Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation
ABSTRACT Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast e...
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American Society for Microbiology
2025-01-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02430-24 |
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| author | Yainitza Hernandez-Rodriguez A. Makenzie Bullard Rebecca J. Busch Aidan Marshall José M. Vargas-Muñiz |
| author_facet | Yainitza Hernandez-Rodriguez A. Makenzie Bullard Rebecca J. Busch Aidan Marshall José M. Vargas-Muñiz |
| author_sort | Yainitza Hernandez-Rodriguez |
| collection | DOAJ |
| description | ABSTRACT Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches. |
| format | Article |
| id | doaj-art-5fe12d14d7e74ec4907551eab5d2a472 |
| institution | Kabale University |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj-art-5fe12d14d7e74ec4907551eab5d2a4722025-01-07T14:05:19ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-01-0113110.1128/spectrum.02430-24Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformationYainitza Hernandez-Rodriguez0A. Makenzie Bullard1Rebecca J. Busch2Aidan Marshall3José M. Vargas-Muñiz4Department of Biological Sciences, Florida Gulf Coast University, Ft. Myers, Florida, USAMolecular Biology, Microbiology, and Biochemistry Program, School of Biological Science, Southern Illinois University, Carbondale, Illinois, USADepartment of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USADepartment of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USADepartment of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USAABSTRACT Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.https://journals.asm.org/doi/10.1128/spectrum.02430-24Hortaea werneckiitransformationmorphologyectopic integrationCRISPR/Cas9 |
| spellingShingle | Yainitza Hernandez-Rodriguez A. Makenzie Bullard Rebecca J. Busch Aidan Marshall José M. Vargas-Muñiz Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation Microbiology Spectrum Hortaea werneckii transformation morphology ectopic integration CRISPR/Cas9 |
| title | Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation |
| title_full | Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation |
| title_fullStr | Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation |
| title_full_unstemmed | Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation |
| title_short | Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation |
| title_sort | strategies for genetic manipulation of the halotolerant black yeast hortaea werneckii ectopic dna integration and marker free crispr cas9 transformation |
| topic | Hortaea werneckii transformation morphology ectopic integration CRISPR/Cas9 |
| url | https://journals.asm.org/doi/10.1128/spectrum.02430-24 |
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