The ability to form botulinum neurotoxin is restricted to six phylogenetically 
and physiologically distinct bacteria (Clostridium botulinum Groups I-IV and 
some strains of C. baratii and C. butyricum). The botulinum neurotoxin is the 
most potent toxin known, with as little as 30-100 ng potentially fatal, and is 
responsible for botulism, a severe neuroparalytic disease that affects humans, 
animals, and birds. In order to minimize the hazards presented by the botulinum 
neurotoxin-forming clostridia, it is necessary to extend understanding of the 
biology of these bacteria. Analyses of recently available genome sequences in 
conjunction with studies of bacterial physiology are beginning to reveal new and 
exciting information on the biology of these dangerous bacteria. At the whole 
organism level, substantial differences between the six botulinum 
neurotoxin-forming clostridia have been reported. For example, the genomes of 
proteolytic C. botulinum (C. botulinum Group I) and non-proteolytic C. botulinum 
(C. botulinum Group II) are highly diverged and show neither synteny nor 
homology. It has also emerged that the botulinum neurotoxin-forming clostridia 
are not overtly pathogenic (unlike C. difficile), but saprophytic bacteria that 
use the neurotoxin to kill a host and create a source of nutrients. One 
important feature that has contributed to the success of botulinum 
neurotoxin-forming clostridia is their ability to form highly resistant 
endospores. The spores, however, also present an opportunity to control these 
bacteria if escape from lag phase (and hence growth) can be prevented. This is 
dependent on extending understanding of the biology of these processes. 
Differences in the genetics and physiology of spore germination in proteolytic 
C. botulinum and non-proteolytic C. botulinum have been identified. The 
biological variability in lag phase and its stages has been described for 
individual spores, and it has been shown that various adverse treatments extend 
different stages of lag phase. For example, heat treatment primarily extended 
germination, while incubation at a chilled temperature primarily extended 
outgrowth. The neurotoxin gene is present within a cluster of associated genes, 
and can be located on the chromosome, a plasmid or a bacteriophage. Two basic 
types of neurotoxin cluster have been identified. Evolution of the neurotoxin 
gene and cluster has occurred independently of the organism, and involved a 
series of recombination events but is still poorly understood. Factors affecting 
the regulation of neurotoxin formation also remain poorly understood, and will 
be the focus of much future research.