Isolation and characterisation of deletion mutants involving the transfer genes of P-group plasmids in Pseudomonas aeruginosa

Abstract

The P-group plasmids RP1 and R26 are recovered at low frequency following conjugal transfer to B3-lysogens of P. aeruginosa PAO. The rare carbenicillin-resistant transcipients that do arise are usually transfer-defective (Tra-) and may show the loss of other plasmid borne functions, namely kanamycin-resistance (Kmr) and reduced plating of phage G101 (Spp+). The four phenotypic classes that occur among the Tra- derivatives are respectively, Tra- (69-81%), Tra- Spp- (12-30%), Tra- Kms and Tra- Kms Spp- (0.2-1%), of which the latter three are dut to plasmid deletions. This is seen from the sizes of the plasmids carried by these bacteria and from the transductional analysis of the R26-derivatives. Thus, although R26 (MW = 52 X 106 daltons) is too large to be transduced by phage F116L (MW = 40 X 106), this is possible for its Tra- Kms and Tra Kms Spp- derivatives. The phenotypes and frequencies of the various transcipient classes suggests that the gene order Km.. Tra.. Spp occurs in both RP1 and R26, and that Spp is more closely linked to Tra than is Km. These conclusions are supported by the sizes of the plasmid mutants since deletions spanning the loci Km Tra Spp, Km Tra, and Tra Spp involve the loss of DNA of MW 8-17 X 106, 5-13 X 106 AND 1-9 X 106 DALTONS RESPECTIVELY. Whilst all the transcipients displayed the incompatibility properties of the parent plasmids (Inc+), only some retanied plasmid surface exclusion (Sfx+). Moreover, a strict correlation existed between the Sfx and Spp phenotypes such that the transcipients were either wild type, Sfx- Spp-, or displayed an intermediate phenotype for both characters. Thir are different manifestations of the same gene function. The deletion map of these various markers in both RP1 and R26 therefore seems to be Km.. Tra.. Sfx/Spp.. Inc.