R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa

Abstract

The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10(-3)--10(-5) per donor cell for markers in the 0-60 min region of the chromosome. Little or nor chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3-5 min after initiation of mating. These results, combined with linkage studies, indicated that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min. R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for ARGB+ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmides of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.

Extension of a Chromosome Linkage Group of Proteus mirabilis

“…Confidence in these experiments was increased by two other observations. Firstly, plates where mating was interrupted at time zero showed none or very few recombinants (see Haas & Holloway, 1976). This proved that no significant second-round mating occurred once it had been interrupted.…”

“…It is not known how plasmid D mobilizes the P . mirabilis chromosome but the behaviour of the latter plasmid differed in a number of respects from that of R68.45 (see Haas & Holloway, 1976). Thus, donor ability of plasmid D was a stable property which differed from that of R68.45-carrying strains where continuous supervision and selection was required; plasmid D in apparently unchanged form (see above) was present and stable in all recombinants, but R68.45 became unstable after involvement in chromosomal transfer with partial or complete loss of plasmid markers in a substantial proportion of Ps.…”

“…This was done according to the solid medium technique of Haas & Holloway (1976). Briefly, 0.1 ml of recipient was spread on plates containing 10 ml minimal agar medium (appropriately supplemented when necessary) prior to the addition at zero time of 0.1 ml of the donor culture prepared as above.…”

Relationship of Group P1 Plasmids Revealed by Heteroduplex Experiments: RP1, RP4, R68 and RK2 Are Identical

“…These plasmids are characterized by their broad host-range among Gram-negative bacteria (Datta & Hedges, 1972;Beringer, 1974). Some of their derivatives can be used to mobilize chromosomal genes in different bacterial species (Stanisich & Holloway, 197 1 ;Beringer, 1974 ;Towner & Vivian, 1976 ;Lacy & Leary, 1976 ;Haas & Holloway, 1976;Meade & Signer, 1977). In this study, the molecular relationships of the P1 incompatibility group plasmids RPl, RP4, RP8, R68, R68.45 and RK2 were tested.…”

“…R68 was first used in early studies for gene mobilization in Pseudomonas aeruginosa (Stanisich & Holloway, 1971) and probably originates from the same source as RPl (Holloway & Richmond, 1973). R68.45 is a derivative of R68 that shows enhanced sex factor ability; it was isolated from P. aeruginosa (Haas & Holloway, 1976). RK2 was described by Ingram et al (1973) and used for physical and genetic studies by Meyer et al (1977); this plasmid was isolated in the same hospital as RPl and R68 (M. Richmond,personal communication).…”