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 years 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 U.S. 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 AHAs closest relatives and to infer the most recent common ancestor (MRCA) shared with its nearest wild 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 occurred early in the lineages 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 wild 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 jointly shape bacterial evolution, and they offer a general framework for understanding domestication and laboratory adaptation across microbial systems. SignificanceHuman-mediated species domestication has been central to the development of civilization, with well-known examples in plants and animals. Microbes have also been domesticated, often unintentionally and before the advent of modern microbiology. The military and pharmaceutical applications of botulinum neurotoxin led to the sustained cultivation and eventual domestication of a high toxin-producing Clostridium botulinum strain that remains widely used today. We reconstruct the genomic changes along this trajectory and identify a key early mutation that generated a hypermutator phenotype, increasing genetic variation available for human-directed selection. These findings reveal fundamental evolutionary processes shaping bacterial genomes under long-term human control. This genome-resolved example of microbial domestication offers a general framework for understanding laboratory adaptation in both evolutionary and applied microbiology.