title: Use of reporter genes to identify recessive trans-acting mutations specifically involved in the regulation of Aspergillus nidulans penicillin biosynthesis genes.
abstract: Starting from three amino acid precursors, penicillin biosynthesis is catalyzed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC), and aat (penDE). To identify trans-acting mutations which are specifically involved in the regulation of these secondary metabolism genes, a molecular approach was employed by using an Aspergillus nidulans strain (AXTII9) carrying acvA-uidA and ipnA-lacZ gene fusions integrated in double copies at the chromosomal argB gene. On minimal agar plates supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), colonies of such a strain stained blue, which is indicative of ipnA-lacZ expression. After mutagenesis with UV light, colonies were isolated on agar plates with lactose as the carbon source, which produced only a faint blue color or no color at all. Such mutants (named Prg for penicillin regulation) most likely were defective in trans-acting genes. Control experiments revealed that the mutants studied still carried the correct number of gene fusions. In a fermentation run, mutants Prg-1 and Prg-6 exhibited only 20 to 50% of the ipnA-lacZ expression of the wild-type strain and produced only 20 to 30% of the penicillin produced by the wild-type strain. Western blot (immunoblot) analysis showed that these mutants contained reduced amounts of ipnA gene product, i.e., isopenicillin N synthase. Both mutant Prg-1 and mutant Prg-6 also differed in acvA-uidA expression levels from the wild type. Segregation analysis indicated that for both mutants the Prg phenotype resulted from mutation of a single gene. Two different complementation groups, which were designated prgA1 and prgB1, were identified. However, the specific activity of the aat (penDE) gene product, i.e., acyl coenzyme A:6-aminopenicillanic acid acyltransferase, was essentially the same for the mutants as for the wild-type strain, implying that the last step of the penicillin biosynthetic pathway is not affected by the trans-acting mutations identified.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität München, Federal Republic of Germany.
authors.forename: A A
authors.initials: AA
authors.lastname: Brakhage
authors.forename: J
authors.initials: J
authors.lastname: Van den Brulle
mesh_terms: D000217:Acyltransferases / Q000032:analysis; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D003433:Crosses, Genetic; D005285:Fermentation; D005697:Galactosides / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005808:Genes, Recessive; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D007211:Indoles / Q000378:metabolism; D007763:Lac Operon; D016296:Mutagenesis; D010088:Oxidoreductases / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012641:Selection, Genetic; D015533:Transcriptional Activation
chemical_list: D005697:Galactosides; D007211:Indoles; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside