Biological and biophysical characteristics of phages isolated from Clostridium botulinum type C and D strains, and physicochemical properties of the phage DNAs.

Abstract

Toxin-converting phages CE beta and d-16 phi and non-converting phages CE gamma and d-1', isolated from toxigenic strains C-468 and D-CB16 of Clostridium botulinum types C and D, respectively, were characterized biologically and biophysically. DNAs isolated from these four phages were studied physicochemically. Phages CE beta, d-16 phi, CE gamma and d-1' were adsorbed to their susceptible cells at the constant rates of 1.1 x 10(-8), 3.3 x 10(-8), 1.4 x 10(-8) and 1.4 x 10(-8) ml/min, and grew after latent periods of 35, 45, 35 and 35 min at the burst sizes of 38, 85, 35 and 35, respectively. Converting phages were more susceptible than non-converting phages to physical and chemical treatments such as temperature, pH, time, UV-irradiation and organic solvents. GC% of phage DNAs were determined by melting temperature, buoyant density and HPLC analysis to be 26, 26, 29 and 29% for CE beta, d-16 phi, CE gamma and d-1' phage DNAs, respectively. The restriction digestion profiles of phage DNAs with seven endonucleases were compared by agarose gel electrophoresis, revealing that the two converting phage DNAs were similar in regard to five enzyme digestion profiles, but different in the other two enzymatic profiles. Non-converting phage DNAs produced the same restriction digestion profiles with all seven endonucleases. The molecular sizes determined from the size of restriction enzyme digestion fragments were about 110 kb for converting phage CE beta and d-16 phi DNAs and 65 kb for non-converting phage CE gamma and d-1' DNAs. Dot blot hybridization experiments revealed that DNA homology between the converting phages CE beta and d-16 phi was 50-75%, while that between non-converting phages CE gamma and d-1' was about 100%. Converting phage and non-converting phage DNAs did not hybridize at all.