RESUMO
The DNAs of bacteriophage c-st, known to realize the lysogenic conversion of toxinogenicity among C. botulinum types C and D strains, and the nucleic acid of a virulent mutant of bacteriophage CB propagated in type A C. botulinum cells were purified and examined. Heterogeneity of phage c-st preparations was observed during purification, manifesting by formation of several bands in isopiknic CsCl gradient during centrifuging. An extra nucleic acid fraction was detected in some DNA preparations of phage c-st; the origin of this fraction is discussed. Plasmid extrachromosomal elements were for the first time found in the cells of nontoxigenic type C C. botulinum A02 strain, known as the indicator for c-st phage. The sensitivity of phage c-st DNA to 25 restriction endonucleases was examined. Analysis of the results of restriction analysis of c-st and CB phage DNAs and plasmid nucleic acids, revealed earlier in type A C. botulinum strains, disclosed several DNA modification enzymes with different recognition sites in type C C. botulinum. At least two of these activities are not found in type A strains. According to restriction analysis, total size of phage c-st DNA is about 160 kbp and of phage CB DNA 35 kbp. Individual EcoRI and HindIII restricts of phage c-st DNA, containing the initial site of botulinum toxin CI gene, were recognized by radioisotope labeled oligonucleotide probe Enzyme immunoassay revealed slight expression of the N-terminal region of bntc I gene in E. coli recombinant variants. These data can be used in further investigation of C. botulinum genetics.
Assuntos
Bacteriófagos/genética , Cromossomos Bacterianos , Clostridium botulinum/genética , DNA Viral/genética , Bacteriófagos/patogenicidade , Sequência de Bases , Clostridium botulinum/virologia , Primers do DNA , Eletroforese em Gel de Ágar , Técnicas Imunoenzimáticas , Plasmídeos , Mapeamento por Restrição , Virulência/genéticaRESUMO
Plasmid profiles in fifteen toxigenic and six nontoxigenic strains of Clostridium botulinum type A were investigated. The electrophoretic patterns of nucleic acids from the bacterial lysates were shown to vary in relation to the mode of bacterial cells lysis and the stage of microbial population growth. Two toxigenic strains harbour a unique 3.9 MD plasmid, five strains have a plasmid of about 6 Md and seven other strains carry an 11.5 Md plasmid. The nontoxigenic derivatives of two toxigenic strains harbouring a 6 Md plasmid have lost this extrachromosomal element. The analysis of the derivatives has not indicated the 6 Md plasmid to be connected with bacteriocin production or antibiotic resistance in Clostridium botulinum. Nontoxigenic variants of three other strains possess an 11.5 Md plasmid similar to their toxigenic parents' one. One of the studied toxigenic strains harboured no plasmids, as well as its nontoxigenic derivative. The functions of all studied plasmids remain unknown. Physical maps of the 11.5 and 6 Md plasmids were constructed on the basis of restriction analysis. The obtained results can be used for identification and differentiation of Clostridium botulinum strains and in further molecular biological experiments with this organism.
