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The Identification of Novel Diagnostic Marker Genes for the Detection of Beer Spoiling Pediococcus damnosus Strains Using the BlAst Diagnostic Gene findEr.

Behr J, Geissler AJ, Schmid J, Zehe A, Vogel RF - PLoS ONE (2016)

Bottom Line: DMG identification settings can be modified easily and installing and running BADGE does not require specific bioinformatics skills.On the basis of an example with relevance for beer brewing, being one of the oldest biotechnological processes known, we show a straightforward procedure, from phenotyping, genome sequencing, assembly and annotation, up to a discriminant marker gene PCR assay, making comparative genomics a means to an end.The value and the functionality of BADGE were thoroughly examined, resulting in the successful identification and validation of an outstanding novel DMG (fabZ) for the discrimination of harmless and harmful contaminations of Pediococcus damnosus, which can be applied for spoilage risk determination in breweries.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Freising, Germany.

ABSTRACT
As the number of bacterial genomes increases dramatically, the demand for easy to use tools with transparent functionality and comprehensible output for applied comparative genomics grows as well. We present BlAst Diagnostic Gene findEr (BADGE), a tool for the rapid prediction of diagnostic marker genes (DMGs) for the differentiation of bacterial groups (e.g. pathogenic / nonpathogenic). DMG identification settings can be modified easily and installing and running BADGE does not require specific bioinformatics skills. During the BADGE run the user is informed step by step about the DMG finding process, thus making it easy to evaluate the impact of chosen settings and options. On the basis of an example with relevance for beer brewing, being one of the oldest biotechnological processes known, we show a straightforward procedure, from phenotyping, genome sequencing, assembly and annotation, up to a discriminant marker gene PCR assay, making comparative genomics a means to an end. The value and the functionality of BADGE were thoroughly examined, resulting in the successful identification and validation of an outstanding novel DMG (fabZ) for the discrimination of harmless and harmful contaminations of Pediococcus damnosus, which can be applied for spoilage risk determination in breweries. Concomitantly, we present and compare five complete P. damnosus genomes sequenced in this study, finding that the ability to produce the unwanted, spoilage associated off-flavor diacetyl is a plasmid encoded trait in this important beer spoiling species.

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Related in: MedlinePlus

DMG evaluation and validation.20 strains of P. damnosus were tested for the presence of the predicted DMGs and already published DMGs using PCR. Note that the published DMGs horA and horC were not predicted by BADGE, as both genes are present in two of three non-spoiling genomes. Still, all strains were tested for their presence and results are shown for comparison. A positive reaction is indicated with a dot. Significant correlation to spoilage potential is indicated using the colors red (very high) and orange (moderate to high). Genome sequenced strains are underlined.
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pone.0152747.g004: DMG evaluation and validation.20 strains of P. damnosus were tested for the presence of the predicted DMGs and already published DMGs using PCR. Note that the published DMGs horA and horC were not predicted by BADGE, as both genes are present in two of three non-spoiling genomes. Still, all strains were tested for their presence and results are shown for comparison. A positive reaction is indicated with a dot. Significant correlation to spoilage potential is indicated using the colors red (very high) and orange (moderate to high). Genome sequenced strains are underlined.

Mentions: Fasta files containing all sequences of each chosen DMG were further processed with Clone Manager 9. DMG sequences were aligned and consensus sequences were generated. Based on the consensus sequence, DMG specific primer pairs were designed (S7 Table). These primers, as well as primers for the published DMGs horC, horA and hitA for comparison, were used for PCR evaluation and validation. 15 additional strains of P. damnosus with known spoilage potential were tested. Fig 4 illustrates the PCR results and shows that 5 of the 10 chosen DMGs are useful for the discrimination of beer spoiling and non-spoiling strains of P. damnosus. Table 4 shows the results of Spearman´s rank correlation and Fisher´s exact test for the tested DMGs. ParA, tetR, tnpA, npxA and especially fabZ show a significant relation to beer spoilage potential and spoilage ability. The established marker genes horC, horA and hitA did not show a significant relation to both classifications.


The Identification of Novel Diagnostic Marker Genes for the Detection of Beer Spoiling Pediococcus damnosus Strains Using the BlAst Diagnostic Gene findEr.

Behr J, Geissler AJ, Schmid J, Zehe A, Vogel RF - PLoS ONE (2016)

DMG evaluation and validation.20 strains of P. damnosus were tested for the presence of the predicted DMGs and already published DMGs using PCR. Note that the published DMGs horA and horC were not predicted by BADGE, as both genes are present in two of three non-spoiling genomes. Still, all strains were tested for their presence and results are shown for comparison. A positive reaction is indicated with a dot. Significant correlation to spoilage potential is indicated using the colors red (very high) and orange (moderate to high). Genome sequenced strains are underlined.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814128&req=5

pone.0152747.g004: DMG evaluation and validation.20 strains of P. damnosus were tested for the presence of the predicted DMGs and already published DMGs using PCR. Note that the published DMGs horA and horC were not predicted by BADGE, as both genes are present in two of three non-spoiling genomes. Still, all strains were tested for their presence and results are shown for comparison. A positive reaction is indicated with a dot. Significant correlation to spoilage potential is indicated using the colors red (very high) and orange (moderate to high). Genome sequenced strains are underlined.
Mentions: Fasta files containing all sequences of each chosen DMG were further processed with Clone Manager 9. DMG sequences were aligned and consensus sequences were generated. Based on the consensus sequence, DMG specific primer pairs were designed (S7 Table). These primers, as well as primers for the published DMGs horC, horA and hitA for comparison, were used for PCR evaluation and validation. 15 additional strains of P. damnosus with known spoilage potential were tested. Fig 4 illustrates the PCR results and shows that 5 of the 10 chosen DMGs are useful for the discrimination of beer spoiling and non-spoiling strains of P. damnosus. Table 4 shows the results of Spearman´s rank correlation and Fisher´s exact test for the tested DMGs. ParA, tetR, tnpA, npxA and especially fabZ show a significant relation to beer spoilage potential and spoilage ability. The established marker genes horC, horA and hitA did not show a significant relation to both classifications.

Bottom Line: DMG identification settings can be modified easily and installing and running BADGE does not require specific bioinformatics skills.On the basis of an example with relevance for beer brewing, being one of the oldest biotechnological processes known, we show a straightforward procedure, from phenotyping, genome sequencing, assembly and annotation, up to a discriminant marker gene PCR assay, making comparative genomics a means to an end.The value and the functionality of BADGE were thoroughly examined, resulting in the successful identification and validation of an outstanding novel DMG (fabZ) for the discrimination of harmless and harmful contaminations of Pediococcus damnosus, which can be applied for spoilage risk determination in breweries.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Freising, Germany.

ABSTRACT
As the number of bacterial genomes increases dramatically, the demand for easy to use tools with transparent functionality and comprehensible output for applied comparative genomics grows as well. We present BlAst Diagnostic Gene findEr (BADGE), a tool for the rapid prediction of diagnostic marker genes (DMGs) for the differentiation of bacterial groups (e.g. pathogenic / nonpathogenic). DMG identification settings can be modified easily and installing and running BADGE does not require specific bioinformatics skills. During the BADGE run the user is informed step by step about the DMG finding process, thus making it easy to evaluate the impact of chosen settings and options. On the basis of an example with relevance for beer brewing, being one of the oldest biotechnological processes known, we show a straightforward procedure, from phenotyping, genome sequencing, assembly and annotation, up to a discriminant marker gene PCR assay, making comparative genomics a means to an end. The value and the functionality of BADGE were thoroughly examined, resulting in the successful identification and validation of an outstanding novel DMG (fabZ) for the discrimination of harmless and harmful contaminations of Pediococcus damnosus, which can be applied for spoilage risk determination in breweries. Concomitantly, we present and compare five complete P. damnosus genomes sequenced in this study, finding that the ability to produce the unwanted, spoilage associated off-flavor diacetyl is a plasmid encoded trait in this important beer spoiling species.

Show MeSH
Related in: MedlinePlus