Nitrous acid was used to modify traditional de Man, Rogosa, Sharpe medium to evaluate whether the addition of sodium nitrite to MRS medium could improve the rate of growth and density of various lactic acid bacteria and nontarget species. Yogurt and Cheddar cheese were inoculated with individual bacterial species followed by the recovery and enumeration of the species using the pour plate method to compare the sensitivity between nitrous acid-modified MRS (mMRS) and traditional MRS. Lactobacillus delbrueckii ssp. bulgaricus were recovered at significantly higher counts from cheese in nitrous acid mMRS than MRS, whereas no significant difference was observed for other species and food systems. Growth curves were also generated for multiple lactic acid bacteria as well as nonstarters in both mMRS and MRS to measure the selectivity of nitrous acid mMRS. The selectivity evaluation of nitrous acid mMRS demonstrated that 5 of the tested lactic acid bacterial species (Bifidobacterium longum, Streptococcus salivarius, Lactococcus lactis, Lactobacillus acidophilus, and Lactobacillus delbrueckii ssp. bulgaricus) grew to significantly higher densities more rapidly in mMRS broth than in traditional MRS. Nontarget bacteria Enterococcus faecalis and Bacillus cereus revealed a more prolific growth rate and higher optical density readings in traditional MRS compared with mMRS. It was determined that nitrous acid mMRS is a viable alternative medium for culturing selected lactic acid bacteria, and offers an improved formulation of MRS for use in standard evaluation methods and optimization of probiotic and other dairy cultures.

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn2+-binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPRmediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

RNA can be modified in over 100 distinct ways, and these modifications are critical for function. Pseudouridine synthases catalyse pseudouridylation, one of the most prevalent RNA modifications. Pseudouridine synthase 7 modifies a variety of substrates in Saccharomyces cerevisiae including tRNA, rRNA, snRNA, and mRNA, but the substrates for other budding yeast Pus7 homologues are not known. We used CRISPR‐mediated genome editing to disrupt Candida albicans PUS7 and find absence leads to defects in rRNA processing and a decrease in cell surface hydrophobicity. Furthermore, C. albicans Pus7 absence causes temperature sensitivity, defects in filamentation, altered sensitivity to antifungal drugs, and decreased virulence in a wax moth model. In addition, we find C. albicans Pus7 modifies tRNA residues, but does not modify a number of other S. cerevisiae Pus7 substrates. Our data suggests C. albicans Pus7 is important for fungal vigour and may play distinct biological roles than those ascribed to S. cerevisiae Pus7.

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