Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans. A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C. perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M. alligatoris genome. The nagH gene was detected by PCR in the closest relative of M. alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters. The hyaluronidase activity in the cellular fraction of M. alligatoris and M. crocodyli SP4 broth cultures was equivalent to 10 ؊16 U of Streptomyces hyalurolyticus hyaluronidase CFU ؊1 . Negligible activity was present in the cell-free supernatant fraction. No chondroitinase activity was detected. There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C. perfringens, in the M. alligatoris genome. The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C. perfringens is absent. The gene was not detected by PCR in any other mycoplasma. Potent cell-associated sialidase activity was present in M. alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M. crocodyli. The presence of hyaluronidase and sialidase in M. alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M. crocodyli is consistent with its comparatively attenuated virulence. This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M. alligatoris.Mycoplasma alligatoris causes a lethal invasive disease in adult alligators (Alligator mississippiensis) and closely related caimans (Caiman latirostris). The pathology observed as early as 1 week after infection by instillation via the glottis includes necrotizing pneumonia, severe pericarditis, necrotizing myocarditis, lymphocytic interstitial nephritis, lymphocytic periportal hepatitis, splenic hyperplasia, pyogranulomatous meningitis, and necrotizing synovitis (9,10,11,13,18,33). Hatchling alligators also rapidly developed disseminated M. alligatoris mycoplasmosis after intratracheal instillation, and the lesions are similar to those of adults and include acute multifocal brainstem hemorrhage (35). Mycoplasma crocodyli (23), the closest known relative of M. alligatoris (98% 16S rRNA gene similarity [9]), causes a similar necrotizing synovitis and occasionally subacute pneumonia in Nile crocodiles (Crocodylus niloticus) (27,28). The severity of the lesions correlates with the numbers of M. alligatoris cells in affected tissues, suggesting that a spreading factor(s) contributes to the virulence. The extracellular matrix (ECM)-degrading enzymes that act as bacterial spreading factors include hyaluronidases, sialidases, and mucinases (26...