Gene disruption of the pcbAB gene encoding ACV synthetase in Cephalosporium acremonium
| Autor: | Stephen Wyatt Queener, Paul L. Skatrud, Victor J. Chen, Niamh O'Callaghan, Jo Ann Hoskins, John S. Wood, Cathleen A. Cantwell |
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| Rok vydání: | 1990 |
| Předmět: |
Lactams
Transcription Genetic ACV synthetase Genes Fungal Molecular Sequence Data Restriction Mapping chemistry.chemical_compound Restriction map Plasmid Transformation Genetic Tyrocidine Sequence Homology Nucleic Acid Genetics Amino Acid Sequence Peptide Synthases Gene Peptide sequence Southern blot biology Acremonium General Medicine biology.organism_classification Molecular biology Anti-Bacterial Agents Blotting Southern chemistry biology.protein Hybridization Genetic Plasmids |
| Zdroj: | Current genetics. 18(6) |
| ISSN: | 0172-8083 |
| Popis: | Plasmid pPS 96 was used to disrupt the genomic region immediately upstream of pcbC in C. acremonium by homologous integration. Approximately 4% of the C. acremonium transformants obtained with pPS 96 were unable to produce beta-lactam antibiotics. All transformants obtained with other plasmids and isolates which had not been exposed to transforming DNA retained the ability to produce beta-lactams. Enzyme analysis showed that ACV synthetase activity was missing in the beta-lactam-minus pPS 96 transformants. Southern hybridization analysis confirmed the presence of multiple copies of pPS 96 in all beta-lactam-minus transformants analyzed. However, predictable alterations of the targeted region were not detected. Transformation of antibioticminus transformants with plasmid pZAZ4, carrying a wild-type copy of the region targeted for disruption, resulted in restoration of the ability to produce beta-lactams in greater than 80% of the transformants recovered. Location of the pcbAB gene upstream from pcbC was confirmed by comparing the amino acid sequence of internal peptides from purified ACV synthetase with that deduced from the DNA sequence of the region targeted for disruption. The direction of transcription of the pcbAB gene is opposite that of the pcbC gene. Further analysis of amino acid sequence data from ACV synthetase revealed regions of strong similarity with the peptide synthetases responsible for production of tyrocidine and gramicidin S in Bacillus brevis. |
| Databáze: | OpenAIRE |
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