The goal of my research was to address current limitations in the understanding of BSR LAB within the brewing industry – specifically, the incomplete working model of BSR LAB stress mechanisms, the poor understanding of the role that plasmids play in beer-spoilage ability, and the under-appreciation of the power of next-generation sequencing (NGS) technologies to investigate these questions. The central hypotheses which guided the experimental work for this thesis are: (1) that the genetics of hop-tolerance is variable in BSR LAB, with the few traditional hop-tolerance genes not being adequate predictors of either hop-tolerance or beer-spoilage ability and (2) that the beer-spoilage phenotype is variable and influenced by both the total genetic content of BSR LAB and other physiological stresses found in beer besides hop-stress.
I began by investigating the differences in transcriptional activity and prevalence of known hop-stress tolerance genes in BSR LAB (Chapters 2 and 3). As hop-tolerance genes are known to be located on plasmids, the role that the entire plasmid profile of a BSR LAB has on its
beer-29
spoilage ability was also examined (Chapter 4) in order to elucidate if other plasmids that do not harbor hop-tolerance genes are also important. In addition, the presence and/or absence of dissolved CO2 (dCO2) in beer was explored to determine its affect on BSR LAB’s ability to grow in beer, and the relationship of dCO2 stress with other beer stresses and BSR LAB stress-tolerance mechanisms (Chapter 5). The NGS technology of RNAseq (sequencing of RNA transcripts) was used to profile the transcriptional response of a dCO2-tolerant BSR LAB organism when grown in beer with or without the presence of dCO2 (Chapter 6). This technology was also used to determine the transcriptional response of two unique BSR LAB in response to growth limiting concentrations of hops, in hopes of separating and defining the concepts of hop-tolerance and beer-spoilage (Chapter 7). Lastly, large-scale genomic comparisons were made amongst beer spoiling and non-beer-spoiling Lactobacillus brevis and other lactic acid bacteria isolates with available genomes, in order to help corroborate suspected beer-spoilage-specific genetic elements and identify the degree of genetic similarity amongst BSR LAB (Chapter 8).
My data in toto point to the need for the brewing industry to perform detailed examination of other beer-stresses apart from hops (e.g., dCO2) on BSR LAB and to detail how these stresses interact and synergistically affect BSR LAB. My results also call attention to the power of NGS technologies to answer questions of (i) what genetic elements must be expressed; i.e., are critical for growth in different beer and brewery conditions and (ii) how prevalent these genes are amongst beer-spoiling and non-beer-spoiling organisms and, thus, how useful they may be for detection approaches as related to microbial quality control in a brewery setting.
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