Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides
Mucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attentio...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Oxford University Press
2017-07-01
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| Series: | G3: Genes, Genomes, Genetics |
| Subjects: | |
| Online Access: | http://g3journal.org/lookup/doi/10.1534/g3.117.041095 |
| _version_ | 1818923944078475264 |
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| author | Alexis Garcia Gloria Adedoyin Joseph Heitman Soo Chan Lee |
| author_facet | Alexis Garcia Gloria Adedoyin Joseph Heitman Soo Chan Lee |
| author_sort | Alexis Garcia |
| collection | DOAJ |
| description | Mucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. Nonetheless, the importance of these fungi as pathogens and model systems has increased. M. circinelloides is one of a few genetically tractable organisms in the basal fungi, but it is far from a robust genetic system when compared to model fungi in the subkingdom Dikarya. One problem is the organism is resistant to drugs utilized to select for dominant markers in other fungal transformation systems. Thus, we developed a blaster recyclable marker system by using the pyrG gene (encoding an orotidine-5′-phosphate decarboxylase, ortholog of URA3 in Saccharomyces cerevisiae). A 237-bp fragment downstream of the pyrG gene was tandemly incorporated into the upstream region of the gene, resulting in construction of a pyrG-dpl237 marker. To test the functionality of the pyrG-dpl237 marker, we disrupted the carRP gene that is involved in carotenoid synthesis in pyrG− mutant background. The resulting carRP::pyrG-dpl237 mutants exhibit a white colony phenotype due to lack of carotene, whereas wild type displays yellowish colonies. The pyrG marker was then successfully excised, generating carRP-dpl237 on 5-FOA medium. The mutants became auxotrophic and required uridine for growth. We then disrupted the calcineurin B regulatory subunit cnbR gene in the carRP::dpl237 strain, generating mutants with the alleles carRP::dpl237 and cnbR::pyrG. These results demonstrate that the recyclable marker system is fully functional, and therefore the pyrG-dpl237 marker can be used for sequential gene deletions in M. circinelloides. |
| first_indexed | 2024-12-20T02:17:28Z |
| format | Article |
| id | doaj.art-6a217c2a965441a9b27d9dffbbb7bb75 |
| institution | Directory Open Access Journal |
| issn | 2160-1836 |
| language | English |
| last_indexed | 2024-12-20T02:17:28Z |
| publishDate | 2017-07-01 |
| publisher | Oxford University Press |
| record_format | Article |
| series | G3: Genes, Genomes, Genetics |
| spelling | doaj.art-6a217c2a965441a9b27d9dffbbb7bb752022-12-21T19:56:55ZengOxford University PressG3: Genes, Genomes, Genetics2160-18362017-07-01772047205410.1534/g3.117.0410954Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloidesAlexis GarciaGloria AdedoyinJoseph HeitmanSoo Chan LeeMucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. Nonetheless, the importance of these fungi as pathogens and model systems has increased. M. circinelloides is one of a few genetically tractable organisms in the basal fungi, but it is far from a robust genetic system when compared to model fungi in the subkingdom Dikarya. One problem is the organism is resistant to drugs utilized to select for dominant markers in other fungal transformation systems. Thus, we developed a blaster recyclable marker system by using the pyrG gene (encoding an orotidine-5′-phosphate decarboxylase, ortholog of URA3 in Saccharomyces cerevisiae). A 237-bp fragment downstream of the pyrG gene was tandemly incorporated into the upstream region of the gene, resulting in construction of a pyrG-dpl237 marker. To test the functionality of the pyrG-dpl237 marker, we disrupted the carRP gene that is involved in carotenoid synthesis in pyrG− mutant background. The resulting carRP::pyrG-dpl237 mutants exhibit a white colony phenotype due to lack of carotene, whereas wild type displays yellowish colonies. The pyrG marker was then successfully excised, generating carRP-dpl237 on 5-FOA medium. The mutants became auxotrophic and required uridine for growth. We then disrupted the calcineurin B regulatory subunit cnbR gene in the carRP::dpl237 strain, generating mutants with the alleles carRP::dpl237 and cnbR::pyrG. These results demonstrate that the recyclable marker system is fully functional, and therefore the pyrG-dpl237 marker can be used for sequential gene deletions in M. circinelloides.http://g3journal.org/lookup/doi/10.1534/g3.117.041095Mucor circinelloidesURA blasterpyrG blasterrecyclable genetic markermucormycosis |
| spellingShingle | Alexis Garcia Gloria Adedoyin Joseph Heitman Soo Chan Lee Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides G3: Genes, Genomes, Genetics Mucor circinelloides URA blaster pyrG blaster recyclable genetic marker mucormycosis |
| title | Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides |
| title_full | Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides |
| title_fullStr | Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides |
| title_full_unstemmed | Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides |
| title_short | Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides |
| title_sort | construction of a recyclable genetic marker and serial gene deletions in the human pathogenic mucorales mucor circinelloides |
| topic | Mucor circinelloides URA blaster pyrG blaster recyclable genetic marker mucormycosis |
| url | http://g3journal.org/lookup/doi/10.1534/g3.117.041095 |
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