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

Comparative genomic and plasmid analysis of beer-spoiling and non-beer-spoiling Lactobacillus brevis isolates

Beer-spoilage-related lactic acid bacteria (BSR LAB) belong to multiple genera and species; however, beer-spoilage capacity is isolate-specific and partially acquired via horizontal gene transfer within the brewing environment. Thus, the extent to which genus-, species- or environment- (i.e., brewery-) level genetic variability influences beer-spoilage phenotype is unknown. Publicly available Lactobacillus brevis genomes were analyzed via BlAst Diagnostic Gene findEr (BADGE) for BSR genes and assessed for pangenomic relationships. Also analyzed were functional coding capacities of plasmids of LAB inhabiting extreme niche environments. Considerable genetic variation was observed in L. brevis isolated from clinical samples, whereas 16 candidate genes distinguish BSR and non-BSR L. brevis genomes. These genes are related to nutrient scavenging of gluconate/pentoses, mannose, and metabolism of pectin. BSR L. brevis isolates also have higher average nucleotide identity and stronger pangenome association to one another, though isolation source (i.e., specific brewery) also appears to influence the plasmid coding capacity of BSR LAB. Finally, it is shown that niche-specific adaptation and phenotype are plasmid-encoded for both BSR and non-BSR LAB. The ultimate combination of plasmid-encoded genes dictates the ability of L. brevis to survive in the most extreme beer environment, namely, gassed/pressurized beer.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Next-generation sequencing approaches for improvement of lactic acid bacteria-fermented plant-based beverages

Plant-based beverages and milk alternatives produced from cereals and legumes have grown in popularity in recent years due to a range of consumer concerns over dairy products. These plant-based products can often have undesirable physiochemical properties related to flavour, texture, and nutrient availability and/or deficiencies. Lactic acid bacteria (LAB) fermentation offers potential remediation for many of these issues, and allows consumers to retain their perception of the resultant products as natural and additive-free. Using next-generation sequencing (NGS) or omics approaches to characterize LAB isolates to find those that will improve properties of plant-based beverages is the most direct way to product improvement. Although NGS/omics approaches have been extensively used for selection of LAB for use in the dairy industry, a comparable effort has not occurred for selecting LAB for fermenting plant raw substrates, save those used in producing wine and certain types of beer. Here we review the few and recent applications of NGS/omics to profile and improve LAB fermentation of various plant-based substrates for beverage production. We also identify specific issues in the production of various LAB fermented plant-based beverages that such NGS/omics applications have the power to resolve

Transcriptional activity and role of plasmids of <em>Lactobacillus brevis </em>BSO 464 and <em>Pediococcus claussenii </em>ATCC BAA-344T during growth in the presence of hops

Whole-transcriptome analysis was performed on beer-spoilage organisms Lactobacillus brevis BSO 464 (Lb464) and Pediococcus claussenii ATCC BAA-344T (Pc344) when grown in growth-limiting concentrations of hop extract. This was done to delineate the hops-specific component of the total transcriptional response for these bacteria when growing in beer. The transcriptome of highly hop-tolerant isolate Lb464 had fewer genes with differential expression in response to a stronger challenge (i.e., higher bitterness units) of hop extract than did Pc344, highlighting the variable nature of hop-tolerance in beer-spoilage-related lactic acid bacteria. As Lb464 can grow in pressurized/gassed beer and Pc344 cannot, this indicates that the genetic and physiological response to hops alone does not dictate the overall beer-spoilage virulence of an isolate. The general response to hops in both isolates involves pathways of acid tolerance and intracellular pH homeostasis, with glutamate and citrate metabolism, and biogenic amine metabolism as additional major responses to the presence of hop extract by Lb464 and Pc344, respectively. A Pc344 chromosomal ABC transporter (PECL_1630) was more strongly expressed than the plasmid-located, hop-tolerance ABC transporter horA. PECL_1630 is suggested to be involved in import of ATP into the cell, potentially assisting the total bacterial community when facing hop stress. This transporter is found in other beer-related P. claussenii suggesting a putative species-specific beer-spoilage-related genetic marker. Lb464 and Pc344 each contain eight plasmids and transcription from almost all occurs in response to both hops and beer. However, as evident by both transcriptional analysis and plasmid variant analysis, each bacterium harbors one plasmid that is critical for responding to hops and beer stress. For both bacteria, complex transcriptional regulation and cooperation between chromosomal and plasmid-based genes occurs in response to the growth challenges imposed by hops or beer

Author response: Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance

Distinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments. DOI: http://dx.doi.org/10.7554/eLife.04634.00

Detection of a hop-tolerance gene horA insertion variant in lactic acid bacteria that results in a truncated horA lacking the walker B motif necessary for transport function

<p>The hop-tolerance gene <i>horA</i> frequently found in beer-spoilage lactic acid bacteria (LAB) was investigated for sequence variability. Although the <i>horA</i> gene was found to have less sequence variability relative to the LAB hop-tolerance gene <i>horC</i>, a sequence insertion in <i>horA</i> in some isolates resulted in early truncation of HorA translation. This truncated HorA was found in LAB both capable and incapable of growth in beer. Protein modeling revealed that the truncated HorA may retain some capacity to bind and sequester hop iso-α-acids but lacks the transport function essential for moving hop compounds out of the cell. Sequence analysis of LAB plasmids that contain <i>horA</i> revealed a high level of conservation in all of the genes comprising the <i>horA</i> gene cassette. Assessing whether a LAB isolated in a brewery setting is capable of making a full-length protein with intact hop transport function or a truncated HorA requires redesign of commonly used <i>horA</i> polymerase chain reaction primers to target detection of <i>horA</i> both with and without the sequence insertion.</p

Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance.

Distinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments

Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance

Distinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments.ISSN:2050-084