Role of DNase in recovery of plasmid DNA from Clostridium perfringens

Abstract

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.

Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis

“…Plasmid DNA was routinely isolated from 1 liter of culture by the method of Anderson and McKay (2). In some cases, 0.7% (vol/vol) diethylpyrocarbonate (Sigma) was added before sodium dodecyl sulfate lysis to inactivate nucleases (5,23,29,46). The purified plasmid band from a CsCl-ethidium bromide density gradient was dialyzed against 10 mM Tris-1 mM EDTA buffer at pH 8.0, precipitated with ethanol, and resuspended in 100 ,ll of the buffer.…”

Molecular genetics and pathogenesis of Clostridium perfringens.

“…perfringens plasmids (3,13,14,18,70,92,113,118,149,158,162,167,168,192). Plasmid profiling also has been used for strain differentiation in C. perfringens (118,149).…”

Recent advances in the genetics of the clostridia

“…Autolysin activity might prevent regeneration of the bacterial cell wall and unless special precautions are taken to limit activity [13] transformation may be difficult to demonstrate. Nucleases, which might, perhaps, interfere more directly with protoplast transformation, have been documented [26][27][28][29] in strains of C. acetobutylicum, C. perfringens and C. thermohydrosulfuricum that have been successfully transformed (see Table 3). Restriction endonucleases have been detected in the last two species as well as several other commonly used laboratory strains (Table 2).…”