Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia
Steven D. Brown,Shilpa Nagaraju,Sgar Utturkar,Sashini De Tissera,Simón Segovia,Wayne Michell,Miriam L. Land,Asela Dassanayake,Michael Köpke
2014 March 21 Biotechnology for Biofuels 2014, 7:40
Clostridium 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.
Results A 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.
Single 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.