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The Aspergillus nidulans cnxABC Locus Is a Single Gene Encoding Two Catalytic Domains Required for Synthesis of Precursor Z, an Intermediate in Molybdenum Cofactor Biosynthesis

1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês

10.1074/jbc.272.45.28381

1083-351X

Shiela E. Unkles, Jacqueline Smith, Ghassan Kanan, Lindsey Jane Millar, Immanuel S. Heck, David H. Boxer, James R. Kinghorn,

Fungal and yeast genetics research

The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3–4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function. The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3–4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function. More than 3 decades ago, Pateman, Cove, and co-workers (see Refs.1Cove D.J. Pateman J.A. Nature. 1963; 198: 262-263Crossref PubMed Scopus (50) Google Scholar and 2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar; reviewed in Refs. 3Cove D.J. Biol. Rev. 1979; 54: 291-327Crossref PubMed Google Scholar, 4Scazzocchio C. J. Less Common Metals. 1974; 36: 461-464Crossref Scopus (12) Google Scholar, 5Scazzocchio C. Coughlan M.P. Molybdenum and Molybdenum-containing Enzymes. Pergammon Press, Oxford, UK1980: 489-515Google Scholar, 6Tomsett A.B. Boddy L. Marchant R. Read D.J. Nitrogen, Phosphorus and Sulphur Utilisation by Fungi. Cambridge Press, Cambridge, UK1989: 33-58Google Scholar) first isolated mutants defective in the synthesis of the molybdenum pterin cofactor. Using the lower eukaryotic model, the ascomycetous fungus Aspergillus nidulans, they isolated a class of chlorate-resistant mutants, which were unable to utilize either nitrate or purines such as adenine, hypoxanthine, and xanthine as sole sources of nitrogen. This inability to grow on nitrate and hypoxanthine was due to the pleiotropic loss of NADPH-nitrate reductase and NADH-xanthine dehydrogenase (purine hydroxylase I) activity, respectively. They speculated that such mutants were defective in the synthesis of a cofactor common to both nitrate reductase and xanthine dehydrogenase and accordingly designated the mutants cnx (common component fornitrate reductase and xanthine dehydrogenase). Five cnx loci (namely cnxABC, cnxE,cnxF, cnxG, and cnxH) were originally observed by Pateman et al. (2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar) on the basis of complementation tests in heterokaryons grown with nitrate as the sole nitrogen source. Later, a sixth locus, cnxJ, was identified, albeit with a different growth phenotype by Arst et al.(7Arst Jr., H.N. Tollervey D.W. Sealy-Lewis H.M. J. Gen. Microbiol. 1982; 128: 1083-1093PubMed Google Scholar). The cnxABC locus showed a complex overlapping pattern of complementation, i.e. growth on nitrate in heterokaryon combinations of different pairwise (cnxA,cnxB, cnxC) mutant backgrounds (2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar, 8Hartley M.J. Genet. Res. 1970; 16: 123-125Crossref PubMed Scopus (9) Google Scholar). Mutants in cnxA and cnxC phenotypically complement each other, vis à vis growth ofcnxA/cnxC heterokaryons on nitrate as a sole nitrogen source, while cnxB mutants fail to complement eithercnxA or cnxC mutants. Since cnxA,cnxB, and cnxC mutants were shown to be genetically tightly linked, it was unclear whether cnxABCconsisted of one gene in which cnxA and cnxCmutants exhibited intragenic complementation or two distinct genes,cnxA and cnxC, with cnxB mutants lacking both activities. Virtually nothing is known about the function of the A. nidulans cnx genes and their role in the synthesis of the molybdopterin cofactor. We describe here the molecular and biochemical characterization of cnxABC, the first cloned A. nidulans cnx locus reported thus far. The wild-type strain used was G051 (biA1). Strains α8 (biA1 cnxA9), G055 (biA1 cnxB11), α23 (yA2 wA3 cnxC2), and G832 (yA2 pyroA4 cnxC3) were mutants described originally by Pateman and Cove (2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar). Strains GH140 (biA1 cnxA140ts), GH95 (yA2 pyroA4 cnxA95), GH1281 (biA1 cnxB1281) and GH2O4 (biA1 cnxB204) were selected in this study on the basis of resistance to 150 mm chlorate as described by Cove (9Cove D.J. Biochim. Biophys. Acta. 1966; 113: 51-56Crossref PubMed Google Scholar). 1G. J. M. M. Kanan, unpublished observations. cnxA95and cnxB204 were selected on 10 mm glutamate,cnxA140 ts with 10 mm proline andcnxB1281 with 5 mm uric acid all as sole nitrogen sources. The cnxA95, cnx204, andcnxB1281 mutations were generated usingN-methyl-N′-nitro-N-nitrosoguanidine, while cnxA140 ts was synthesized after 1,2,7,8-diepoxyoctane chemical mutagenesis (2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar). Assignment of mutations to cnx loci was carried out by the heterokaryotic complementation test, i.e. growth of pairwise heterokaryons on nitrate as the sole nitrogen source against the representativecnx mutants, cnxA9, cnxB11,cnxC3, cnxE3, cnxF7, cnxG4, and cnxH4, as described by Pateman et al. (2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar). The temperature-sensitive mutant cnxA140 ts shows mutant phenotypes of resistance to chlorate and nongrowth on nitrate as the sole nitrogen source at 37 °C but near wild type phenotypes of sensitivity to chlorate and growth on nitrate at 25 °C with a mutant phenotype on hypoxanthine at both temperatures. Additionally, this mutant has a wild-type nitrate reductase temperature stability profile from cells grown at the permissive temperature of at 25 °C.1 Standard Aspergillus growth media, handling techniques (10Clutterbuck A.J. King R.C. Handbook of Genetics. 1. Plenum Publishing, New York1974: 447-510Google Scholar), and transformation (11Johnstone I.L. Hughes S.G. Clutterbuck A.J. EMBO J. 1985; 4: 1307-1311Crossref PubMed Scopus (156) Google Scholar) were as described previously. For expression analysis, cultures were grown at 30 °C for 16 h in liquid minimal medium (9Cove D.J. Biochim. Biophys. Acta. 1966; 113: 51-56Crossref PubMed Google Scholar) containing the sole nitrogen sources stated in the figure legends. Standard procedures were used for propagation of cosmids and for subcloning and propagation of plasmids in Escherichia coli strain DH5α. Conditions employed here for A. nidulans Southern and Northern blot analysis were as described previously (12MacCabe A.P. Riach M.B.R. Unkles S.E. Kinghorn J.R. EMBO J. 1990; 9: 279-287Crossref PubMed Scopus (102) Google Scholar). The nucleotide sequence of the wild-typecnxABC gene was determined in both strands using a Sequenase version 2 DNA sequencing kit according to the manufacturer's instructions (Amersham Corp.). For primer extension analysis, mRNA was prepared from mycelium grown in minimal medium containing 10 mm sodium nitrate as the sole nitrogen source at 25 °C for 18 h, using a Quickprep mRNA Purification Kit (Pharmacia Biotech Inc.). Messenger RNA (2 μg) was hybridized with 5′32P-end-labeled primer PE-1 (5′-GAAGTCGCTTGAGCTGCG-3′, position +47) at 52 °C for 1 h and reverse transcribed using a primer extension system as recommended (Promega). The extension product was compared on a denaturing sequencing gel with a DNA sequence ladder prepared using the same end-labeled primer with pSTA502 as template. Genomic DNA was prepared from mycelia grown in liquid culture for 16–18 h at 25 °C using a Nucleon BACC2 Kit (Scotlab). DNA was cleaved with EcoRI, and around 100 ng was amplified using 2.5 units/μl Dynazyme (Flowgen) or 2.5 units/μl Taq DNA polymerase (Boehringer Mannheim), a 1 μm concentration of each primer, and 100 μmdNTPs. Cycling conditions were 94 °C for 1 min, 50 °C for 20 s, and 72 °C for 50 s for one cycle, followed by 30 cycles of 94 °C for 10 s, 50 °C for 20 s, and 72 °C for 50 s. The entire cnxABC coding region was amplified in four overlapping sections using primers P1 and P2 (5′-CGTTGTCGAGCAGAATC-3′ and 5′-TATGCTCTTCATAACCGC-3′, positions −27 to +687), primers P3 and P4 (5′-GTAAATCTCAGTCTGGAC-3′ and 5′-CATGCCAATAACGTCAAG-3′, positions +601 to +1263), primers P5 and P6 (5′-TGAGGCAGCTCGAACAG-3′ and 5′-GACGTCGGTTGGAGAAG-3′, positions +1176 to +1876), and primers P7 and P8 (5′-ATATGACGCTGATTGATG-3′ and 5′-TGTCATACACATCCAGG-3′, positions +1796 to +2344). Following removal of PCR primers and unincorporated nucleotides by the Glassmax DNA Isolation Spin Cartridge System (Life Technologies), the amplified DNA was sequenced directly in a single strand only by automated DNA sequencing using an ABI 373 A automated fluorescent sequencing apparatus and the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) with the PCR primers at a concentration of 10 μm. Additional primers used for sequencing were PE-2 (5′-CCGGTAGTGACATATCGC-3′, position +140), AR5 (5′-GATTACCTTAGGATCAG-3′, position +280), AR3 (5′-TGGATTCCAAGGCCGAG-3′, position +975), AR8 (5′-TTGTATACATGATTCAC-3′, position +1404), and CR9 (5′-ATGACGCTGAGATGAAG-3′, position +2051). Sequences were compared with the wild type using Sequencher (Gene Codes Corp.). Upon identification of a putative mutation, the relevant primer pair was used to reamplify the digested genomic DNA, and the product was sequenced again to confirm the mutational change. Conidial suspensions of around 106 viable cells were made in 0.9% (w/v) sodium chloride, 0.1% (v/v) Tween 80, and 1 μl of the strain under test was spotted on the surface of agar minimal medium containing 10 mmsodium nitrate as the sole nitrogen source. A second 1-μl aliquot of the standard strain was spotted on top of the test strain, and the medium was incubated at 37 °C. Form A dephospho and compound Z levels were analyzed, as a measure of precursor Z and molybdopterin, respectively, according to the method described by Johnson and Rajagopalan (13Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 110-116Crossref PubMed Google Scholar, 14Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 117-125Crossref PubMed Google Scholar) with modifications. A conidial suspension in 20 ml of 0.9% saline, 0.1% Tween 80 (approximately 5 × 106 conidia/ml) was used to inoculate 400 ml of minimal medium supplemented with the stated sole nitrogen sources after Cove (9Cove D.J. Biochim. Biophys. Acta. 1966; 113: 51-56Crossref PubMed Google Scholar). Cells were grown for 16 h at 30 °C and 250 rpm and harvested by filtration through sterile muslin cloth. After washing with 50 ml of sterile distilled water, the cells were pressed dry and frozen in liquid nitrogen. Frozen cell material (1.5 g) was homogenized by sonication (5 × 15 s) in 3 ml of 100 mm Tris-HCl buffer (pH 7.2), and the cell debris was removed by centrifugation (20 min at 14,000 rpm). Of the resulting supernatant, 1 ml was combined with 125 μl of I2/KI (1%/2%) in 1 m HCl and then left in darkness for 10 h at room temperature. 138 μl of 1% ascorbic acid and 0.5 ml of 1m Tris-HCl were added before centrifugation at 14,000 rpm for 10 min at room temperature to remove any particulate matter. The supernatant was mixed with 13 μl of 1 m MgCl2and 2 units of alkaline phosphatase and incubated for a further 12 h in darkness at room temperature. The samples were subsequently applied to QAE-Sephadex A-25 (Sigma) columns (acetate form, 0.5-ml bed volume) and washed with 5 ml of distilled water. Form A dephospho was eluted with 5 ml of 10 mm acetic acid, adjusted to pH 7.0 with NH4OH, and stored frozen until HPLC analysis. Compound Z was eluted with 8 ml of 10 mm HCl. The HCl eluates were applied to Florisil (Sigma) columns (250 mg of Florisil mesh 100–200, washed with 12 ml of 10 mm HCl; bed volume, 0.6 ml). The columns were then washed with 1 ml of 10 mm HCl, and compound Z was eluted with 1.7 ml of 22.5% acetone. The acetone eluates were rotoevaporated until dry. The compound Z samples were dissolved in 300 μl of H2O, of which 100 μl was injected for HPLC analysis. Analysis of form A dephospho and compound Z by reversed phase HPLC was performed using a Hypersil ODS column (250 × 4.6 mm, 5 μm). Form A dephospho (500 μl of the acidic acid eluate) was eluted with 10% methanol, 50 mm ammonium acetate (pH 6.7, 1 ml/min) and detected using a Shimadzu RF-551 fluorescence detector set to 370/450 nm (emission/excitation). Compound Z was eluted with 5% methanol, 50 mm triethylammonium acetate (pH 7.0, 1 ml/min) and detected by fluorescence at 350/450 nm (emission/excitation). Total protein was estimated using the bicinchoninic acid (BCA) method, with bovine serum albumin as a standard. Previous classical genetics has shown that thecnxABC locus is approximately 3 recombination map units from the brlA locus on linkage group VIII. Using a 4.8-kbBamHI DNA fragment of pILJ421 (11Johnstone I.L. Hughes S.G. Clutterbuck A.J. EMBO J. 1985; 4: 1307-1311Crossref PubMed Scopus (156) Google Scholar) containing thebrlA gene as a hybridization probe, two cosmids were isolated from the chromosome 8-specific cosmid library (15Brody H. Griffith J. Cuticchia A.J. Arnold J. Timberlake W.E. Nucleic Acids Res. 1991; 19: 3105-3109Crossref PubMed Scopus (165) Google Scholar). Since neither of these cosmids complemented phenotypically (i.e.growth on nitrate as sole nitrogen source) cnx mutant straincnxA9, cnxB11, or cnxC 3 in transformation experiments (see “Experimental Procedures”), the cosmids were used to generate end probes to walk to neighboring cosmids. Eight overlapping cosmids were identified, and one of these, W26G08, was found to complement all three mutant strains, i.e. cnxA9, cnxB11, and cnxC3, at transformation frequencies in excess of 100 transformants/μg of cosmid DNA. Phenotypic complementation of the mutants using EcoRI-,XbaI-, and BamHI-digested and isolated fragments of cosmid W26G08 indicated that the complementing region was located on a 6.5-kb EcoRI-XbaI fragment, estimated by restriction endonuclease mapping to be around 65 kb from thebrlA gene. This DNA fragment, the restriction map of which is shown in Fig. 1 A, was subcloned into vector pUC18, resulting in recombinant plasmid pSTA502.Figure 1Restriction endonuclease map of recombinant plasmid pSTA502 and DNA and deduced amino acid sequence of the A. nidulans cnxABC locus. A, shaded arearepresents the region of the plasmid in which the DNA sequence has been determined in both strands. The bars below themap indicate the positions of subclones generated for transformation to localize the complementing region of pSTA502.B, numbers on the right refer to nucleotides relative to the adenosine of the start codon, numbered 1, and numbers on the left refer to amino acid residues. The vertical arrow indicates the transcriptional start point. Potential CCAAT and TATA motifs in the promoter region areunderlined. A possible receptor site for the NIRA control protein is in boldface type. The intron is shown in lowercase with the 5′ and 3′ consensus sequences in boldface type and underlined and potential lariat motifsunderlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Restriction endonuclease map of recombinant plasmid pSTA502 and DNA and deduced amino acid sequence of the A. nidulans cnxABC locus. A, shaded arearepresents the region of the plasmid in which the DNA sequence has been determined in both strands. The bars below themap indicate the positions of subclones generated for transformation to localize the complementing region of pSTA502.B, numbers on the right refer to nucleotides relative to the adenosine of the start codon, numbered 1, and numbers on the left refer to amino acid residues. The vertical arrow indicates the transcriptional start point. Potential CCAAT and TATA motifs in the promoter region areunderlined. A possible receptor site for the NIRA control protein is in boldface type. The intron is shown in lowercase with the 5′ and 3′ consensus sequences in boldface type and underlined and potential lariat motifsunderlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To define more precisely the regions of pSTA502 responsible for the complementation of the three cnx mutant alleles, further subclones of pSTA502 were obtained (Fig. 1 A). Subclones pSTA503, pSTA505, and pSTA507 did not complement cnxA9,cnxB11, or cnxC3 alleles. Subclones pSTA504 and pSTA506 complemented the cnxC3 mutation at high frequency, but only pSTA506 complemented cnxB11 and cnxA9, although at an efficiency much reduced compared with the original recombinant plasmid, pSTA502. Therefore, the minimum region necessary for complementation resides on the stretch of pSTA502 to the right of the PstI site (Fig. 1 A). The DNA sequence of a 3619-base pair stretch of pSTA502 encompassing the complementing region was determined in both strands (Fig. 1 B). A long open reading frame is interrupted by a single intron 198 base pairs long (shown in lowercase), the precise location of which was determined by sequencing a reverse transcriptase-PCR product generated by primers P5 and P6 straddling the proposed intron position. 3S. E. Unkles, unpublished observations. The intron is bounded by canonical 5′- and 3′-splice sequences and has an internal motif corresponding to that required for lariat formation in fungi (16Gurr S.J. Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, UK1987: 93-139Google Scholar). A single transcriptional start point was determined to be at position −73 relative to the proposed translational start,3 which is the first ATG in frame following the transcriptional start point and is surrounded by a recognized translational initiation sequence context for A. nidulans(17Arst Jr., H.N. Sheerins A. Mol. Microbiol. 1996; 19: 1019-1024Crossref PubMed Scopus (21) Google Scholar). Perusal of the noncoding region upstream of the ATG suggests potential TATA motifs at positions −241 and −315, and CCAAT motifs at positions −447 and −509. Additionally, a potential receptor site for NIRA (CCGCGG) (18Punt P.J. Strauss J. Smit R. Kinghorn J.R. van den Hondel C.A.M.J.J. Scazzocchio C. Mol. Cell. Biol. 1995; 15: 5688-5699Crossref PubMed Scopus (115) Google Scholar), the product of the nirA gene, which mediates nitrate induction of systems required for nitrate assimilation including nitrate reductase, nitrite reductase (13Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 110-116Crossref PubMed Google Scholar), and nitrate uptake (19Unkles S.E. Hawker K.L. Grieve C. Campbell E.I. Montague P. Kinghorn J.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 204-208Crossref PubMed Scopus (190) Google Scholar), is present at position −146. Comparison of the cnxABC deduced amino acid sequence to the Swissprot data base indicated that the inferred A. nidulansprotein was homologous to two E. coli proteins (Fig.2), encoded by the moaA gene (similar to the first section of the A. nidulans sequence) and the moaC gene (similar to the second segment) (20Rivers S.L. McNairn E. Blasco F. Giordano G. Boxer D.H. Mol. Microbiol. 1993; 8: 1071-1081Crossref PubMed Scopus (91) Google Scholar). ThecnxABC intron lies between the two regions. The A. nidulans CNXA domain has an 80-residue N-terminal extension in comparison with the E. coli MOAA protein. This N-terminal extension has no similarity to other proteins in the data base. The start of the CNXC domain is not clear from comparison with the MOAC protein, since there is a stretch of around 120 amino acid residues following the MOAA-similar region without similarity to any other proteins,3 including E. coli MoCo biosynthetic proteins (21Gems D.H. Clutterbuck A.J. Curr. Genet. 1993; 24: 520-524Crossref PubMed Scopus (51) Google Scholar). Conserved cysteine residues of potential functional significance in CNXA are in boldface type (Fig.2) and are discussed below under “Determination of MutantcnxA Sequences.” Fragments PCR1, PCR2, PCR3, and PCR4 were amplified using pSTA502 as the template and used in genetic transformation experiments in attempts to repair phenotypically the growth defects of mutant strains cnxA9, cnxB11, andcnxC3 on nitrate as the sole source of nitrogen (Fig.3). Such transformation experiments were carried out using one of the above amplified DNA fragments with and without the presence of the vector pHELP, an autonomously replicatingA. nidulans plasmid that greatly enhances transformation frequencies of co-transformed plasmids (see Ref. 21Gems D.H. Clutterbuck A.J. Curr. Genet. 1993; 24: 520-524Crossref PubMed Scopus (51) Google Scholar and “Experimental Procedures”). It was observed, not unexpectedly perhaps, that the frequency of nitrate utilizing complementers was at least 100-fold greater when PCR fragments were co-transformed with the vector pHELP. Since the frequencies using PCR fragments alone tended to be low, the inclusion of pHELP sharpened the differences between positive and negative phenotypic complementation, although the overall trend with the different PCR fragments was similar. PCR1 complemented phenotypically each recipient mutant strain, i.e. cnxA9,cnxB11, or cnxC3, at high frequency using vector pHELP (1000–3000 nitrate utilizing transformants/μg of PCR fragment). PCR2 complemented only the cnxA9 mutation, while PCR3 complemented the cnxC3 mutation only. No complementing transformants were obtained for the cnxB11 mutation with either fragment PCR2 or PCR3. Fragment PCR4 did not complement thecnxC3 mutation. The results indicate, in conjunction with the homologies observed above, that the cnxA section of thecnxABC locus is equivalent to moaA and that thecnxC locus is equivalent to the E. coli moaCgene, but phenotypic complementation of the cnxB11 mutant requires the presence of both cnxA- andcnxC-encoding DNA fragments. Finally, the observation that transformation with pHELP permits complementation of thecnxC3 mutant by the apparently promoterless fragment PCR3 encoding CNXC domain suggests that the transcriptional activity may initiate from within pHELP itself. Complementation by PCR3 is less likely to be due to integration at the homologous locus by double cross-over, since fragment PCR4, which covers the region of thecnxC3 mutation but does not contain the entire CNXC domain, does not complement the cnxC3 mutant following transformation with pHELP. In an attempt to resolve conclusively the question of how three genetically complementing classes of mutants could be obtained in a single gene (i.e. the cnxABC gene), the DNA sequence of the original Pateman et al. mutants (2Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar) (i.e. cnxA9,cnxB11, cnxC2, and cnxC3) as well ascnx mutants isolated and characterized during this study (cnxA95, cnxA140 ts,cnxB204, and cnxB1281) were determined following PCR amplification. (The origin of the mutants is cited under “Experimental Procedures.”) The positions of the mutational changes and the DNA sequence of the wild type compared with the mutant are shown in Fig. 4. ThecnxA9, cnxA140 ts, andcnxA95 mutations are the result of either single base pair changes or duplication of short stretches of nucleotides in multiples of three in the cnxA-encoding domain. Therefore, neither type of event changes the reading frame, inter alia grossly affects the sequence of the protein. One of the original Pateman and Cove mutants, cnxA9, is an insertion of three amino acids (between residues 345 and 346) into a region highly similar betweenA. nidulans cnxA and E. coli moaA protein products and in which Rajagopalan and colleagues have recognized cysteine residues (highlighted in boldface type, Fig. 2), which are involved in the binding of an iron-sulfur prosthetic group in the E. coli MOAA protein (22Rajagopalan K.V. Solomonson L.P. Barber M.J. Cannons A.C. Nitrogen Assimilation: Fourth International Symposium. USF Printing Services, Tampa, FL1997Google Scholar). The mutant cnxA95is a single base pair alteration resulting in the change of one of these conserved cysteine residues, cysteine 345, to tyrosine, supporting the suggestion that this is an important functional site. The phenotypically temperature sensitive allele,cnxA140 ts, is a duplication resulting in the addition of seven amino acids between residues 139 and 140, just preceding a short homologous amino acid stretch at residue 147 (Fig.2). The DNA sequence changes in the cnxB11, cnxB1281, and cnxB204 mutants all occur within thecnxA-encoding domain of the gene and are either duplications (not in multiples of three) or single base pair deletions. The result of these duplications or deletions is a change in reading frame, positioning a stop codon in frame downstream of the change. Therefore, both cnxA and cnxC domains of the protein are markedly disrupted by the cnxB mutations. A further mutant,cnxB35, is possibly a translocation within thecnxA domain between primers P3 and P4.3 A PCR product was obtained from this mutant with primer combinations P1 and P2, P5 and P6, and P7 and P8, but not with P3 and P4 or P3 and P6. (See “Experimental Procedures” for primer details). The cnxC2 mutation results from the deletion of a single base pair near the end of the CNXC-encoding domain, while thecnxC3 mutation is the consequence of a four-base pair deletion eight codons after the position of the intron incnxABC. This mutation is the most proximal cnxCmutation and hence helps to delimit the start of the CNXC protein at least to before this point. Both cnxC mutations, therefore, disrupt only the CNXC domain. Interestingly, the cnxC2mutation indicates an essential role in protein function for the C-terminal 20 amino acid residues. The resu