Susceptibility of Pediococcus isolates to antimicrobial compounds in relation to hop-resistance and beer-spoilage

<p>Abstract</p> <p>Background</p> <p>Though important in the context of food microbiology and as potential pathogens in immuno-compromised humans, bacterial isolates belonging to the genus <it>Pediococcus </it>are best known for their association with contamination of ethanol fermentation processes (beer, wine, or fuel ethanol). Use of antimicrobial compounds (e.g., hop-compounds, Penicillin) by some industries to combat <it>Pediococcus </it>contaminants is long-standing, yet knowledge about the resistance of pediococci to antimicrobial agents is minimal. Here we examined <it>Pediococcus </it>isolates to determine whether antibiotic resistance is associated with resistance to hops, presence of genes known to correlate with beer spoilage, or with ability to grow in beer.</p> <p>Results</p> <p>Lactic acid bacteria susceptibility test broth medium (LSM) used in combination with commercially available GPN3F antimicrobial susceptibility plates was an effective method for assessing antimicrobial susceptibility of <it>Pediococcus </it>isolates. We report the finding of Vancomycin-susceptible <it>Pediococcus </it>isolates from four species. Interestingly, we found that hop-resistant, beer-spoilage, and beer-spoilage gene-harbouring isolates had a tendency to be more susceptible, rather than more resistant, to antimicrobial compounds.</p> <p>Conclusion</p> <p>Our findings indicate that the mechanisms involved in conferring hop-resistance or ability to spoil beer by <it>Pediococcus </it>isolates are not associated with resistance to antibiotics commonly used for treatment of human infections. Also, Vancomycin-resistance was found to be isolate-specific and not intrinsic to the genus as previously believed.</p

Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13–Mms2 is required for DNA damage repair but not nuclear factor κB (NF-κB) activation, whereas Ubc13–Uev1A is involved in NF-κB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination

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

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.ISSN:2050-084

HPLC analysis of beer during Pc344-358 growth.

<p>Estimated concentrations of each compound were determined over time and plate counts were used to measure bacterial growth. Triplicate growth curves were analyzed, and standard deviations are indicated with error bars. Cellobiose and maltose could not be differentiated on the HPLC column, and are thus grouped together. Dextrin, ethanol, and glucose data are not included, as no change in concentration was found, or it was too low to detect (i.e., glucose).</p

Transcriptome Sequence and Plasmid Copy Number Analysis of the Brewery Isolate <i>Pediococcus</i><i> claussenii</i> ATCC BAA-344^T during Growth in Beer

<div><p>Growth of specific lactic acid bacteria in beer leads to spoiled product and economic loss for the brewing industry. Microbial growth is typically inhibited by the combined stresses found in beer (e.g., ethanol, hops, low pH, minimal nutrients); however, certain bacteria have adapted to grow in this harsh environment. Considering little is known about the mechanisms used by bacteria to grow in and spoil beer, transcriptome sequencing was performed on a variant of the beer-spoilage organism <i>Pediococcus</i><i>claussenii</i> ATCC BAA-344^T (Pc344-358). Illumina sequencing was used to compare the transcript levels in Pc344-358 growing mid-exponentially in beer to those in nutrient-rich MRS broth. Various operons demonstrated high gene expression in beer, several of which are involved in nutrient acquisition and overcoming the inhibitory effects of hop compounds. As well, genes functioning in cell membrane modification and biosynthesis demonstrated significantly higher transcript levels in Pc344-358 growing in beer. Three plasmids had the majority of their genes showing increased transcript levels in beer, whereas the two cryptic plasmids showed slightly decreased gene expression. Follow-up analysis of plasmid copy number in both growth environments revealed similar trends, where more copies of the three non-cryptic plasmids were found in Pc344-358 growing in beer. Transcriptome sequencing also enabled the addition of several genes to the <i>P</i><i>. claussenii</i> ATCC BAA-344^T genome annotation, some of which are putatively transcribed as non-coding RNAs. The sequencing results not only provide the first transcriptome description of a beer-spoilage organism while growing in beer, but they also highlight several targets for future exploration, including genes that may have a role in the general stress response of lactic acid bacteria. </p> </div