Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene

Summary A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial -glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of -glucuronidase activity.     

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: Complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene

Summary Three cellulose-negative (Cel^-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize (1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.     

Transformation of Aspergillus oryzae using the A. niger pyrG gene

Summary A transformation system for Aspergillus oryzae based on the orotidine-5-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per g of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Cloning of the Agrobacterium tumefaciens C58 trpE gene by complementation in Escherichia coli

Summary The trpE gene of Agrobacterium tumefaciens C58 was cloned from a gene library by complementation in Escherichia coli. It was shown to be unlinked to trpD gene in this organism. It was also shown that the nontumorigenic phenotype of tryptophan auxotrophs of A. tumefaciens could be complemented by addition of exogenous tryptophan. The role of bacterially synthesised tryptophan in the process of tumour formation is discussed.Abbreviations Ap ampicillin - Cm chloramphenicol - Gent gentamycin - Km kanamycin - dATP deoxyadenosine 5-triphosphate - IAA indole acetic acid - NB nutrient broth - MinAB minimal Agrobacterium medium     

Arginine and proline genes ofAspergillus niger

Summary Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. Thearg genes belong to six different linkage groups and thepro genes to two. Onearg-mutant could be complemented by transformation with theA. nidulans arg B ⁺ gene, and thisA. niger gene thus appeared to be homologous to theA. nidulans arg B. We isolated anA. niger strain with theargB gene tightly linked with thenicA1 marker. This strain is very suitable as acceptor for transformation with anargB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using thenicA1 marker gene.     

Effect of thedpy-20 androl-6 cotransformation markers on α-tubulin gene expression inC. elegans transformants

An -1 tubulin::lacZ fusion gene was introduced into the germline ofCaenorhabditis elegans, using eitherrol-6 ordpy-20 genomic DNA as a cotransformation marker. Distinct patterns in cellular specificity of the -1 tubulin::lacZ fusion gene expression were observed, depending on the cotransformation marker used. For therol-6 marker, the tubulin fusion gene was expressed in several neurons in the head and tail ganglia and a set of 38–39 ventral cord motor neurons along the body length of the animal during larval and adult development. In contrast, for thedpy-20 marker system, not only were fewer neurons stained in the head and tail region, but also the staining of ventral cord motor neurons was extremely reduced both in number and intensity. Thedpy-20 marked-mediated suppression of the -1 tubulin gene expression was observed both in thecis andtrans configurations. Similar down-regulation in the ventral cord motor neurons was observed when the -2 tubulin::lacZ fusion gene construct was tested in these experiments using thedpy-20 marker. In controls, where the tubulin fusion gene was directly injected to obtain transformants without any marker DNA, the cellular staining pattern was close to the fusion gene expression observed with therol-6 marker DNA. These results underline the importance of the choice of transformation marker system in generation of the transgenic animals, and reveal a down-regulation of the -tubulin fusion gene expression in the ventral cord motor neurons in transgenic animals when thedpy-20 gene was used as a cotransformation marker.     

Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism

Summary A selection procedure using Mn²⁺ is described. A high percentage of the Mn²⁺ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of -galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur ⁺ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%–70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.     

Cloning, characterization of the acyl-CoA : 6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene

Summary The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5 region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39 240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of -galactosidase with symmetric variants of - and -centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the -centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the -centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for -centered trp operator variants with exchanges in positions 3, 4 and 5.     

Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus niger niaD gene encoding nitrate reductase

Summary A transformation system for Gibberella fujikuroi based on the Aspergillus niger nitrate reductase gene (niaD) was developed. A strain (designated SG140) carrying a non-reverting niaD mutation (niaD11) was generated by screening mutagenised cells for non-growth on nitrate as sole nitrogen source. Transformation frequencies of 1–2 transformants per g DNA were observed when strain SG140 was transformed to nitrate utilisation. Southern blot analyses of niaD⁺ transformants showed that the vector DNA sequences were integrated into the chromosomal DNA. The results demonstrate that the A. niger niaD gene is expressed in G. fujikuroi.     

Molecular cloning,expression and structure of the endo-1,5-α-l-arabinase gene of Aspergillus niger

Secretion of endo-1,5--l-arabinase A (ABNA) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing l-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA ⁺ RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-tranformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized. Correspondence to: L. H. de Graaff     

Mitochondrial protein import: isolation and characterization of the Saccharomyces cerevisiae MFT1 gene

Summary Mitochondrial targeting of an Atp2-LacZ fusion protein confers a respiration-defective phenotype on yeast cells. This effect has been utilized to select strains that grow on nonfermentable carbon sources, some of which have decreased levels of hybrid protein localized to the organelle. Many of the mutants obtained were also temperature-sensitive for growth on all media. The recessive mft (mitochondrial fusion targeting) mutants have been assigned to three complementation groups. MFT1 was cloned and sequenced: it encodes a 255 amino acid protein that is highly basic and has no predicted membrane-spanning domains or organelle-targeting sequences. The MFT1 gene is 91% identical to an open reading frame 3 of the SIR3 gene. Evidence is presented that these two closely related genes could represent a recent gene duplication.The sequence reported here has been listed in the EMBL Data Library with Accession Number X55360.     

Cloning of the Aspergillus niger gene encoding α-l-arabinofuranosidase A

Using l-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of -l-arabinofuranosidase A by an Aspergillus niger d-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an -l-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding -l-arabinpfuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Enlarged map ofAgrobacterium tumefaciens C58 and the location of chromosomal regions which affect tumorigenicity

Summary A revised and enlarged genetic map of theAgrobacterium tumefaciens C58 chromosome has been produced with the help of plasmid R68.45. Apart from the location of several auxotrophic markers, the map also shows the position of two independent genes,ctu1 andctu2, which, when mutated, block the tumorigenesis of the bacterium. Of these two, onlyctu1 is complemented by the C58 chromosomalvir region cloned by Douglas et al. (1985). The same mutant was complemented by a chromosomal gene or genes located nearleu ofRhizobium meliloti and known to affect the nodulation properties of that bacterium. It has also been observed that C58 tryptophan auxotrophs invariably lose tumorigenicity. Prototrophic revertants and mutants supplied with extra tryptophan for about two weeks after infection produce normal tumours. These investigations suggest that for successful tumorigenesis a continuous supply of tryptophan is needed (to be converted into auxin IAA?) at least during the early stages.     

Cloning of the trp genes from the archaebacterium Methanococcus voltae: nucleotide sequence of the trpBA genes

Summary A cosmid bank of Methanococcus voltae DNA was obtained in Escherichia coli after ligation of partially HindIII-digested M. voltae DNA in the HindIII site of the transferable cosmid pVK100. The bank was used to perform complementation experiments with E. coli auxotrophic mutants. Five cosmids complementing trpA shared three adjacent HindIII fragments of 2.1, 2.3 and 14 kb. Two of these cosmids also complemented trpD and carried an additional 4.2 kb HindIII fragment. The trpA- and trpD-complementing regions were more precisely localized using Tn5 mutagenesis. A 1.7 kb PstI fragment, cloned into pUC9 in both orientations, was responsible for the trpA complementation. This fragment was sequenced and an open reading frame (ORF) of 852 nucleotides (ORFtrpA) encoding a 284 amino acid polypeptide of mol. wt. 31938 was found. The amino acid sequence was compared with that of the subunit of tryptophan synthase (trpA gene product) from nine eubacterial species and to the N-terminal part of the tryptophan synthase of Saccharomyces cerevisiae (TRP5 gene product). Similarity varied from 24% (Brevibacterium lactofermentum) to 35% (S. cerevisiae). The nucleotide sequence of the region upstream from M. voltae ORFtrpA was determined and revealed the presence of an ORF of 1227 nucleotides (ORFtrpB) encoding a 409 amino acid polypeptide of mol. wt. 44634. The polypeptide sequence was similar to the subunit of tryptophan synthase (trpB gene product) from six eubacterial species and to the C-terminal part of the tryptophan synthase of S. cerevisiae. Similarity varied from 49% (S. cerevisiae, B. lactofermentum) to 58% (Pseudomonas aeruginosa). This high conservation supports the hypothesis of a common ancestor for the trpA and trpB genes of archaebacteria, eubacteria and eucaryotes. M. voltae ORFtrpA and ORFtrpB, which are transcribed in the same direction, are separated by a 37 bp AT-rich region. Immediately upstream from ORFtrpB, the 3 end of an ORF homologous to E. coli and Bacillus subtilis trpF was found. As the trpD-complementing region was located upstream from the trpFBA sequenced region, the organization of trp genes in the archaebacterium might thus be trpDFBA. Such an organization resembles that of enteric eubacteria, in which the trpEDCFBA genes are grouped in a single operon. However, M. voltae ORFtrpA and ORFtrpB do not overlap, in contrast with what is found in most eubacteria.     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.