Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia

Abstract

BackgroundClostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published.ResultsA closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G + C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a phophoenolpyruvate synthase) and substrate utilization pathway (mannose and aromatics utilization) that might explain phenotypic differences between C. autoethanogenum and C. ljungdahlii.ConclusionsSingle molecule sequencing will be increasingly used to produce finished microbial genomes. The complete genome will facilitate comparative genomics and functional genomics and support future comparisons between Clostridia and studies that examine the evolution of plasmids, bacteriophage and CRISPR systems.

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

“…The 16S RNA sequences between the three strains are 99% similar; also, the gene arrangement and sequence in the Wood-Ljungdahl operon are almost identical (6). For C. autoethanogenum (7,8) and C. ljungdahlii (9), complete genome sequences are available, and a comparative genomic analysis suggested some key differences (7). C. autoethanogenum can grow on CO as the sole carbon and energy source, forming mainly ethanol and acetic acid but also 2,3-butanediol, lactic acid, and some H 2 as fermentation products (6,10).…”

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

“…Because of infrequent homologous recombination in C. acetobutylicum, the implementation of an effective screening tool, such as CRISPR, for the selection of edited cells can enhance screening of mutant cells. CRISPR systems have been identified in ϳ70% of members of Clostridium, including the solventogenic clostridia (30,48,60). While a native CRISPR system has been recently unveiled in C. acetobutylicum strain GXAS18-1 (61), no such homologs are detected in C. acetobutylicum strains DSM792, DSM1731, or EA2018 (60).…”

“…CRISPR systems have been identified in ϳ70% of members of Clostridium, including the solventogenic clostridia (30,48,60). While a native CRISPR system has been recently unveiled in C. acetobutylicum strain GXAS18-1 (61), no such homologs are detected in C. acetobutylicum strains DSM792, DSM1731, or EA2018 (60). Using the editing template of a double-stranded DNA in a plasmid containing the SpCRISPR-Cas9 machinery to replace cac1502 via DCHR, site-specific genome editing was successfully demonstrated.…”

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

“…In the genome of the acetogens Clostridium ljungdahlii (15) and Clostridium autoethanogenum (79), which like A. woodii lack cytochromes and menaquinone but are not sodium ion dependent, the metVF genes are associated with neither an rnfC2 gene nor with hdrABC or mvhD genes. Cell extracts of C. autoethanogenum do not catalyze the reduction of methylene-H 4 F with NADH or NADPH (18).…”