Computational protocol: Draft Genome Sequence of the Strict Anaerobe Clostridium neopropionicum X4 (DSM 3847T)

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Protocol publication

[…] Clostridium neopropionicum X4 was first isolated in 1982 from a mesophilic industrial anaerobic digester treating vegetable cannery wastewaters (). Later, it was further characterized as an obligately anaerobic endospore-forming bacterium, being the first representative of propionate fermentation from ethanol with simultaneous acetate production (, ). Tholozan et al. () reported the formation of [2-13C]propionate, [1-13C]acetate, [2-13C]propanol, and traces of [3-13C]butyrate as products of [1-13C]ethanol fermentation, suggesting the absence of a randomizing pathway (). The respective reactions of ethanol conversion to these metabolic end products are all exergonic (). Meanwhile, C. neopropionicum was suggested to be assigned to the genus Tyzzerella ().The MasterPure complete DNA purification kit (Epicentre, Madison, WI, USA) was used to isolate chromosomal DNA. Illumina Nextera XT shotgun sequencing libraries were generated from the extracted DNA, and sequencing was performed with an Illumina MiSeq machine, as recommended by the manufacturer (Illumina, San Diego, CA, USA). Sequencing resulted in 2,669,816 300-bp paired-end reads. Reads were trimmed using Trimmomatic 0.35 () to remove sequences with quality scores <20 (Illumina 1.9 encoding) and adaptor sequences. The remaining 2,530,048 reads were used for the de novo assembly performed with the SPAdes genome assembler software version 3.6.2 (). The assembly resulted in 29 contigs (>500 bp) and an average coverage of 182-fold. The genome of C. neopropionicum probably consists of a single circular chromosome (3.19 Mb), with an overall G+C content of 40.17%.Automatic gene prediction was performed by using the software tool Prodigal (). Genes coding for rRNA and tRNA were identified using RNAmmer () and tRNAscan (), respectively. The Integrated Microbial Genomes-Expert Review (IMG-ER) system () was used for automatic annotation, which was subsequently manually curated by using the Swiss-Prot, TrEMBL, and InterPro databases ().The genome harbored 6 rRNA genes, 58 tRNA genes, 2,090 protein-coding genes with predicted functions, and 839 genes coding for hypothetical proteins. Genome analysis revealed the presence of several genes encoding alcohol dehydrogenases, including also an adhE gene (CLNEO_13930), which codes for a bifunctional aldehyde/alcohol dehydrogenase and a gene (CLNEO_02960) that putatively represents the NADP+-dependent ethanol dehydrogenase gene postulated by Tholozan et al. (). Genes encoding key enzymes of the acrylate pathway (propionate-coenzyme A [CoA] transferase [Pct], lactoyl-CoA dehydratase [Lcd], and acrylyl-CoA reductase [Acr]) were also identified and show high sequence similarity to the corresponding genes described for Clostridium propionicum. These genes are organized in two clusters (lcdCAB-pct and acrCBA), which showed an arrangement identical to that in C. propionicum (). Whole-genome comparison based on computed average nucleotide identity (ANI) revealed a similarity of >80% between C. neopropionicum and C. propionicum, which is clearly in contrast to earlier findings of only 18% DNA/DNA homology (). However, our analysis and former 16S rRNA gene-based studies () suggest that C. neopropionicum and C. propionicum are highly related. […]

Pipeline specifications

Software tools Trimmomatic, SPAdes, Prodigal, RNAmmer, tRNAscan-SE
Applications Genome annotation, WGS analysis
Chemicals Ethanol, 1-Propanol, Carbon Dioxide