Conversion of polyploid and alloploid Saccharomyces sensu stricto strains to leu2 mutants by genome DNA editing.

A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2- mutants produced in this study will be deposited in several repository organizations. KEY POINTS: • All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains • Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains • The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.

Community structures of total bacterial DNA, cultivable bacteria and prototrophs in bulk soil and rhizospheres

Aims: It has been hypothesized that root exudates can be a nutritional factor influencing the bacterial community structure as well as the occurrence of prototrophs and auxotrophs in rhizospheres. The present study was performed to examine the community structures of total bacterial DNA, cultivable bacteria and prototrophs in 3 soil samples with different levels of abundance of root exudates. Methodology and results: Denaturing gradient gel electrophoresis (DGGE) was performed to examine the community structures of total bacterial DNA, cultivable bacteria and prototrophs in 3 soil samples including bulk soil, rhizosphere of a single plant species and rhizosphere of multiple plant species. For clustering analysis, a dendrogram generated from the DGGE patterns revealed the different bacterial community structures in these soil samples. Both rhizospheres claded together, separating from bulk soil. The DGGE patterns of cultivable bacteria showed particular fingerprints corresponding to kinds of media and soil samples. Nutrient agar (NA) medium, isolation medium for prototroph (IMP) and IMP supplemented with soil extracts were used for bacterial cultivations. Prototrophs were isolated and examined by random amplified polymorphic DNA (RAPD) and 16S rRNA gene sequence analysis. The genetic diversity of prototrophs in 3 soil samples was similar (approximately 5% to 10% similarities) and most of them (13 of 28 strains) were members of Pseudomonas with 97% to 100% identities. Conclusion, significance, and impact of study: The present study provides a strong evidence of the influence of root exudates and plant species on bacterial community structures.