Role of nucleases in the isolation of plasmid deoxyribonucleic acid from Pseudomonas cepacia 4G9

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.

“…As had been reported by others (13,25,26), we also encountered difficulties in plasmid isolation from P. cepacia (24.8, 7.4, 5.8, 4.9, and 3.5 kb). using standard methodology.…”

Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 92%

See 1 more Smart Citation

Plasmids of Pseudomonas cepacia strains of diverse origins

Lennon

DeCicco

1991

“…(ii) Electrophoretic assay. The in vitro electrophoretic assay described by Williams et al (34) was modified to evaluate the DNase activity associated with C. acetobutylicum protoplasts during transformation. The reaction mixture consisted of 125 ,ul of a twice-washed protoplast suspension, 50 pul of dye-buoyant density-purified plasmid pUB110 in CBM containing 0.3 M sucrose (final concentration, 7.5 Rg/ml), and 325 pI of CBM containing 0.3 M sucrose.…”

Section: Subtilismentioning

confidence: 99%

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Lin

Blaschek

1984

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 550C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycinresistant (Kmr) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Kmr transformants.

“…Extracellular DNase is produced by strains of all five C. perfringens types (20) and may be responsible for variable agarose electrophoresis plasmid band patterns. The variable and irreproducible isolation of plasmid DNA from the gram-negative bacteria Serratia marcescens and Pseudomonas cepacia 4G9 was attributed to nuclease activity (21)(22)(23)(24). The objective of this study was to examine the role played by endogenous DNase in the recovery of plasmid DNA from C. perfringens cleared lysates.…”

mentioning

confidence: 99%

Role of DNase in recovery of plasmid DNA from Clostridium perfringens

Blaschek¹,

Klacik²

1984

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.