Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping

Summary TheAspergillus nidulans gene coding for acetamidase (amdS) was introduced intoA. niger by transformation. Twelve Amd⁺ transformants were analysed genetically. TheamdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed)A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for theamdS insert have the Amd⁺ phenotype. We exploited the opportunity for two-way selection inA. niger: transformants can be isolated based on the Amd⁺ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologousamdS gene inA. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI betweenpabA1 andcnxA1, providing evidence for a single transformational insert. The results also show that theamdS transformants ofA. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use ofamdS transformants for mitotic gene mapping in other fungi.     

Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa

Summary Theqa-2 gene ofNeurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. TheQUTE gene ofAspergillus nidulans corresponds to theqa-2 gene ofN. crassa. The plasmid pEH1 containing theQUTE gene fromA. nidulans was used to transform aqa-2 ^– strain ofN. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to theQUTE gene probe. Northern blot analyses indicated thatQUTE mRNA was produced in the transformants. The functional integrity of theQUTE gene inN. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-typeN. crassa. The evidence that theQUTE structural gene ofA. nidulans is inducible when introduced into theN. crassa genome implies that theN. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5 to theQUTE gene.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Announcement

Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [¹⁴C]propionate and [¹⁴C]p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.     

ATP and adenylate energy charge during phosphate-mediated control of antibiotic synthesis.

The addition of inorganic phosphate or guanosine-5′-monophosphate to a phosphate-limited mycelial system of $\text{Streptomyces griseus}$ inhibited candicidin production. Accompanying the inhibition was a rapid increase in intracellular ATP concentration. Adenylate energy charge increased only slightly indicating that ATP is a more likely intracellular effector than energy charge in mediating phosphate control of antibiotic biosynthesis.     

Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Summary Genetic transformation of Nicotiana tabacum protoplasts was achieved by incubation of protoplasts with a plasmid DNA-calcium phosphate coprecipitate, followed by fusion of the protoplasts in the presence of polyvinyl alcohol and subsequent exposure to high pH. A derivative of the plasmid pBR322 containing a chimaeric gene, consisting of the nopaline synthase promoter, the coding region of the aminoglycoside phosphotransferase gene of Tn5 and the polyadenylation signal region of the octopine synthase gene, was used for these transformation experiments. This chimaeric gene confers resistance of transformed plant cells to kanamycin. This novel transformation procedure reproducibly yielded transformants at frequencies of approximately 0.01%. Aminoglycoside phosphotransferase II activity was detected in both transformed calli and in regenerated plants. DNA from some of the transformed clones was analyzed by Southern blot hybridization. The input DNA appears to be integrated into high molecular weight cellular DNA. Genetic analysis of one of the kanamycin resistant plants shows that the chimaeric gene is transmitted to the progeny as a single dominant trait in a Mendelian fashion. As a comparison the input DNA was also introduced into tobacco protoplasts using Agrobacterium tumefaciens and Ti-plasmid derived gene vectors.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

NUT1, a major nitrogen regulatory gene inMagnaporthe grisea, is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^– mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^– transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^– transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

Biosynthesis of candicidin by phosphate-limited resting cells ofStreptomyces griseus

Summary A phosphate-limited resting cell system ofStreptomyces griseus in a synthetic medium has been developed in which biosynthesis of the polyene macrolide, candicidin, is linear for at least 36 h without cell growth. Glucose and to a lesser degree sucrose, but not lactose, support antibiotic synthesis. Glucose is utilized at a constant rate for antibiotic synthesis without affecting mycelial dry weight. Acetate and propionate, the building units of the macrolide aglycone, stimulate candicidin biosynthesis in cultures supplemented with glucose but do not support its synthesis in the absence of glucose. Maximal stimulation of candicidin biosynthesis was produced by 40 mM propionate or 250 mM acetate. The biosynthetic intermediate, methyl malonate, and the analog, 1-propanol, were more stimulatory than propionate at the same concentration.     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Plant defense gene promoter enhances the reliability of<Emphasis Type="Italic"> shiva-1</Emphasis> gene-induced resistance to soft rot disease in potato

PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.