Multiplex PCR for Putative<i>Lactobacillus</i>and<i>Pediococcus</i>Beer-Spoilage Genes and Ability of Gene Presence to Predict Growth in Beer

Haakensen, Monique; Schubert, Alison; Ziola, Barry

doi:10.1094/asbcj-2008-0314-01

Cited by 29 publications

(45 citation statements)

References 14 publications

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“…Despite several reports on the roles of hitA, horA, and horC in hop tolerance and beer spoilage ability in specific BSOs (14,15), these plasmid-based genes are not found in every BSO, nor are they in a consistent combination when present (16,17). This indicates that hop tolerance and, by extension, the overall ability to grow in beer are not mediated solely by the products of these three hop tolerance genes.…”

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confidence: 72%

“…The presence or absence of each of the eight L. brevis BSO 464 plasmids was determined through the use of two multiplex PCRs (see Table S1 in the supplemental material). Multiplex PCR B contained primers for the 16S rRNA gene to verify the presence of bacterial DNA (16,21). All primers were designed and ordered using the Oligoanalyzer program provided by Integrated DNA Technology (IDT).…”

Section: Plasmid Variantsmentioning

confidence: 99%

“…The hop resistance genes are proposed to counteract the action of hops by removing iso-␣-acids from the cell through ATP-binding-cassette-type multidrug resistance transporters, such as HorA (9), or through PMF-dependent multidrug transporters, such as HorC (10) and its transcriptional regulator HorB (14). Involvement of HitA in hop resistance is believed to be through divalent cation transport (11).Despite several reports on the roles of hitA, horA, and horC in hop tolerance and beer spoilage ability in specific BSOs (14, 15), these plasmid-based genes are not found in every BSO, nor are they in a consistent combination when present (16,17). This indicates that hop tolerance and, by extension, the overall ability to grow in beer are not mediated solely by the products of these three …”

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confidence: 99%

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Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

Bergsveinson

Baecker

Pittet

et al. 2015

Appl Environ Microbiol

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Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate. Beer is a very harsh environment for bacterial growth due to unique physiochemical attributes. Specifically, the presence of antimicrobial hop compounds (15 to 55 ppm iso-␣-acids) and ethanol (0.5 to 14% [vol/vol]) (1), coupled with the low availability of nutrients, a microaerophilic environment, low pH, and, finally, pressure in the packaged product due to CO 2 content, together mean that contaminating microorganisms must employ compensatory mechanisms to survive in the milieu. When successful microbial growth in beer does occur, the result is a spoiled product with unappealing off flavors and an altered sensory profile, ultimately leading to issues with brand confidence and economic loss for brewers globally (2).Isolated beer-spoiling organisms (BSOs) frequently belong to the genus Lactobacillus or Pediococcus, both of which are classified as lactic acid bacteria (LAB) (3, 4). The ability to spoil beer, however, is not intrinsic to all members of a given species in each genus and is instead isolate specific. This indicates that BSOs have undergone a degree of genetic specialization relative to non-BSO LAB isolates of the same genus and species. LAB are a diverse group of organisms, and a given species can be isolated from a number of environments. For example, Lactobacillus plantarum isolates can be found, not only in spoiled beer, but also in spoiled meats and the human intestinal microbiome (5). Due to the varied nature of these environments and the frequency with which specific bacteri...

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confidence: 72%

Section: Plasmid Variantsmentioning

confidence: 99%

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confidence: 99%

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Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

Bergsveinson

Baecker

Pittet

et al. 2015

Appl Environ Microbiol

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“…In our case, we became interested in a particular L. rhamnosus isolate due to its ability to spoil beer. Among five L. rhamnosus isolates tested, only one (ATCC 8530) was able to grow in beer (1). We therefore performed further analyses on this isolate and found that it was among a few lactic acid bacteria (LAB) that showed increased resistance to hops in the presence of ethanol (2).…”

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Genome Sequence of Lactobacillus rhamnosus ATCC 8530

et al. 2012

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Lactobacillus rhamnosus is found in the human gastrointestinal tract and is important for probiotics. We became interested in L. rhamnosus isolate ATCC 8530 in relation to beer spoilage and hops resistance. We report here the genome sequence of this isolate, along with a brief comparison to other available L. rhamnosus genome sequences. Lactobacillus rhamnosus is an organism of interest for human health and probiotics. In our case, we became interested in a particular L. rhamnosus isolate due to its ability to spoil beer. Among five L. rhamnosus isolates tested, only one (ATCC 8530) was able to grow in beer (1). We therefore performed further analyses on this isolate and found that it was among a few lactic acid bacteria (LAB) that showed increased resistance to hops in the presence of ethanol (2). This finding was particularly intriguing considering the antimicrobial properties of both compounds and because hops has been suggested as a replacement for antibiotics to control LAB contamination in the fuel ethanol industry (6). To get a better understanding of how L. rhamnosus ATCC 8530 grows in beer and resists hops, we sequenced its genome for comparative analysis with other sequenced LAB.Sequencing was done using the Roche 454 Genome Sequence FLX platform. Paired and unpaired reads were obtained during two different runs, giving a final coverage of ϳ30ϫ. A total of 520,000 reads were assembled using Newbler GS De Novo assembler, giving 34 contigs split among six scaffolds. The scaffolds were then ordered according to the L. rhamnosus Lc 705 genome, and gaps were closed by sequencing PCR amplicons via the ABI 3700xl platform. The final circularized genome is 2,960,339 bp with a GϩC content of 46.8%.Genome annotation was done via the Institute for Genome Sciences Annotation Engine, which predicted 2,904 coding sequences (CDS), 60 tRNA, and five rRNA operons. Of the predicted CDS, ϳ700 are hypothetical proteins with unknown function. Unlike L. rhamnosus Lc 705 and HN001, no plasmids are found in this isolate.Comparative analysis with L. rhamnosus ATCC 8530 and the four other currently available L. rhamnosus genome sequences (3, 4) showed that L. rhamnosus ATCC 8530 is least similar to the probiotic isolates GG and HN001. In contrast, the intestinal isolate L. rhamnosus LMS2-1 is very similar to ATCC 8530, with most (Ͼ90%) predicted proteins having Ն99% identity. Preliminary analyses indicate that only four regions are different between L. rhamnosus ATCC 8530 and LMS2-1, all related to mobile elements (L. rhamnosus ATCC 8530 has no predicted bacteriophagerelated genes). The majority of L. rhamnosus ATCC 8530 was also conserved with the dairy industry isolate L. rhamnosus Lc 705, except for one distinct location missing in the former isolate and several bacteriophage-related regions not found in the latter isolate, respectively.Considering the high degree of similarity between L. rhamnosus ATCC 8530 and LMS2-1, further analyses will be done to determine if the latter isolate can grow in beer, as well as to show a co...

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“…The type strain (ATCC BAA-344), which we sequenced, produces exopolysaccharide (referred to as rope, or slime), which may be involved in biofilm formation and persistence in a brewery. This bacterium spoils beer in 7 to 8 days and contains the known beer spoilage-associated gene horA (7). Ethanol tolerance for lactic acid bacteria is midrange (9), and hops resistance is high (V. Pittet, B. R. Bushell, and B. Ziola, unpublished data).…”

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confidence: 99%

Complete Genome Sequence of the Beer Spoilage Organism Pediococcus claussenii ATCC BAA-344T

Pittet

Abegunde

Marfleet

et al. 2012

Journal of Bacteriology

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Multiplex PCR for PutativeLactobacillusandPediococcusBeer-Spoilage Genes and Ability of Gene Presence to Predict Growth in Beer

Cited by 29 publications

References 14 publications

Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

Genome Sequence of Lactobacillus rhamnosus ATCC 8530

Complete Genome Sequence of the Beer Spoilage Organism Pediococcus claussenii ATCC BAA-344T

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