In vivo evidence for the role of DNA-ligase in radiation resistance of Clostridium botulinum 33A

No DNA rejoining could be detected in radiation sensitive C. botulinum 51B. DNA ligase is known to exhibit some distinct properties, particularly a specific requirement for Mg++ ion and inability to rejoin DNA breaks produced by irradiation under oxic conditions. The present study provides in vivo evidence so far lacking for the role of DNA-ligase in recovery of 33A spores from radiation damage. The spores were frozen at -750C during irradiation to arrest all enzymatic activity. After irradiation, during thawing the spores were exposed to various chemical and physical treatments known to affect DNA-ligase. It was first shown that the process of recovery of irradiated spores has the properties of an enzymatic process, with a temperature optimum at 30-450C, a pH optimum at pH 5-6 and a distinct sensitivity to inhibition by 3-4M urea. More specific observations implicating DNA-ligase are the following: (i) removal of cations by EDTA (or other chelators) substantially inhibits spore recovery; (ii) inhibition of recovery by EDTA treatment can be fully reversed by supplementation with 0.04M Mg++, but not with Mn++, Ca++ or Zn++; and (iii) spores irradiated under oxic conditions were not affected by EDTA or Mg++, as would be expected if DNA-ligase is involved. These in vivo observations implicate DNA-ligase in the recovery of radiation damaged spores. The understanding of the nature of spore recovery gained from this study provides a basis for evaluation of various food formulations used in radiation preservation with respect to their content of Mg++, presence of natural Mg++ binding compounds, and the possibility of deliberate addition of Mg++ binding food additives such as, e.g., citrate, polyphosphate, sequestrene etc