Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was dete rmined to have 32,743 bp with a G+C content of 59.8%, Sequence analysis pre dicted a total of 29 open reading frames, with approximately half of them c ontributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile eleme nts, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetic ally tagged with the Omega Str(r)/Spc(r) gene cassette by homologous recomb ination, Intrastrain transfer of pRA2-encoded genetic markers between isoge nic mutants of P. alcaligenes NCIB 9867,were observed at high frequencies ( 2.4 x 10(-4) per donor). This transfer aas determined to be mediated by a n atural transformation process that required cell-cell contact and was compl etely sensitive to DNase I (1 mg/ml), Efficient transformation was also obs erved when pRA2 DNA was applied directly onto the cells, while transformati on with foreign plasmid DNAs was not observed. pRA2 could be conjugally tra nsferred into Pseudomonas putida RA713 and KT2440 recipients only when plas mid RK2/RP4 transfer functions were provided in trams, Plasmid stability an alysis demonstrated that pRA2 could be stably maintained in its original ho st, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 gener ations of nonselective growth. Disruption of the pRA2 pac25I restriction en donuclease gene did not alter plasmid stability, while the pRA2 minireplico n exhibited only partial stability, This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.