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Growth of bacteria in beer leads to turbidity and off-flavors, resulting in a spoiled and unpalatable product and thus economic loss. The most common beer-spoilage organisms (BSOs) are lactic acid bacteria (LAB), with Lactobacillus and Pediococcus species being the most problematic. Because of the harsh environment (low nutrients, antimicrobial compounds ethanol and hops, anaerobic), only select isolates are able to sustain growth in and spoil beer. To begin understanding the phenomenon of LAB adapting to overcome stresses in beer, ethanol tolerance, hop resistance, and nutrient acquisition mechanisms were investigated. First, ethanol tolerance was analyzed in the context of beer-spoilage ability, and it was found that it is intrinsically high in LAB, thus leading to the conclusion that LAB ability to spoil beer is not dependent on ethanol resistance levels. This was then followed by genome sequencing of the BSO Pediococcus claussenii ATCC BAA-344T (Pc344) to elucidate mechanisms being used to resist hops and acquire low abundance or alternative nutrients. Subsequent analysis of Pc344 and Lactobacillus brevis BSO 464 via reverse transcription quantitative PCR demonstrated the variability found among BSOs in the presence of beer-spoilage-related genes and their use during growth in beer. Further analysis of Pc344 was performed via RNA-sequencing to get a global view of gene expression during mid-logarithmic growth in beer. It was found that several alternative nutrients were being used by Pc344 to sustain growth, and that hop resistance was enabled by a variety of mechanisms including oxidative stress response and pH control. Finally, genomic comparison of BSOs determined that conservation is only present for closely related organisms and that no specific genes/proteins are indicative of an isolate’s beer-spoilage potential. It is more likely that horizontal gene transfer plays a major role in LAB adaption for growth in beer, and that plasmids are very important for this evolution, as was demonstrated by plasmid-variants of Pc344. The main conclusions of this thesis are therefore that hop resistance is the main factor determining ability to grow in beer, and that transfer of genetic elements is the driving force behind LAB evolving into BSOs.

The brewing industry has considerable economic impact worldwide; therefore, demand exists for a better understanding of the organisms that cause beer-spoilage. Low nutrient levels, depleted oxygen levels, high alcohol levels, and the presence of antimicrobial hop compounds all play a role in making beer an inhospitable environment for most microorganisms. Nonetheless, there are bacteria that are resistant to all of these selective pressures. The most common beer-spoilage bacteria are the Gram-positive lactic acid bacteria <i>Lactobacillus</i> and <i>Pediococcus</i>. It is currently believed that hop-resistance is the key factor(s) permitting <i>Lactobacillus</i> and <i>Pediococcus</i> bacteria to grow in beer. However, it is likely that in addition, ethanol-tolerance and the ability to acquire nutrients also play roles in the beer-spoilage ability of <i>Lactobacillus</i> and <i>Pediococcus</i> isolates. The ability of <i>Lactobacillus</i> and <i>Pediococcus</i> to grow in beer was assessed and correlated to the presence of previously described beer-spoilage related genes, as well as with the presence of novel genes identified in this study. Molecular and culture-based techniques for detection and differentiation between <i>Lactobacillus</i> and <i>Pediococcus</i> isolates that can and cannot grow in beer were established and described in detail. Interestingly, beer-spoilage related proteins were often found to share homology with multi-drug transporters. As such, the presence of these beer-spoilage associated genes was also compared to the ability of isolates to grow in the presence of a variety of antibiotics and, unexpectedly, beer-spoiling bacteria were found to be more susceptible to antibiotics than were non beer-spoiling isolates of the same genus. Additionally, it was found that isolates of <i>Lactobacillus</i> and <i>Pediococcus</i> that can grow in beer do not group phylogenetically. In order to fully appreciate the relationship of speciation with beer-spoilage, phylogenetic and whole genome/proteome studies were conducted to clarify the taxonomy of the <i>Lactobacillus</i> and <i>Pediococcus</i> genera. Through the research in this thesis, a greater understanding of the mechanism(s) enabling bacteria to grow in beer has been gained and taxonomy of the genera <i>Lactobacillus</i> and <i>Pediococcus</i> has been clarified.

The brewing industry has considerable economic impact worldwide; therefore, demand exists for a better understanding of the organisms that cause beer-spoilage. Low nutrient levels, depleted oxygen levels, high alcohol levels, and the presence of antimicrobial hop compounds all play a role in making beer an inhospitable environment for most microorganisms. Nonetheless, there are bacteria that are resistant to all of these selective pressures. The most common beer-spoilage bacteria are the Gram-positive lactic acid bacteria <i>Lactobacillus</i> and <i>Pediococcus</i>. It is currently believed that hop-resistance is the key factor(s) permitting <i>Lactobacillus</i> and <i>Pediococcus</i> bacteria to grow in beer. However, it is likely that in addition, ethanol-tolerance and the ability to acquire nutrients also play roles in the beer-spoilage ability of <i>Lactobacillus</i> and <i>Pediococcus</i> isolates. The ability of <i>Lactobacillus</i> and <i>Pediococcus</i> to grow in beer was assessed and correlated to the presence of previously described beer-spoilage related genes, as well as with the presence of novel genes identified in this study. Molecular and culture-based techniques for detection and differentiation between <i>Lactobacillus</i> and <i>Pediococcus</i> isolates that can and cannot grow in beer were established and described in detail. Interestingly, beer-spoilage related proteins were often found to share homology with multi-drug transporters. As such, the presence of these beer-spoilage associated genes was also compared to the ability of isolates to grow in the presence of a variety of antibiotics and, unexpectedly, beer-spoiling bacteria were found to be more susceptible to antibiotics than were non beer-spoiling isolates of the same genus. Additionally, it was found that isolates of <i>Lactobacillus</i> and <i>Pediococcus</i> that can grow in beer do not group phylogenetically. In order to fully appreciate the relationship of speciation with beer-spoilage, phylogenetic and whole genome/proteome studies were conducted to clarify the taxonomy of the <i>Lactobacillus</i> and <i>Pediococcus</i> genera. Through the research in this thesis, a greater understanding of the mechanism(s) enabling bacteria to grow in beer has been gained and taxonomy of the genera <i>Lactobacillus</i> and <i>Pediococcus</i> has been clarified.