Integration of Replication-defective R68.45-like Plasmids into the Pseudomonas aeruginosa Chromosome

Abstract

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.

Salicylic Acid Produced by the Rhizobacterium Pseudomonas aeruginosa 7NSK2 Induces Resistance to Leaf Infection by Botrytis cinerea on Bean

“…With selection for tetracycline, pME3356 was integrated into the chromosome in a first crossing-over event. In a second crossing-over event, obtained after enrichment for tetracycline sensitive cells (28), plasmids were excised and lost because of their inability to replicate in P. aeruginosa. SA-negative mutants of strain MPFM1 were identified by their inability to form orange halos on casamino acids medium with chrome azurol S (3,31).…”

Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa

“…1) in the pBR325 derivative pME327 (Table 1). The origin of transfer (oriT) of the broad-host-range plasmid RP1 was then inserted into pME327::fl-Hg by cointegration in vivo with the replicationdeficient mini-RP1 plasmid pME28 (39). A pME327::Ql-Hg:: pME28 cointegrate thus obtained contained the pME28 insertion in the pBR325 vector moiety, i.e., outside the arc operon.…”

“…A pME327::Ql-Hg:: pME28 cointegrate thus obtained contained the pME28 insertion in the pBR325 vector moiety, i.e., outside the arc operon. The cointegrate was then introduced into E. coli S17-1, which carries the RP4 (RP1) transfer genes in the chromosome (43) and mobilizes pME28 derivatives at high frequencies (39). The S17-1 (pME327::Ql-Hg::pME28) donor was mated with P. aeruginosa PAQ1, with delayed selection for Hg resistance on minimal medium containing 25 ,ug of HgCl2 per ml.…”

The global regulator GacA of Pseudomonas fluorescens CHA0 is required for suppression of root diseases in dicotyledons but not in Gramineae

“…Cells of Pseudomonas with a chromosomally-integrated plasmid were selected on plates containing tetracycline and checked for b-galactosidase activity. Strains from which the vector had been excised via a second homologous recombination event were obtained by enrichment for tetracycline sensitivity (Reimmann et al, 1988), and b-galactosidase activity was used to verify the presence of the insert.…”