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Journal of Bacteriology, January 2000, p. 81-90, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of the Endogenous Plasmid from Pseudomonas alcaligenes NCIB 9867: DNA Sequence and Mechanism of Transfer

Stephen M. Kwong,1 Chew Chieng Yeo,1 Antonius Suwanto,1,2 and Chit Laa Poh1,*

Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore 119260, Singapore,1 and Department of Biology, Faculty of Science and Mathematics, Bogor Agricultural University, Jl. Raya Pajajaran, Bogor 16144, Indonesia2

Received 6 July 1999/Accepted 30 September 1999

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the Omega Strr/Spcr gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 × 10-4 per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

* Corresponding author. Mailing address: Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore. Phone: 65 8743674. Fax: 65 7766872. E-mail: micpohcl{at}nus.edu.sg.

Journal of Bacteriology, January 2000, p. 81-90, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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