Enzyme-based DNA extraction from zoospores of ruminal fungi

Abstract

We report here a rapid, efficient and simple method for the extraction of high molecular weight DNA from zoospores of Neocallimastix frontalis EB188. This anaerobic fungus, isolated from bovine digesta, effectively degrades plant fiber in vitro (Barichievich et al. 1990. Appl. Env. Micro. 56:43-48). Our interest in ruminal fungi stems from their ability to degrade wood materials (Joblin et al. 1989. FEMS Micro. Lett. 56:119-122) and their potential use in biomass saccharification. Zoospore DNA synthesis is of particular interest to our laboratory. It is these motile zoospores which colonize and degrade plant materials (Mountfort 1987. FEMS Micro. Rev. 46:501-508). To detail fully this metabolic event, it will be necessary to extract nucleic acids from zoospores. Such procedures have not been reported in the literature. Using acetone drying and enzymatic removal of cell walls, we have isolated high molecular weight DNA from very small amounts of culture. The procedure takes less than one hour and DNA yields are high. The DNA is readily cut with restriction endonucleases and religated efficiently but is otherwise stable. Electrophoretic analysis of the DNA confirmed the presence of repetitive sequences. This procedure will aid the study of DNA replication, DNA repair and DNA RFLP analysis of various strains using small (<1 >ml) cultures.

Anaerobic gut fungi: Advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production

“…However, genomic DNA and mRNA can be extracted from gut fungal isolates using modified techniques established for common organisms such as S. cerevisae and E. coli . These methods tend to be strain specific and include mechanical disruption using beads (Denman et al, ) or grinding under liquid nitrogen (Brookman et al, ; Brownlee, ), enzymatic lysis with zymolyase, lyticase or proteinase K (Brownlee, ; Tsai and Calza, ), and solubilization with detergents such as CTAB or SDS (Brookman and Nicholson, ; Brookman et al, ; Brownlee, ).…”

“…While nuclei are present in the rhizomycelium of polycentric fungi, they are difficult to isolate and are easily contaminated with polysaccharides. Thus, there is an optimal harvest time to capture quality DNA from zoospores of actively growing monocentric and polycentric fungi of around 2–3 days (Tsai and Calza, ). Genomic isolations earlier than this will yield little DNA due to low free zoospore concentration.…”

“…At this time, zoospores are still being formed and are trapped within the zoosporangia creating multiple physical barriers, which reduces lysis efficiency. Similarly, extractions from over‐mature zoospores (>120 h) will yield low amounts of degraded DNA due to inherent instabilities in their AT‐rich genome (Brownlee, ; Nicholson et al, ; Youssef et al, ) and morphological changes in the zoospore cell wall, reducing cell lysis efficiency (Tsai and Calza, ). The physiology and growth conditions of these fungi also lead to significant polysaccharide contamination of DNA isolated by chemical extraction methods, which can interfere with subsequent molecular reactions (Brownlee, , ).…”

High-quality RNA extraction and the regulation of genes encoding cellulosomes are correlated with growth stage in anaerobic fungi

“…The fungus used in this study, A. robustus , is a polycentric fungus with multinucleate rhizomycelia ( Haitjema et al., 2017 ; Mondo et al., 2017 ). This range could vary for other fungal strains or microbial pairings as a result of biological differences, such as the presence or absence of rhizoidal structures, as nuclei are present in the rhizomycelium of polycentric fungi ( Tsai and Calza, 1992 ; Haitjema et al., 2014 ). Therefore, future studies should be conducted to determine whether this range for capturing sufficient RNA quantity/quality is generalizable across more anaerobic fungal genera.…”