Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability

Abstract

Plasmid R68.45 has been useful in promoting the transfer of chromosomal markers in bacteria of many genera. In donors harboring R68.45, chromosomemobilizing ability (Cma) may be lost without the loss of other plasmid markers. However, Cma can be somewhat stabilized by maintenance of the donors in the presence of kanamycin (Km). We isolated variants of R68.45 from four bacterial species of three genera. Plasmid variants isolated included those without Cma, without transfer function (Tra) and Cma, or without Tra, Cma, and Km resistance. In Erwinia carotovora subsp. atroceptica EA153, the loss of plasmid markers is dependent on the culture medium on which the donors are maintained. Restriction endonuclease analyses of the variant plasmids revealed that most are deletion mutants of R68.45. In all cases when the uncertainty in the ends of the deletions was not too great, one end of the deletion was shown to originate within or near the direct DNA duplication in R68.45 which is required for Cma and which maps close to the Km resistance determinant. Furthermore, the types of deletions observed are consistent with what might be expected for deletions generated by tandemly repeated insertion sequences. Therefore, we suggest that the DNA duplication is the source of much of the instability observed in R68.45. However, data are presented for E. carotovora subsp. atroceptica EA153 which suggest that another region of R68.45 may also play a role in its stability in this species. Plasmid R68.45 was derived from plasmid R68 in Pseudomonas aeruginosa PAO (16). Both of these plasmids confer resistance to ampicillin (Ap), kanamycin (Km), and tetracycline (Tc); both promote their own transfer (Tra); and both have chromosome-mobilizing ability (Cma), but the activities of the latter are distinctly different (8, 9). R68 has low Cma in P. aeruginosa PAO, whereas R68.45 efficiently mobilizes genes from multiple chromosomal origins in this strain (18). Chromosome mobilization from multiple origins results in what appears to be a nonpolarized transfer. This nonpolarized transfer is expressed in the bacteria of many genera, whereas Cma of R68 is much more restricted and, when present, occurs in a polarized manner from one or a few chromosomal origins. In fact, to our knowledge, there are only two bacterial species, Erwinia carotovora subsp. atroceptica (4) and Proteus mirabilis (10), in which R68.45 has been reported not to have Cma. Recent studies have shown that the Cma of R68.45 is associated with a DNA insertion of approximately 2.05 kilobases (kb). This insertion creates a direct repeat of a neighboring t Paper 82-6-J of the scientific journal series, Kansas Agricultural Experiment Station, Manhattan, KS 66506.

Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans

“…The pGSS33 probe never hybridized with MRIOO or MR400 chromosomal DNAs (data not shown). A search for pHRH1 plasmid was carried out in these strains but was unsuccessful regardless of which techniques were employed (Casse et ai, 1979;Kado andLiu, 1981: Currier andMorgan. 1982).…”

“…The Casse ef al. method (1979), the Kado and Liu method (1981) and the in situ lysis method described by Currier and Morgan (1982) were used to search for the presence of pHRHi plasmid in D. fructosovorans MR100 and MR400 cells.…”

Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment

“…Gene rearrangement was observed more frequently in situ than in vitro and may be a result of environmental stress imposed by biological or physical factors found only in the environment. R68.45 is known to be unstable after conjugal transfer (7,13). This instability does not necessarily result in the loss of the whole plasmid; only portions of the plasmid may be deleted (14).…”

The istA gene of insertion sequence IS21 is essential for cleavage at the inner 3′ ends of tandemly repeated IS21 elements in vitro.

“…The prototype plasmid carrying (IS21)2, R68.45, forms cointegrates with other replicons at high frequencies (10-3 to 10-5) and, by transient integration into the bacterial chromosome, promotes effective chromosome transfer in a wide range of bacteria (Haas and Holloway, 1976;Riess et al, 1983;Reimmann et al, 1988;Haas and Reimmann, 1989). The parental plasmid R68, in contrast, has very poor chromosome mobilizing ability (Haas and Holloway, 1976;Currier and Morgan, 1982;Haas and Riess, 1983). The stimulation of cointegrate formation by (IS21)2 is due to a hyperactive IS21 -IS21 junction.…”