetic recombination analysis of nrt mutants

Genetic recombination analysis between nrtA1 and 10 mutant strains with the nrt phenotypes (i.e chlorate resistant, bromate sensitive and nitrate /nitrite utilizing) indicated that four strains designated nrtA2, nrtA3, nrtA4 and nrtA5 were found to map in the same locus, designated nrtA locus i.e no chlorate sensitive recombinants were observed (Table 2). The remaining six nrt mutants were found to locate in at least another locus (non-nrtA) since recombinant progeny i.e non-parental types or chlorate sensitive recombinants were observed (Table 2). In order to confirm these findings sexual pair-wise crosses were performed (Fig. 2a,b) between a designated nrtB (yA2 pyroA4 nrtB1) and the other five non-nrtA strains (Table 2). Genetic recombination analysis of such crosses indicate that all of the six non-nrtA mutant strains are indeed located in a second locus designated nrtB locus, which is genetically unlinked with the nrtA locus i.e no chlorate sensitive recombinants progeny were observed (Table 2). Other interesting phenomena were noted in the generated progeny from crosses between nrtA and nrtB mutant strains, but not between nrtA with nrtA or nrtB with nrtB strains. These phenomena include the appearance of chlorate resistant bromate sensitive nitrate and nitrite non-utilizers (Fig. 2c). In addition, other recombinants have shown high resistance to both toxic analogues and grown strongly on nitrate, nitrite and hypoxanthine and these were designated as RR strains (Fig. 2d). Furthermore, some progeny have shown complete sensitivity to chlorate but high resistance to bromate and these were designated as SR strains (Table 2).

[FIGURE 1 OMITTED]

3.3 Net nitrate uptake activities

Results presented in Table 3 indicate that all tested nrtA mutant strains that grown for 8 h under nitrate inducible conditions have shown approximately similar net transport basal levels (i.e. a round 3.5-4.5 nmols.min.mg) compared with 11.77 [+ or -] 1.34 nmols.min.mg in the wild-type strain. However, the net nitrate uptake level in the older cells (i.e. 16 h old) has reached approximately a wild-type (12.44[+ or -]0.16) level (i.e. a round 10 to 13 nmols.min.mg) in all nrtA strains. However, the young (8 h) nrtB strains showed a double fold increase in uptake level (i.e. in the range of 6 to 8 nmols.min.mg) as compared to nrtA levels, whereas, a wild-type and nrtA levels (i.e. in the range of 11 to 12 nmols.min.mg) were obtained with the older cells that have been grown for 16 h. Furthermore, three strains (nrtRR2; nrtRR3 and nrtRR4) have shown double fold increase in net uptake activity (i.e. in the range of 7.5 to 9.8 nmols.min.mg) as compared to either nrtA or nrtB strains that have grown for 8 h. However, an uptake level in the range of 12 to 14.7 nmols.min.mg was obtained in the older (16 h) cells (Table 3). Moreover, surprisingly, the two cold sensitive strains (nrtRR1cs and nrtRR5cs) that have grown for 8 h have generated an uptake level of approximately 10 nmols.min.mg which is approximately three folds more than that of nrtA, 2 folds more than the nrtB and a wild-type level. Whereas, a level of a round 14 nmols.min.mg was obtained with the older cells and this is similar to that obtained by the other nrtRR strains (Table 3).

[FIGURE 2 OMITTED]

3.4 Disappearance of nitrate reductase specific activity in wild-type and nrt mutant strains

3.4.1 Enzymatic levels at zero time after assay in young and old cells

Results presented in Table 4 indicate that all tested strains that were grown for either 8 or 16 h have shown nitrate reductase activity. However, the enzyme activity was much higher in younger (8 h) cells. The highest specific activity at zero time for cells of 8 h old was obtained with the strain nrtRR4 (i.e approximately 4-folds increase (378.65[+ or -]3.26) as compared to wild-type enzyme activity (91.17[+ or -]3.62)). This was followed by strains nrtRR5cs and nrtA2 which showed approximately 2-folds increase in activity. However, strains nrtRR2; nrtRR3 and nrtRR1cs all have shown similar activity i.e. approximately 1.5-folds increase as compared to wild-type level. In addition, the strains nrtA1; nrtA4 and nrtB1 showed approximately half-wild-type level of enzyme activity, whereas, nrtA3 strain has generated a wild-type level (Table 4). Concerning levels of enzyme activity (at zero time) for cells that have been grown for 16 h, obtained results indicate that the wild- type enzymatic activity has been declined to 29% as compared to that in cells grown for only 8 h. However, the enzymatic activity in strain y[A.sub.2]pyro[A.sub.4]nrtRR4 has decreased to 82% only whereas, that in strains: nrtRR1cs; nrtA2; nrtRR5cs; nrtRR3 and nrtB2 has reached an activity of 76%; 68%; 66%; 64% and 60% respectively. The other nrtA strains have shown a reduction in enzymatic activity in the range of 18% to 41%.

3.4.2 Disappearance of enzymatic activity after 24 min in young (8 h) cells

As clearly seen in Table 4, all tested strains (including the wild-type) that were grown for 8 h showed decreased nitrate reductase specific activity in the range of 2% to 5% after 24 min incubation at 35[degrees]C. However, the exception for this was strain nrtB2 where, the detected activity has reached 9% of the initial percentage of specific activity.

3.4.3 Disappearance of enzymatic activity after 24 min in old (16 h) cells

As compared to enzymatic activity that has been detected at zero time of incubation at 35[degrees]C (i.e assayed immediately after extraction) all tested strains that were grown for 16 h showed a decline in nitrate reductase specific activity in the range of 2% to 3% after 24 min incubation of the crude extract at 35[degrees]C (Table 4).

3.5 Disappearance of nitrate reductase (NR) activity under various growth conditions

It is clearly seen from results presented in figures 3, 4 and 5 that the enzyme NR was induced by nitrate but completely repressed by ammonium. However, when induced cells were incubated for further 30, 60 and 90 min in presence or absence of (10 mM) nitrate (acts as a substrate and inducer for the NR enzyme) rather than with 10 mM chlorate (Toxic analogue of nitrate) or 10 mM ammonium (repressor for the enzyme) the enzyme activity was mostly restrained in tested strains. As indicated in Figure 3, the greatest loss of enzyme activity in tested strains was detected when ammonium (whether alone or in combination with nitrate) was added to induced cells. The enzyme activity after 90 min further incubation has reached approximately 1%-3% (with ammonium) and 3%-5% (with ammonium and nitrate) in all tested strains. Furthermore, chlorate alone or in combination with nitrate was the second most effective reducing agent of enzyme activity where, the wild-type activity has declined (after 90 min) to 7% (Fig 3a) with chlorate alone whereas, that of nrtA strains has reached 12% to 16% (except nrtA3 strain: 3%; Fig 3d). The presence of chlorate along with nitrate has slightly restrained the enzyme activity as compared with that obtained with chlorate alone. The activity obtained after 90 min with all strains has ranged from 21% (wild-type; Fig 3a) to 39% with nrtA strains (except nrtA3: the activity was 7.5%; Fig 3d). Surprisingly, the absence of nitrate with the induced cells during the incubation period has restrained the wild-type enzyme activity to 45% as compared to that (29%) obtained with nrtA strains (except nrtA1 strain which showed 58% activity; Fig 3b). In conclusion, the addition of growth inhibitor or repressor (whether alone or in combination with the inducer (nitrate)) to the induced cells during the incubation period has reflected the following descending order of restrained enzyme activity: presence of N[O.sub.3.sup.-]> absence of N[O.sub.3.sup.-]> N[O.sub.3.sup.-] + Cl[O.sub.3.sup.-]> Cl[O.sub.3.sup.-]> N[O.sub.3.sup.-] + N[H.sub.4.sup.+]> N[H.sub.4.sup.+]. Results presented in figure 4 indicate that the addition of nitrate to induced cells has restrained the enzyme activity in nrtB strains to approximately 69% after 90 min incubation whereas, that of the wild-type has reached 59% (Fig 4a). The addition of ammonium to induced cells has caused rapid loss of enzyme activity (after 90 min) where, such activity has reached only 1%-2% in all strains. However, when nitrate in addition to ammonium was added the activity has reached a level in the range of 2% (with nrtB1; Fig 4b) to 5% (with nrtB2; Fig 4d). The addition of chlorate to nrtB strains has decreased the NR activity to approximately 5%-8% after the indicated incubation period. However, when chlorate and nitrate were used the detected enzymatic activity has reached only 12% of its original activity. The absence of nitrate from the media has restrained approximately 30% of the original enzymatic activity. Results presented in figure 5 indicate that the enzyme activity of nrtRR strains was restrained to approximately 44%-68% of its original activity when nitrate was added to the induced cells during the further incubation periods. However, in absence of nitrate the obtained enzyme activity was in the range of 37%-39% (except nrtRR5; 23%; Fig 5f). The addition of chlorate has restrained an enzyme activity in the range of 3% (with nrtRR1 and nrtRR3; Fig 5b and d) to 17% (obtained with nrtRR5; Fig 5f). However, when nitrate in combination with chlorate was added the activity has reached a range of 6%-9% in nrtRR1 and nrtRR2 (Fig 5b and c) and 30%-39% in the rest of nrtRR strains (Fig 5d,e and f). The greatest loss of enzyme activity was obtained when ammonium either alone or in combination with nitrate was used, where such activity has reached approximately 1%-2% with ammonium and 2%-5% with nitrate and ammonium. In conclusion, obtained results indicate that the greatest preservation of NR activity under various growth conditions was shown in nrtRR strains then in nrtB, whereas, the greatest loss of enzymatic activity was obtained with the nrtA strains. Furthermore, the addition of ammonium to induced cells before enzyme assay followed by chlorate (alone) rather than in combination with nitrate had caused rapid loss of enzyme activity where, further synthesis of the enzyme was not possible. However, the presence or absence of inducer (nitrate) has preserved approximately 40% (in absence of inducer) to 68% (in presence of inducer) of enzyme activity after further incubation of induced cells for 90 min.

Discussion

Wild-type strains were treated with the chemical mutagen NTG which preferentially induce single mutations in GC nucleotide base pairs (Kinghorn et al., 2005). After mutagenesis with NTG (Adelberg, et al., 1965), mutants were isolated on the basis of resistance to (200 mM) chlorate (Cove, 1976a; Kanan, et al., 2002) and /or bromate (200 mM) toxicity with arginine as a sole source of nitrogen (Kanan, 2002; Kanan and Al-Najjar, 2010). This approach takes advantage of the fact that mutations in the untranscribed strand are much less efficiently repaired than those in the transcribed strand, thereby favoring changes, for example, in G-rich codons rather than C-rich residues (Kinghorn, et al., 2005). It has been previously reported that chlorate itself is not toxic, but it render toxic when it is converted to chlorite by the action of the molybdoenzyme nitrate reductase (Cove, 1976 a,b). A possible explanation for chlorate toxicity is that it mimics nitrate and leads to shutdown of nitrogen metabolism as chlorate can not act as a nitrogen source.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

However, we are not aware of any published work which indicates the action of bromate on cells but this may lead to the suggestion that bromate toxicity arise as a result of mimicking nitrite in being the toxic analogue of nitrite, though it may alter genes that act as nitrite specific transport systems (Kanan, 2002; Kanan and Al- Najjar, 2010). Genetic analysis of mutants with nrt (nitrate transporter) phenotypes and non-parental recombinant progeny generated from pair-wise sexual crosses between nrtA and nrtB strains have shown the below given growth patterns: Firstly, resistance to chlorate but not to bromate (i.e. nrt strains; with the RS phenotype). Secondly, sensitivity to chlorate and resistance to bromate (i.e. brn strains; with the SR phenotype). Thirdly, resistance to both toxic analogues (i.e. cbrn strains; with the RR phenotype). Since chlorate (toxic analogue of nitrate) mimics nitrate and bromate (toxic analogue of nitrite) mimics nitrite these findings may suggest that resistance to both selective agents i.e. RR phenotypes may specify a bispecific system that is responsible for the transport of nitrate as well as nitrite. The strains with RR phenotypes may occur due to a complex locus that co-segregates as one single gene or two tightly linked genes (Kanan, 2002). This locus is independent from either the nitrate specific system i.e. represented by strains with the RS phenotypes or the nitrite specific system specified by strains with SR phenotypes. This suggestion would agree with the findings of Galvan and his co-workers (1996) and that of Kanan (2002). Previously conducted transport studies on A. nidulans strains carrying the nrtA mutations resulted in reduced net nitrate uptake up to 4-folds in conidia and young mycelial cells (8 h). In contrast, such mutations seemed to have no effect on older cells (16 h) with regard to nitrate transport (Brownlee and Arst, 1983; Kinghorn, et al., 2005). These findings suggest that nrtA gene encodes a differentially regulated component expressed in spores and young mycelia but not in older cells. Also, this is attributed to the presence of more than one nitrate and /or nitrite uptake systems that show resistance to chlorate but complete sensitivity to bromate (RS) (Brownlee and Arst, 1983; Unkles, et al., 2001; Galvan, et al., 1996; Trueman, et al., 1996; Kanan, 2002). This residual level is due to the nitrate uptake contribution by NrtB transporter i.e due to increased NrtB expression or possibly to a gain of function mutation in another transporter (Unkles, et al., 1991; Kinghorn, et al., 2005). In contrast, nrtB mutants possessed net nitrate uptake capacity between 6 to 8 nmols. min. mg. and the nrtRR strains yielded high level of net uptake i.e. a round 10-11 nmols.min.mg. These results may suggest that the selection of nitrate transport mutants on the basis of resistance to chlorate toxicity (200 mM) and sensitivity to bromate (200 mM) yields nrtA mutants which all appear to be loss of function. This is in contrast to nrtB mutants which are sensitive to chlorate toxicity at a range of concentrations with various single nitrogen sources (Unkles, et al., 2001; Kinghorn, et al., 2005) or the nrtRR mutants that show high resistance to both toxic substances with various nitrogen sources (Kanan, 2002). The presence of nitrate and nitrite non-utilizers in recombinants generated from crosses between mutant strains with nrtA phenotypes may suggest that double mutants would lead to a loss of growth on nitrate and nitrite. One unique feature of high affinity nitrate transporters is the presence of a consensus A G N G N M G, residues 164 to 172 in transmembrane 5 (Tm) (Trueman, et al., 1996; Forde, 2000), this motif sequence is partially present in Tm5 of E. coli NarK for nitrite efflux, a dissimilar sequence in fungi may suggest that this motif is involved in nitrate/nitrite substrate recognition i.e. in being nitrate signature (Trueman, et al., 1996; Forde, 2000). One clear hotspot for mutations was observed with the above nitrate signature of Tm5, where residues Gly167, Ala169, Gly170 and Gly172 were represented by a total of ten substitutions (Kinghorn, et al., 2005). This high frequency of change, suggests that this motif is crucial for function and the substitution mutations result in loss of function (Kinghorn, et al., 2005). Furthermore, altered mutational hotspot regions in any of the 12 transmembrane domains and /or the C-terminal tail may be required for NrtA insertion into the membrane and /or protein stability causing reduction of transport activity or abolition of function (Kinghorn, et al., 2005). The Aspergillus nitrate uptake system was found to be different from other nitrate-inducible nitrate uptake systems (with km's around 250 [micro]M) in other related fungi, such as Neurospora crassa (Schloemer and Garrett, 1974; Quesada, et al., 1994) or Penicillium chrysogenum (Goldsmith, et al., 1973). The Aspergillus nitrate uptake system requires a functional nitrate reductase activity in order to be active. In Aspergillus nidulans as well as in plants and other related fungi nitrate uptake system is induced by nitrate along with nitrate reductase. As clearly seen in results, nitrate reductase activity rapidly disappeared in medium in which the absence of inducer, the presence of inhibitor and the repression by ammonium caused rapid loss of enzyme activity, in which further synthesis of the enzyme was not possible. These findings agreed with that shown previously for A. nidulans (Cove, 1966), Neurospora crassa (Subramanian and Sorger, 1972). Furthermore, it seems to be that the presence of inducer and chlorate inhibit the utilization of various nitrogen sources and that the nitrate reductase protein is required for this inhibition, since mutant strains with altered nitrate reductase have shown resistance to these inhibitors (Kanan, 2002; Kanan, et al., 2002). Moreover, the presence of ammonium represses the system and prevents the synthesis of nitrate reductase and leads to rapid loss of its activity and this is agreed with that obtained previously for Penicillium chrysogenum (Goldsmith, et al., 1973). Chlorate strongly inhibited net nitrate uptake and nitrate reductase activity, a process separated and distinct from but dependent upon, nitrate reductase reaction. nrtA expression was subjected to control by the positive regulatory areA gene, mediating nitrogen metabolite repression, but was not under the control of the positive acting regulatory gene nirA, mediating nitrate induction. This suggests that there is only one mechanism of inactivation operating under the various growth conditions. However when cells were transferred to medium lacking a nitrogen source this result in a less decay of enzyme activity as compared to that in presence of inhibitors. This indicates that starvation for nitrogen in itself does not cause inactivation. However, it is not known if this loss of enzyme activity is due to proteases action on the enzyme.

Acknowledgment

We appreciate the strains sent to us by Dr. J. R. Kinghorn, School of biological and medical sciences, University of St. Andrews, St. Andrews, Scotland-U.K.

References

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Corresponding Author: Khalid M. Al-Batayneh, Chair, Department of Biological Sciences, Yarmouk University, Irbid- Jordan E-mail: albataynehk@hotmail.com Fax: +962 2 7211117

(1) Khalid M. Al-Batayneh, (1) Emad I. Hussein, (1) Wesam Al Khateeb, (2) Khalaf Alhussaen, (2) Ibrahim H. Bashaireh and (1) Ghassan J.M. Kanan

(1) Department of Biological Sciences, Yarmouk University, Irbid, Jordan.

(2) Department of Plant Production and Protection, Faculty of Agriculture, Jerash University, Jerash, Jordan.

(3) Nursing School, Philadelphia University, Amman, Jordan.

Khalid M. Al-Batayneh, Emad I. Hussein, Wesam Al Khateeb, Khalaf Alhussaen, Ibrahim H. Bashaireh and Ghassan J.M. Kanan; Effect of various growth conditions on nitrate uptake and nitrate reductase activity in Aspergillus nidulans nitrate assimilation defective mutants.
Table 1: Isolation of nrt, brn and cbrn (nitrate transporters) mutant
strains after treatment with the chemical mutagen
N-methyl-N-nitro-N-nitrozoguanidine (NTG).

Treated wild-type strain                  biA1

Selection temperature                     25[degrees]C   37[degrees]C

N-source                                  Arginine       Arginine
Total Screened colonies                   96             96
Total confirmed chlorate resistant but    60             76
  bromate sensitive nitrate
  assimilation defective mutants
  (nrt; cnx and niaD with RS phenotype)
Generated nrt mutant with RS phenotype    23             31
Other nitrate assimilation defective      37             45
  mutant strains (i.e cnx and niaD
  mutants with RS-phenotype
Confirmed chlorate sensitive but          8              13
  bromate resistant mutants (brn
  strains with SR phenotype)
Confirmed chlorate and bromate            3              5
  resistant mutants (cbrn strains
  with RR phenotype)

Treated wild-type strain                  yA2pyroA4

Selection temperature                     25[degrees]C   37[degrees]C

N-source                                  Arginine       Arginine
Total Screened colonies                   96             96
Total confirmed chlorate resistant but    71             67
  bromate sensitive nitrate
  assimilation defective mutants
  (nrt; cnx and niaD with RS phenotype)
Generated nrt mutant with RS phenotype    27             19
Other nitrate assimilation defective      44             48
  mutant strains (i.e cnx and niaD
  mutants with RS-phenotype
Confirmed chlorate sensitive but          18             17
  bromate resistant mutants (brn
  strains with SR phenotype)
Confirmed chlorate and bromate            7              12
  resistant mutants (cbrn strains
  with RR phenotype)

Mutagenized strains were inoculated into chlorate master plates
(glucose supplemented minimal medium, pH 6.5) with potassium
chlorate (200 mM) and arginine (10 mM) as sole N-source and
incubated at the indicated selection temperature. Confirmed mutant
strains were tested at both temperatures (25[degrees]C and
37[degrees]C) on potassium chlorate (200 mM) with arginine
(10 mM); potassium bromate (200 mM) with arginine (10 mM)) and on
non-selective media (sodium nitrate, sodium nitrite and hypoxanthine
(10 mM). For putative thermo-sensitive and cryso-sensitive mutants
analysis was repeated at least three times at both temperatures
(25[degrees]C and 37[degrees]C) for conformation of results.
The obtained cnx and niaD mutants specify other genes in the nitrate
assimilation pathway but not nitrate transporters.

Table 2: Genetic recombination analysis between various nrt mutant
strains representing two nitrate transport genes.

yA2biA1 nrtA1 crossed                CL[O.sup.-.sub.3]
to 4 nrt mutant strains   Total      R (a)
found to map in           screened   Br[O.sup.-.sub.3]
the same locus            progeny    S (b)

biA1 nrtA2                72         72
biA1 nrtA3                72         72
biA1 nrtA4                72         72
yA2nrtA5                  72         72

Designated yA2pyro
A4 nrtB1 crossed to
non-nrtA strains and
found to map in the
same nrtB locus

biA1 nrtB2                72         72
biA1 nrtB3                72         72
biA1 nrtB4                72         72
yA2pyroA4 nrtB5           72         72
yA2 pyroA4 nrtB6          72         72

yA2biA1 nrtA1 crossed
to 6 designated nrtB
strains (found to
map in non-nrtA
locus)

yA2 PyA4 nrtB1            72         59
biA1 nrtB2                72         62
biA1 nrtB3                72         45
biA1 nrtB4                72         41
yA2 PyroA4 nrtB5          72         48
YA2 PyroA4 nrtB6          72         52

yA2biA1 nrtA1 crossed     Cl[O.sup.-.sub.3]    Cl[O.sup.-.sub.3]
to 4 nrt mutant strains   R                    S
found to map in           Br[O.sup.-.sub.3]    Br[O.sup.-.sub.3]
the same locus            R                    R

biA1 nrtA2                0.0                  0.0
biA1 nrtA3                0.0                  0.0
biA1 nrtA4                0.0                  0.0
yA2nrtA5                  0.0                  0.0

Designated yA2pyro
A4 nrtB1 crossed to
non-nrtA strains and
found to map in the
same nrtB locus

biA1 nrtB2                0.0                  0.0
biA1 nrtB3                0.0                  0.0
biA1 nrtB4                0.0                  0.0
yA2pyroA4 nrtB5           0.0                  0.0
yA2 pyroA4 nrtB6          0.0                  0.0

yA2biA1 nrtA1 crossed
to 6 designated nrtB
strains (found to
map in non-nrtA
locus)

yA2 PyA4 nrtB1            0.0                  13
biA1 nrtB2                0.0                  10
biA1 nrtB3                8                    18
biA1 nrtB4                3                    28
yA2 PyroA4 nrtB5          5                    19
YA2 PyroA4 nrtB6          2                    18

                          Cl[O.sup.-.sub.3]   Br[O.sup.-.sub.3]
yA2biA1 nrtA1 crossed     sensitive           Resistant
to 4 nrt mutant strains   recombinants        recombinants
found to map in
the same locus            No        %         No        %

biA1 nrtA2                0.0       0.0       0.0       0.0
biA1 nrtA3                0.0       0.0       0.0       0.0
biA1 nrtA4                0.0       0.0       0.0       0.0
yA2nrtA5                  0.0       0.0       0.0       0.0

Designated yA2pyro
A4 nrtB1 crossed to
non-nrtA strains and
found to map in the
same nrtB locus

biA1 nrtB2                0.0       0.0       0.0       0.0
biA1 nrtB3                0.0       0.0       0.0       0.0
biA1 nrtB4                0.0       0.0       0.0       0.0
yA2pyroA4 nrtB5           0.0       0.0       0.0       0.0
yA2 pyroA4 nrtB6          0.0       0.0       0.0       0.0

yA2biA1 nrtA1 crossed
to 6 designated nrtB
strains (found to
map in non-nrtA
locus)

yA2 PyA4 nrtB1            13        18        13        18
biA1 nrtB2                10        14        10        14
biA1 nrtB3                18        25        26        36
biA1 nrtB4                28        39        31        43
yA2 PyroA4 nrtB5          19        26        24        33
YA2 PyroA4 nrtB6          18        25        20        28

Sexual crosses were performed between nrtA1 strain and each of 10
strains with the nrt phenotype isolated in this study. Four strains
have mapped in the nrtA locus while, 6 strains have mapped in another
nrt locus designated nrtB. No sensitive recombinants were obtained
from crosses between strains representing the same locus, whereas
such recombinants were shown in crosses between strains representing
unlinked nrt loci. For each tested cross one hybrid cleistothecium
was analyzed and a total of 72 progeny were tested. Progeny were
tested on supplemented glucose minimal medium (pH 6.5) with:
potassium chlorate (200 mM) and either NH4Cl, Uric acid or Arginine
(10 mM) were used as the sole N-sources. The same progeny were tested
on minimal medium with bromate (200 mM) with the same N-sources.
The wild-type and both parents were included in the tests as
controls. (a): (R), denotes resistance. (b): (S), denotes
sensitive. All phenotypic analysis were done at 37[degrees]C.

Table 3: Nitrate uptake rate in wild-type and various nitrate
transport nrt mutant strains.

                       Nitrate uptake rate assayed in mycelial
                       cells of 8 and 16 hours of age: (b)

Tested strain (a)      8 (h)                  16 (h)

biA1(wild-type)        11.77 [+ or -] 1.70    12.44 [+ or -] 0.16
yA2biA1nrtA1            3.44 [+ or -] 1.34    12.45 [+ or -] 0.48
biA1nrtA2               4.59 [+ or -] 5.62    10.11 [+ or -] 3.98
biA1nrtA3               4.20 [+ or -] 1.23    11.46 [+ or -] 0.10
biA1nrtA4               4.71 [+ or -] 0.99    13.35 [+ or -] 0.26
yA2pyroA4nrtBl          8.32 [+ or -] 0.87    12.16 [+ or -] 0.33
biA1nrtB2               6.06 [+ or -] 1.35    10.77 [+ or -] 3.25
biA1nrtRR1 (cs) (c)     9.90 [+ or -] 2.65    14.33 [+ or -] 4.27
biA1nrtRR2              9.81 [+ or -] 3.69    14.67 [+ or -] 2.58
yA2pyroA4nrtRR3         7.43 [+ or -] 0.70    13.46 [+ or -] 0.17
yA2pyroA4nrtRR4         9.30 [+ or -] 0.58    11.96 [+ or -] 0.60
biA1nrtRR5 (cs)        10.70 [+ or -] 0.34    13.44 [+ or -] 0.44

(a) Mycelia were grown in supplemented glucose minimal medium,
with urea (5 mM) as the nitrogen source, at 37[degrees]C for
8 or 16 h as indicated. NaN[O.sub.3] (10 mM) was added 3 h before
harvesting as described in the text. (b) Nitrate uptake rates are
expressed as nanomoles of N[O.sup.-.sub.3] removed per min per
milligram (dry weight) and are the means [+ or -] SD from at least
three independent experiments. c denotes cryso-sensitive mutant
strains.

Table 4: Disappearance of nitrate reductase activity in wild-type and
various nitrate transport mutant strains.

                                Nitrate reductase specific
                                activity assayed after different
                      Age of    time course intervals (min) (b):
                      cells
Tested strain         (h) (a)   0.0

biA1 (wild-type)      8          91.17 [+ or -] 3.62
                      16         27.43 [+ or -] 6.91
yA2biA1nrtA1          8          49.63 [+ or -] 5.62
                      16          9.36 [+ or -] 5.57
biA1nrtA2             8         162.98 [+ or -] 4.75
                      16        110.68 [+ or -] 23.01
biA1nrtA3             8          87.96 [+ or -] 4.11
                      16         36.19 [+ or -] 4.94
biA1nrtA4             8          58.43 [+ or -] 2.11
                      16         24.17 [+ or -] 2.65
yA2pyroA4nrtB1        8          57.50 [+ or -] 1.45
                      16         27.26 [+ or -] 6.39
biA1nrtB2             8          97.56 [+ or -] 6.45
                      16         59.15 [+ or -] 7.88
biA1nrtRR1(cs) (c)    8         115.71 [+ or -] 1.44
                      16         88.96 [+ or -] 24.49
biA1nrtRR2            8         116.11 [+ or -] 1.15
                      16         44.34 [+ or -] 4.04
yA2pyroA4nrtRR3       8         128.69 [+ or -] 3.33
                      16         83.95 [+ or -] 21.87
yA2pyroA4nrtRR4       8         378.65 [+ or -] 3.26
                      16        309.45 [+ or -] 1.75
biA1nrtRR5(cs)        8         166.20 [+ or -] 3.58
                      16        109.19 [+ or -] 16.09

                                Nitrate reductase specific
                                activity assayed after different
                      Age of    time course intervals (min) (b):
                      cells
Tested strain         (h) (a)   8

biA1 (wild-type)      8         22.90 [+ or -] 1.56
                      16         3.16 [+ or -] 0.74
yA2biA1nrtA1          8          8.79 [+ or -] 2.25
                      16         0.99 [+ or -] 0.42
biA1nrtA2             8         43.56 [+ or -] 2.68
                      16        11.01 [+ or -] 1.98
biA1nrtA3             8         31.33 [+ or -] 4.17
                      16         3.48 [+ or -] 0.05
biA1nrtA4             8         13.91 [+ or -] 2.54
                      16         2.83 [+ or -] 0.93
yA2pyroA4nrtB1        8         33.63 [+ or -] 1.78
                      16         3.21 [+ or -] 0.03
biA1nrtB2             8         41.45 [+ or -] 3.68
                      16         6.72 [+ or -] 0.25
biA1nrtRR1(cs) (c)    8         48.91 [+ or -] 6.58
                      16        12.88 [+ or -] 1.76
biA1nrtRR2            8         62.84 [+ or -] 4.75
                      16         4.87 [+ or -] 0.05
yA2pyroA4nrtRR3       8         38.66 [+ or -] 4.21
                      16        11.86 [+ or -] 3.60
yA2pyroA4nrtRR4       8         68.60 [+ or -] 1.65
                      16        40.07 [+ or -] 1.77
biA1nrtRR5(cs)        8         35.36 [+ or -] 2.12
                      16        13.60 [+ or -] 3.14

                                Nitrate reductase specific
                                activity assayed after different
                      Age of    time course intervals (min) (b):
                      cells
Tested strain         (h) (a)   16

biA1 (wild-type)      8          8.79 [+ or -] 4.65
                      16         1.02 [+ or -] 0.62
yA2biA1nrtA1          8          3.34 [+ or -] 1.52
                      16         0.27 [+ or -] 0.13
biA1nrtA2             8         13.97 [+ or -] 3.36
                      16         4.75 [+ or -] 2.13
biA1nrtA3             8          9.58 [+ or -] 5.14
                      16         1.47 [+ or -] 0.11
biA1nrtA4             8          4.28 [+ or -] 4.74
                      16         1.22 [+ or -] 0.43
yA2pyroA4nrtB1        8         11.79 [+ or -] 2.35
                      16         1.16 [+ or -] 0.17
biA1nrtB2             8         13.62 [+ or -] 1.47
                      16         2.51 [+ or -] 0.17
biA1nrtRR1(cs) (c)    8         14.69 [+ or -] 3.62
                      16         4.35 [+ or -] 0.18
biA1nrtRR2            8         11.35 [+ or -] 3.41
                      16         1.29 [+ or -] 0.25
yA2pyroA4nrtRR3       8         18.32 [+ or -] 5.21
                      16         3.67 [+ or -] 0.97
yA2pyroA4nrtRR4       8         27.24 [+ or -] 3.34
                      16        11.21 [+ or -] 1.24
biA1nrtRR5(cs)        8          9.22 [+ or -] 4.14
                      16         3.84 [+ or -] 0.82

                                Nitrate reductase specific
                                activity assayed after different
                      Age of    time course intervals (min) (b):
                      cells
Tested strain         (h) (a)   24

biA1 (wild-type)      8          1.95 [+ or -] 9.58
                      16         0.93 [+ or -] 0.39
yA2biA1nrtA1          8          1.62 [+ or -] 4.56
                      16         0.14 [+ or -] 0.09
biA1nrtA2             8          7.43 [+ or -] 6.25
                      16         2.64 [+ or -] 0.07
biA1nrtA3             8          2.47 [+ or -] 2.15
                      16         0.80 [+ or -] 0.05
biA1nrtA4             8          1.64 [+ or -] 6.21
                      16         0.80 [+ or -] 0.25
yA2pyroA4nrtB1        8          1.88 [+ or -] 3.68
                      16         0.85 [+ or -] 0.25
biA1nrtB2             8          8.56 [+ or -] 4.37
                      16         2.07 [+ or -] 0.13
biA1nrtRR1(cs) (c)    8          5.34 [+ or -] 2.32
                      16         1.95 [+ or -] 0.15
biA1nrtRR2            8          3.68 [+ or -] 3.68
                      16         1.42 [+ or -] 0.47
yA2pyroA4nrtRR3       8          7.41 [+ or -] 7.14
                      16         1.83 [+ or -] 0.14
yA2pyroA4nrtRR4       8         16.81 [+ or -] 1.10
                      16         6.76 [+ or -] 2.65
biA1nrtRR5(cs)        8          4.73 [+ or -] 3.18
                      16         1.89 [+ or -] 0.12

(a) Mycelia were grown at 37[degrees]C in supplemented glucose
minimal medium for 8 and 16 h as indicated above with nitrate
(10 mM) as sodium salt as described in the text. (b) Nitrate
reductase activity is expressed as nanomoles of NADPH oxidized
per min per milligram of protein and values are mean [+ or -] SD of
at least three independent experiments. (c) denotes cryso-sensitive
mutant strains.
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Title Annotation:Original Article
Author:Al-Batayneh, Khalid M.; Hussein, Emad I.; Al Khateeb, Wesam; Alhussaen, Khalaf; Bashaireh, Ibrahim H
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:7JORD
Date:Sep 1, 2010
Words:9008
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