Characterization Of A Cryptic Plasmid PMM1 In Pseudomonas Aeruginosa And Its Temperature-sensitive Derivatives

Pseudomonas aeruginosa (PA) is a representative bacterium in Pseudomonas spp and is widely distributed in nature. PA is an opportunistic pathogen. It can cause seriously acute and chronic infections in human beings, and is the third leading pathogen in nosocomial infections. Although some antibiotics are effective against P.aeruginosa, it bears innate insisitance to many of the clinical important drugs. So it is essential to study P.aeruginosa at the molecular level.Plasmid is a relative simple genetic material. It is easy for us to isolate, purify and manipulate it in vitro. So it is a good material to study the mechanisms of DNA replication, gene structure and gene function. In addition, it provides a convenient tool to investigate gene expression and gene regulation.A new cryptic plasmid from a natural multi-plasmid strain of Pseudomonas had been isolated. The plasmid, designated pMM1, was digested with single restriction endonuclease BamHI, and ligated to the cloning vector pUC19 digested with the same enzyme. The recombinatant plasmid was isolated and sequenced by primer M13, bidirectionally. Sequencing the plasmid pMM1 was continued by the primer walking method. The plasmid pMM1 was 2140 base pairs in length with a base composition of 45.8% G+C. One primary open reading frame (ORF) was identified from plasmid pMM1 with a coding region for a polypeptide of 374 amino acid residues. This putative gene product showed significant homology to functionally characterized Rep proteins in the GenBank database. There were a lot of direct and invert repeats in pMM1.Vector NTI and BlastX analysis showed the minimal replication was located in the fragment containing Rep and its upstream sequence. Sequence analysis revealed this open reading frame (Rep) is highly similar to the rep gene of the RCR group VIII. The minimal replication region of pMM1 containing Rep, dso and sso origin possesses all characteristic features of the rolling-circle mechanism of replication plasmid.We mutagenized pMM1 in vitro by error-prone PCR and followed by introducing them into Pseudomonas aeruginosa cells and selecting for temperature sensitive derivatives. A mutant plasmid, which was stably maintained at 30°C but not at 42°C, was isolated from the cells. Sequencing the plasmid, named pMM8, revealed two substituations in the sso and dso region. Moreover, a Pseudomonas aeruginosa/Escherichia coli shuttle vector pUM8, which is composed of pMM8 and the E.coli cloning vector pUC19 was constructed. This temperature-sensitive cloning vector will be usefull for disrupting genes in this industrially important bacterium.