Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

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