The historical domestication of a Clostridium botulinum strain used for the industrial production of botulinum neurotoxin

Laboratory production of botulinum neurotoxin (BoNT) began more than 90 y ago for medical, military, and later pharmaceutical applications, creating one of the longest-running examples of microbial cultivation under sustained human control. A single Clostridium botulinum Group I lineage, Army Hall A (AHA), was established by the US military in 1942 and later gave rise to Hall A-hyper (HAH), the strain widely used for pharmaceutical BoNT production. We analyzed more than 1,000 C. botulinum genomes to identify AHA’s closest relatives and to infer the most recent common ancestor (MRCA) shared with its nearest lineage. Relative to this MRCA, AHA accumulated nearly 100 genetic changes, including 46 single-nucleotide substitutions, 44 small insertions or deletions, and several large deletions and structural variants that led to the loss of more than 80 genes. A nonsense mutation in mutS generated a hypermutator phenotype that accelerated mutation rates and increased genetic diversity. This event is inferred to have occurred early in the lineage’s domestication and appears to have facilitated laboratory-adaptive traits, including the lack of sporulation and increased BoNT yield. Competition experiments under standard growth conditions confirmed substantial laboratory adaptation, with AHA exhibiting a strong fitness advantage over a close relative. Together, these results reconstruct the genomic trajectory of a bacterium evolving under prolonged human-mediated selection and provide a genome-resolved example of microbial domestication. The findings show how laboratory conditions, industrial selection, and changing mutation rates can shape bacterial evolution, and they offer a general framework for understanding domestication and laboratory adaptation across microbial systems.