AbstractClostridial neurotoxins (CNTs), which include botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT), are the most potent toxins known to science and are the causative agents of botulism and tetanus, respectively. The evolutionary origins of CNTs and their relationships to other proteins remains an intriguing question. Here we present a large-scale bioinformatic screen for putative toxin genes in all currently available genomes. We detect a total of 311 protein sequences displaying at least partial homology to BoNTs, including 161 predicted toxin sequences that have never been characterized. We focus on a novel toxin family from Chryseobacterium piperi with homology to BoNTs. We resequenced the genome of C. piperi to confirm and further analyze the genomic context of these toxins, and also examined their potential toxicity by expression of the protease domain of one C. piperi toxin in human cells. Our analysis suggests that these C. piperi sequences encode a novel family of metalloprotease toxins that are distantly related to BoNTs with similar domain architecture. These toxins target a yet unknown class of substrates, potentially reflecting divergence in substrate specificity between the metalloprotease domains of these toxins and the related metalloprotease domain of clostridial neurotoxins.