Plasmid analysis, comparative genomics and transcriptomics of beer-spoilage lactic acid bacteria emphasizing the role of dissolved carbon dioxide and traditional beer-spoilage markers
Specific isolates of lactic acid bacteria (LAB) are capable of growing in and spoiling beer, and are the cause of product and process contamination, and financial loss for brewers the world over. To date, our understanding of how these contaminants are able to grow in beer is limited to analysis of hop-tolerance mechanisms, with a limited number of putative hop-tolerance genes having been described. In order to demonstrate that these hop-tolerance genes are incomplete descriptors of overall beer-spoilage ability, the transcriptional activity of these genes in two different beer-spoilage related (BSR) LAB isolates, and the prevalence and sequence conservation of hop-tolerance gene horC in BSR LAB with varying beer-spoilage ability is examined. This analysis is followed by work demonstrating that the total plasmid profile of a beer-spoilage LAB, and not just plasmids harboring hop-tolerance genes, contributes to the isolate’s overall beer-spoilage phenotype and highlights redundancy in potential beer-spoilage mechanisms. The next chapter provides evidence that the presence of dissolved CO2 (dCO2) in beer selects for the ability of LAB to spoil packaged beer, and that tolerance to this stress is not correlated with hop-tolerance, indicating that dCO2 stress is an important part of the total beer environment. This is followed by the presentation and analysis of the genome of the rapid beer-spoiling isolate Lactobacillus brevis BSO 464 and subsequent RNA sequencing for this isolate when grown in degassed and gassed beer so as to elucidate which genes are active when grown in beer, and when grown specifically in the presence of dCO2. Global transcriptome sequencing of this L. brevis isolate and Pediococcus claussenii ATCC BAA-344T when each were grown in growth-limiting concentrations of hops was also performed in order to clarify the hop-specific transcriptional response from that of the response when these isolates grow in the total beer environment. Lastly, comparison is made between available genomes of BSR LAB to reveal that the specific brewery environment a BSR LAB is recovered from, influences genetic variability and that comparison within a given LAB species reveals genetic differences that can be exploited as beer-spoilage genetic markers. This comparative analysis reveals that the total plasmid-coding capacity strongly influences individual BSR LAB beer-spoilage phenotype and the environment they are able to grow in. Overall, beer-spoilage ability is shown to be adaptive and acquired incrementally and not solely as a result of the presence of hop-tolerance genes.
Beer-spoilage, Brewing, Genomics, Hop-tolerance, Lactic acid bacteria, Plasmid analysis, RNA sequencing, Transcriptomics
Doctor of Philosophy (Ph.D.)