Dataset Information

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

ABSTRACT: Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containing GH48 gene sequences directly linked to the corresponding taxonomic information. Phylogenetic correlation analysis of GH48 to 16S rRNA gene sequences revealed that GH48 gene sequences with 94% identity belong with high confidence to the same genus. Applying this analysis, GH48 amplicon reads revealed that at mesophilic fermenter conditions, 50-99% of the OTUs appear to belong to novel taxa. In contrast, at thermophilic conditions, GH48 gene sequences from the genus Hungateiclostridium dominated with 60-91% relative abundance. The novel primer combinations enabled detection and relative quantification of a wide spectrum of GH48 genes in cellulolytic microbial communities. Deep phylogenetic correlation analysis and a simplified taxonomic identification with the novel database facilitate identification of cellulolytic organisms, including the detection of novel taxa in biogas fermenters.

SUBMITTER: Rettenmaier R

PROVIDER: S-EPMC7565076 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Publications

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

Rettenmaier Regina R Lo Yat Kei YK Schmidt Larissa L Munk Bernhard B Lagkouvardos Ilias I Neuhaus Klaus K Schwarz Wolfgang W Liebl Wolfgang W Zverlov Vladimir V

Microorganisms 20200825 9

Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containin ...[more]

PMID: 32854333

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Project description:Genes belonging to the same functional group may include numerous and variable gene sequences, making characterizing and quantifying difficult. Therefore, high-throughput design tools are needed to simultaneously create primers for improved quantification of target genes. We developed MetaFunPrimer, a bioinformatic pipeline, to design primers for numerous genes of interest. This tool also enables gene target prioritization based on ranking the presence of genes in user-defined references, such as environment-specific metagenomes. Given inputs of protein and nucleotide sequences for gene targets of interest and an accompanying set of reference metagenomes or genomes, MetaFunPrimer generates primers for ranked genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 publicly available soil metagenomes. We demonstrate computationally that these amoA-AOB primers can cover 94% of the amoA-AOB genes observed in the 1,550 soil metagenomes compared with a 49% estimated coverage by previously published primers. Finally, we verified the utility of these primer sets in incubation experiments that used long-term nitrogen fertilized or unfertilized soils. High-throughput quantitative PCR (qPCR) results and statistical analyses showed significant differences in relative quantification patterns between the two soils, and subsequent absolute quantifications also confirmed that target genes enumerated by six selected primer pairs were significantly more abundant in the nitrogen-fertilized soils. This new tool gives microbial ecologists a new approach to assess functional gene abundance and related microbial community dynamics quickly and affordably. IMPORTANCE Amplification-based gene characterization allows for sensitive and specific quantification of functional genes. There is often a large diversity of genes represented for functional gene groups, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high-throughput primer design for various genes of interest and also allows for ranking gene targets by their presence and abundance in environmental data sets. Primers designed by this tool improve the characterization and quantification of functional genes in broad gene amplification platforms and can be powerful with high-throughput qPCR approaches.

Dataset Information

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

Publications

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

Similar Datasets

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