Previous studies have shown that holotoxins with reduced numbers of GM 1 BS are still able to intoxicate host cells, albeit with attenuated activity. The initial studies (26, 27) used chemical modification and denaturation-renaturation to generate mixed populations of holotoxins predicted to have one or two (nonnative but active) GM 1 BS. In our previous study (22) , we used genetic methods to create populations of chimeric holotoxins with only one or two completely native GM 1 BS and showed that these chimeras were still capable of intoxicating host cells but with attenuated activity.