Retrotransfer kinetics of R300B by pQKH6, a conjugative plasmid from river epilithon

Abstract The kinetics of conjugation, retrotransfer and mobilisation were studied at 5–15 min intervals between strains of Pseudomonas putida using plasmid pQKH6, isolated from river epilithon, and R300B. Transconjugants from the direct conjugation of pQKH6 and mobilisation of R300B by pQKH6 appeared rapidly, reaching maximum densities within 30–60 min of the start of both filter and liquid mating experiments. However, retrotransconjugants only appeared after a delay. This delay was short (approx. 45–60 min) in filter mating and much longer (2–5 h) for liquid mating experiments. Attempts at predicting the time course of retrotransconjugant development from (1) numbers of transconjugants from the conjugation and mobilisation experiments and (ii) mathematical models based on a mass action approach, both failed to reproduce the observed delay. It was concluded that retrotransfer did not proceed by either a one-step mechanism occuring early in conjugation or two separate conjugation and mobilisation steps. The clear demonstration of a delay in retrotransconjugant formation implies that a new mechanism must be sought. The likely importance of retrotransfer in the environment is discussed.     

Single-step conjugative cloning of bacterial gene fusions involved in microbe-host interactions

In vivo expression technology (IVET) is a genetic strategy for isolating genes expressed in vivo. In order to fully exploit this technology, it is necessary to analyse large numbers of IVET-generated gene fusions, which must be recovered from the chromosome of host bacteria. In bacteria for which transductional methods are not available, the recovery of integrated fusion plasmids is problematic and currently limits broad application of IVET. We describe a rapid, single-step, triparental conjugative approach for recovering chromosomally integrated fusion plasmids from both Pseudomonas fluorescens and Salmonella typhimurium. This simple and broadly applicable conjugative cloning system extends the utility of the IVET approach to clinically and agronomically relevant microbes and may be employed to recover non-replicating and integrated plasmids in other systems. Received: 28 March 1997 / Accepted: 23 May 1997     

Shuttle transfer (or retrotransfer) of chromosomal markers mediated by plasmid pULB113

Summary The IncP1 plasmid pULB113 (RP4::miniMu) not only mediates the transfer of chromosomal markers in the classical direction (i.e. from the donor to the recipient cell) but also in the opposite direction (i.e. from the recipient bacterium to the donor). This phenomenon of retrotransfer was observed in homologous matings with Pseudomonas fluorescens, Alcaligenes eutrophus and Salmonella typhimurium. Retrotransconjugants could be discriminated from direct transconjugants by appropriate chromosomal and plasmid markers used to distinguish the mating partners not bearing pULB113. Retrotransfer of chromosomal markers occurred at frequencies equal to, or sometimes greater than, those observed for the direct mobilization, thus allowing the recovery of recipient recessive markers in the donor with linkage values similar to those found in the normal direction. Retrotransfer was also observed in heterospecific matings involving A. eutrophus and pULB113 bearing P. fluorescens: R-primes carrying different selected and unselected markers were recovered in both bacteria. Retrotransfer of shuttle transfer seems to be a specific trait of IncP1 plasmids.     

Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.