The NanI and NanJ sialidases of Clostridium perfringens are not essential for virulence.

The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.

Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function.

The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn4451, consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243-261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598-707). In these mutants, removal of residues 598-707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533-583). Analysis of mutations within the aa 243-261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.