Formation of type II F-primes from unstable Hfrs and their recA-independent conversion to other F-prime types

Abstract

Four E. coli Hfr strains, representing stable (Hfr Cavalli), moderately stable (AB312) and unstable (Ra-1, Ra-2) Hfr states, were used in the isolation of a series of F′ plasmids. Type II F′s were found to be the most prevalent F′ plasmid formed from all of the Hfrs, while the percentages of Δtra F′s increased as the stability of the Hfr increased. Two observations suggested that F′ formation in unstable Hfrs like Ra-2 may proceed through a type II F′ precursor. First, the major F′ products of Ra-2 are tra ⁺ type II F′s and, second, other F′ types (I, II) and classes (tra ⁺, Δtra) from Ra-2 appeared to be deletion derivatives of a larger F′ progenitor. By monitoring the molecular changes that occur when the Ra-2 derived type II F′ pWS200 is transferred from one recA host to another, we have found that all F′ types and classes can be generated from pWS200 in a recA-independent manner. F sequences involved in the genetic conversions of pWS200 include the oriT locus and the directly repeated γδ junctions of F and chromosomal DNA. A model for the formation of F′s in unstable Hfrs is postulated in which a tra ⁺ type II F′ primary excision product is seen to be modified, through recA-independent processes, to other F′ types and classes. This model differs from the current model of F′ formation in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II F′s. These studies have also shown that the formation of δtra F′s is a recA-independent process that can occur from the F′ and Hfr states, that γδ-mediated deletions in pWS200 often demonstrate regional specificity in having endpoints near the ilv operon and that genetic alterations in either replication origin of pWS200 (F oriV, chromosomal oriC) stabilize the replication of this “mini-Hfr” cointegrate.