Characterization of a cryptic plasmid from Pseudomonas sp. and utilization of its temperature-sensitive derivatives for genetic manipulation

A new cryptic plasmid, named pMM101, in an environmental Pseudomonas sp. has been isolated, and its 2140-bp DNA sequence has been determined and analyzed. One open reading frame (Rep(MM)) similar to the rep gene of the rolling-circle replicon (RCR) group VIII has been allocated in the replication region where a putative double-strand origin dso and single-strand origin sso could also be identified. After in vitro mutagenesis by error-prone PCR and introduced into Pseudomonas aeruginosa PAO1, a mutant plasmid which was stably maintained at 30 degrees C but not at 42 degrees C was isolated and analyzed. It had two nucleotide substitutions in the putative sso and dso regions. Replacing one of the two sites with wild type sequence resulted in similar instability at 42 degrees C, indicating either mutation could result in the temperature-sensitive trait. Using the mutant replicon, a Pseudomonas (temperature-sensitive)-Escherichia coli shuttle vector, pUM109, has been constructed and used successfully to disrupt P. aeruginosa genes. This temperature-sensitive vector provides an alternative tool for genetic manipulations in this industrially as well as medically important bacterium.