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

Abstract

The endogenous plasniid pIW2 from l'seudo~~to~taulcs aliger~esN CIU 9867 was deter~iiirietlt o have 32,733 bp tvitli a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading franics, wit11 approsilliately halt of them contributing towards the functions of plasmid replication, ~iiubilizationa, nd stability. The Pac2j I restrictio~i-modifications ystem and two mobile elements, Tn5563 and 1S1633, were physic:llly localized. An addition;il eight open reading frames with unknown funclions were also dcttectcd. p W was ge~~ctically tagged with the ILStrc/Spcc gene cassette by homologous recombination. Intrastr~intr ansfer of pIW-cricotlecl genetic ni:irkers between isoge~iic~ iiutantos fP. alcalige~resN CIU 9867 were obsemetl at high frecli~encics( 2.4 X per donor). 'l'liis transfer was determined to be niediated by a natural transformation process that required cell-cell contact and was co~npletelys ensitive to DNase I (1 nig/ml). Eliitient transform;ltion wvas also observed when p WD NA was applied directly onto the cells, while transfor~nationw ith foreign plasmid DNhs was not observed. pRA2 coold be conjugally trmsferred into Pseudomona~p utida m713 and KT2440 recipients only when plasmid RK2IRP4 transfer functions were provided in trans. Plasmid stiibility analysis tlemonstrated that pRAZ could be stably maintained in its original host, P. alcaligenes NCIU 9867. as well as in P. putida R4713 atter 100 generations of nonselective growth. Disruption of the p l W pac2jl restriction endoriuclease gene did not alter plasmid stability, while the p M minireplicon exhibited only pinrtial stability. This indicates that other pW-yncoded determinants could have significant roles in influencing plasmid stability.