GENESIS OF ESCHERICHIA COLI F PRIME PLASMIDS IN CONJUGATION EXPERIMENTS BETWEEN AN UNSTABLE HFR DONOR AND RECA RECIPIENT

The formation of F-prime (F') plasmids from the unstable, related Escherichia coli Hfrs Ra-1 and Ra-2 was investigated. The donor properties of these atypical Hfrs were first examined in detail using interrupted mating experiments and linkage analysis of the recombinants. Purified Hfr cells of both donor types gave similar transfer kinetics for arg (89'), thyA (60') and his (44') despite their wide separation on the E. coli chromosome and their different positions relative to the Ra-1 and Ra-2 transfer origins. These markers entered a F('-) recA('+) recipient within 4-10 minutes of mating aggregate formation and reached a plateau level within 50 minutes. By contrast, markers in the leu (2')-trp (27') interval were transmitted in gradient fashion from an origin near leu for both Hfrs. Linkage analysis of selected and unselected markers in the recombinants from Ra-1/Ra-2 x F('-) recA('+) matings revealed two regions of high negative interference on the Ra chromosome at 95' and 35'. Genes separated by these regions were coinherited infrequently while genes bracketed by the regions were strongly linked in the recombinants, suggesting a "segmental" transfer of markers from Ra-1 or Ra-2. Further genetic analysis of the Ra donors and recombinants showed that: (i) F is transferred concomitantly to the recipients and chromosomal transfer is F-dependent; (ii) Ra-type transfer is a strain specific phenomenon dependent on the (gamma)(delta) insertion sequence of F. Thus, purified Ra "Hfrs" actually represent a special case of F('+)-mediated transfer. Mating between Ra-2 and a F('-) recA('-) recipient produced type II F' transconjugants as the predominant progeny. The type II F's from the Rha('+) selection were of discrete size classes, with some isolates containing apparent chromosomal and tra gene deletion mutants of a larger type II F' precursor (pWS200). Reasoning that the minor F' types (IA, IB, (DELTA)II) and class (Tra('-)) might derive from a larger Tra('+) type II F' parent, pWS200 was examined after transfer to a second host. Genetic tests, restriction enzyme analysis and DNA:DNA hybridization revealed that all F' types and classes were generated from pWS200 after a single transfer. The genetic alterations of pWS200 were recA-independent events mediated by the oriT, (gamma)(delta), and IS3 sequences of F. Based on these findings, a model for the genesis of F' plasmids is proposed which postulates a Tra('+) type II F' intermediate in the formation of other F' types and classes. This model is distinguished from the current model in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II F's.