Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Summary Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.     

Cloning of an Aspergillus niger invertase gene by expression in Trichoderma reesei

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5^- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.     

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency

Summary The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5 non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5 regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Neisseria gonorrhoeae Heme Biosynthetic Mutants Utilize Heme and Hemoglobin as a Heme Source but Fail To Grow within Epithelial Cells

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding γ-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An fbpA mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme, N. gonorrhoeae strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.     

Sequence of the cloned pyr4 gene of Trichoderma reesei and its use as a homologous selectable marker for transformation

Summary We have cloned and sequenced the Trichoderma reesei pyr4 gene encoding orotidine-5-monophosphate decarboxylase. Comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that T. reesei is closely related to Cephalosporium acremonium and Neurospora crassa. The cloned pyr4 gene has been used as a homologous selectable marker for transformation of T. reesei. The majority of transformants obtained with circular plasmid were mitotically unstable and contained non-integrated plasmid molecules, sometimes in addition to plasmid integrated in the genome, Linearization of plasmid prior to transformation decreased the transformation frequency but increased the proportion of stable transformation obtained.     

An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-β-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species. Received: 5 February / 20 March 2000     

The oliC3 gene of Aspergillus niger: isolation,sequence and use as a selectable marker for transformation

Summary The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycinresistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5 untranslated sequences with those from other fungi.     

Selection for phenylalanine hydroxylase activity in cells transformed with recombinant retroviruses

Cells deficient in phenylalanine hydroxylase (PAH) are tyrosine auxotrophs and will not survive in tyrosine-free media. PAH activity can be constituted in cultured cells by infection with recombinant retroviruses carrying a human PAH cDNA. Mouse hepatoma cells transformed with recombinant PAH will grow in tyrosine-free media since these cells constitutively synthesize the cofactor tetrahydrobiopterin which is essential for PAH activity. NIH3T3 cells transformed with the PAH cDNA express the PAH apoenzyme, but this enzyme is inactive in vivo since these cells do not synthesize biopterin. We describe a method of selection for PAH in the fibroblast-like NIH3T3 cells involving tyrosine-free media supplemented with biopterin, reducing agents, and antioxidants. Cells transformed with the recombinant PAHgene exhibit PAH activity in culture and will grow in the biopterin-supplemented tyrosine-free media. Metabolic selection for PAH activity provides a new selectable marker for gene transfer experiments. This method is shown to be useful in the production of high liters of recombinant retroviruses carrying PAHand provides a model for experiments in somatic gene therapy of phenylketonuria.     

Transformation of Penicillium nalgiovense with the amdS gene of Aspergillus nidulans

Summary Penicillium nalgiovense was transformed with the amdS gene from Aspergillus nidulans as a selectable marker. The vector apparently integrated at multiple sites into the chromosomes of the transformants, which were mitotically stable. A transformation efficiency of 12 transformants/g vector DNA was achieved when the expression phase was prolonged to 8 h.     

Transformation of Trichoderma viride using the Neurospora crassa pyr4 gene and its use in the expression of a Taka-amylase A gene from Aspergillus oryzae

Summary A pyrG mutant of Trichoderma viride, a very efficient cellulase producer, was isolated from among 5-fluoroorotic acid-resistant mutants. The mutation was complemented with the pyr4 gene of Neurospora crassa and used as a selection marker for the transformation of T. viride. A plasmid vector, pDJB1-Taa, carrying both the pyr4 gene and a gene encoding Taka-amylase A from Aspergillus oryzae, was constructed and introduced into protoplasts of T. viride pyrG^-. The transformation frequency was 1–10 transformants (3 on average) per g DNA. One transformant showed highly elevated -amylase production (about 17 times higher than the recipient level) and the integration of more than one copy of the Taka-amylase gene.     

Nature of abortive transformation in Saccharomyces cerevisiae

Disruption mutagenesis by homologous recombination in Saccharomyces cerevisiae is carried out by transforming-DNA fragments containing the target gene disrupted by a selectable marker. A large number of transient (abortive) transformants are often formed that may hinder the isolation of integrants containing the gene disruption. We show that abortive transformants result from re-circularization of the linear transforming-DNA in vivo. Their number was greatly reduced when the cut DNA could not readily re-ligate, either by digestions that gave non-compatible or blunt ends, or by de-phosphorylation. In addition, true integrants could be readily distinguished from abortive transformants through replica plating onto selective media. Enhanced disruption-mutagenesis was also observed when non-compatible ends were generated in an ARS-containing insertion vector.     

Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans

A vector system was constructed that is designed to decrease the number of transformants required to be screened when looking for gene disruption events in filamentous fungi. This vector was used to mutate two genes, an ATP-binding cassette transporter (LmABCt4) and a two-component histidine kinase gene (LmHK1) in the ascomycete Leptosphaeria maculans. The system uses the thymidine kinase gene from the herpes simplex virus as a negative selectable marker. Thymidine kinase expression is regulated by the TrpC regulatory elements from Aspergillus nidulans and should be applicable to other ascomycetous fungi. When thymidine kinase is expressed in the presence of particular thymidine analogues, these analogues are converted to toxic compounds which kill the cell. We also report the transformation of L. maculans using Agrobacterium tumefaciens-mediated DNA delivery.Communicated by U. Kück     

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Summary A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5-phosphate decarboxylase (pyrG) and the vector pA04-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular PA04-2 resulted in the appearance of Pyr⁺ transformants at a frequency of up to 20 per g of DNA, whereas with linear pA04-2 up to 200 transformants per g DNA were obtained. In 75 % of the Pyr⁺ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pA04-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding -galactosidase, and uidA, encoding -glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.     

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl₂ protoplast fusion protocol. Hygromycin B-resistant (HygB^R) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygB^R progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants

We have constructed a dominant selectable marker, PHT1, for transformation of the basidiomycete Coprinus cinereus. PHT1 consists of a bacterial hygromycin B resistance gene fused to the promoter and terminator regions of the C. cinereusβ-tubulin gene. We found in transformation experiments that PHT1 confers hygromycin B resistance to all strains of C. cinereus tested, that it integrates without apparent bias into the genome, and that it is stable through meiotic crosses. We used a plasmid containing this marker, pPHT1, for restriction enzyme-mediated integration (REMI) and found that this technique could increase transformation efficiencies more than seven-fold. In REMI experiments using KpnI, the integrated DNA was flanked by intact KpnI sites in 53% of the cases examined, single-copy insertions represented 60% of the integration events, and most multicopy insertions were oriented head-to-tail. A screen of REMI-generated transformants yielded sporulation-defective mutants at a frequency of 1.2%. Genetic analysis showed that in six of nine mutants examined, the defect in spore formation is most likely a direct result of the pPHT1 insertion, and in three of these mutants a single pPHT1 locus was shown to cosegregate with the sporulation defect. We used semi-random PCR to isolate the genomic DNA adjacent to one pPHT1 insertion in a sporulation-defective mutant and found that we had disrupted the C. cinereusspo11 gene. Thus, REMI, in combination with pPHT1, is a powerful tool for the dissection of the meiotic process in C. cinereus. Received: 26 February / 13 July 1999     

Transformation of Trichoderma harzianum by high-voltage electric pulse

Summary We have developed conditions for an efficient method of genetic transformation in Trichoderma harzianum, using high-voltage electroporation. Transformation was obtained with a plasmid carrying the Escherichia coli, hygromycin B phosphotransferase gene as a dominant selectable marker, and the gpd promoter and trpC terminator from Aspergillus nidulans. The transformation frequency is up to 400 transformants per g of plasmid DNA. The transformants were phenotypically 100% stable; they were also mitotically stable. Hybridization experiments suggest that the transforming DNA might be integrated at the same position in the T. harzianum genome. This report opens possibilities for improving transformation systems that have already been described for fungi, or else for transforming filamentous fungi where the use of polyethylene glycol is not efficient.     

Electrotransformation and Expression of Bacterial Genes Encoding Hygromycin Phosphotransferase and β-Galactosidase in the Pathogenic Fungus Histoplasma capsulatum

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional β-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.