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Type: Thesis
Title: Nitrate assimilation phenotype variability and heterogeneity in Brettanomyces bruxellensis
Author: Zeppel, Ryan James
Issue Date: 2019
School/Discipline: School of Biological Sciences
Abstract: Brettanomyces bruxellensis is a yeast species associated with industrial fermentation ecosystems. The ability to use alternative nutrient sources, such as nitrate, may be advantageous and allow it to outcompete other microbial species. The assimilation of nitrate is conferred by the expression of structural proteins that facilitate nitrate uptake and reduction. The genes (YNR1, YNI1 and YNT1) encoding these structural proteins form the structural nitrate assimilation gene cluster. Expression of these genes is putatively controlled by nitrogen catabolite repression and two Zn(II)2Cys6 transcription factors, which are encoded by YNA1 and YNA2. These two genes are not collocated with the structural nitrate assimilation gene cluster and form the regulatory nitrate assimilation gene cluster. This study explores sequence and ploidy variation at these two nitrate assimilation gene clusters in B. bruxellensis and relates this to variable nitrate assimilation potential across a cohort of forty-one B. bruxellensis strains isolated from a range of industrial fermentative sources. It was found that in some apparent nitrate-negative isolates a subset of the population can switch to a nitrate-positive phenotype, which could be an example of microbial bet-hedging. These strains shared a common haplotype of the structural nitrate assimilation gene cluster. A representative of this cohort of isolates, AWRI1608, was found to switch from nitrate-negative to nitrate-positive at a rate of approximately 1 in 105 cells during incubation on solid media with nitrate as the sole nitrogen source for 7 days. Nitrate-positive colonies were isolated from these plates and were shown to retain their nitrate-positive phenotype over 100 generations in non-selective media, but were near-isogenic with the original isolate and showed no mutations in or near nitrate assimilation genes. AWRI1608 and its nitrate-positive variants were used to develop a method for assessing competitive fitness between isolates using a newly-developed transformation protocol to mark strains with antibiotic-resistance. This study is the first to show nitrate assimilation phenotype heterogeneity in B. bruxellensis.
Advisor: Borneman, Anthony
Curtain, Chris
Dissertation Note: Thesis (MPhil) -- University of Adelaide, School of Biological Sciences, 2019
Keywords: Brettanomyces
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