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Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum


Craig Woods

Claudio Tomi-Andrino

Anne M. Henstra


Sean D. Simpson


The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.
IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.


Woods, C., Humphreys, C. M., Tomi-Andrino, C., Henstra, A. M., Köpke, M., Simpson, S. D., …Minton, N. P. (2022). Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum. Applied and Environmental Microbiology, 88(7),

Journal Article Type Article
Acceptance Date Feb 13, 2022
Online Publication Date Mar 14, 2022
Publication Date 2022-04
Deposit Date Feb 24, 2022
Publicly Available Date Sep 15, 2022
Journal Applied and Environmental Microbiology
Print ISSN 0099-2240
Electronic ISSN 1098-5336
Publisher American Society for Microbiology
Peer Reviewed Peer Reviewed
Volume 88
Issue 7
Public URL
Publisher URL


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