Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides

Abstract

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.

Keywords: Mucor circinelloides; URA blaster; mucormycosis; pyrG blaster; recyclable genetic marker.

Figure 1

Development of a recyclable pyrG marker in M. circinelloides. (A) Construction of a pyrG blaster marker with repeats of 237 bp on 5′ and 3′ end. The short repeat (237 bp) in the 3′ end was tandemly incorporated to enhance the excision of the marker gene. The resulting pyrG blaster marker can be amplified by PCR with M13 forward and reverse primers. (B) Homologous replacement of the carRP gene with the pyrG blaster marker. The loss of the carRP gene results in a failure to produce carotenoids in M. circinelloides. Therefore, the carRPΔ mutants display white colonies as indicated by the red arrows on the plate, whereas wild-type cells form yellow colonies. Although the target gene is disrupted, only a portion of the nuclei contains the carRPΔ allele because the spores are multinucleated with aseptate hyphae. Transformants that stably form white colonies were selected to obtain the carRPΔ mutant with a homozygous karyotype. Gene size is not to scale.

Figure 2

Confirmation of excision of the pyrG marker. The MSL27 (carRPΔ::pyrG-dpl237) strain was spot-inoculated on MMC media containing 5-FOA. After 3 d of incubation, spontaneous resistant mutants emerged forming a sector from colony (data not shown). The 5-FOA resistant mutants went through a vegetative cycle on MMC media containing 5-FOA. The obtained spores were subject to PCR analysis and auxotrophy tests. The excision of the pyrG marker from the carRPΔ::pyrG-dpl237 allele was confirmed by PCR (see text in the section of Excision of pyrG gene). The strains with the pyrG marker excised did not grow on media without uridine. Two independent carRPΔ::dpl237 strains were obtained. (1) MU402 (WT in carRP allele and pyrG⁻); (2) MSL27 (carRPΔ::pyrG-dpl237); (3) MSL28 (carRPΔ::dpl237); (4). MSL29 (carRPΔ::dpl237). Gene size is not to scale.

Figure 3

Southern blot analysis confirms the deletion of the carRP gene and excision of the pyrG marker gene. Southern blotting was performed to ensure the deletion of the carRP gene and excision of the pyrG marker. Genomic DNA of the wild-type strain was digested with BclI and produced a 5011-bp fragment that was recognized by the probe. A double digestion with BclI and EcoRI resulted in a 1322-bp fragment that was recognized by the probe. The digestion of the genomic DNA of the carRPΔ::pyrG-dpl237 mutant with BclI produced a 2501-bp fragment that was recognized by the probe. The double digestion with the enzymes BclI and EcoRI produced an 872-bp fragment recognized by the probe. Finally, the carRPΔ::dpl237 mutant underwent the same digestions, where a 3539-bp fragment was produced after BclI enzyme digestion, and an 870-bp fragment was produced after BclI and EcoRI double digestion. Both fragments were detected during our hybridization procedure by our probe. Gene size is not to scale.

Figure 4

Disruption of the cnbR gene in the carRPΔ::dpl237 mutant and phenotypes of the double mutants. The carRPΔ::dpl237 strain, MSL28, produces white colonies and requires uridine to grow. The cnbRΔ::pyrG strain, MSL7, produces a yeast-locked colony while still retaining the function of the carRP gene, and thus produces yellow yeast-locked progeny. The carRPΔ::dpl237 cnbRΔ::pyrG strain, MAG1, exhibits a yeast-locked phenotype and produces white colonies due to the disruption in both carRP and cnbR genes. Microscopic images were also taken to confirm the phenotype of each strain. Bar, 10 μm.