pλCM System: Observations on the Roles of Transposable Elements in Formation and Breakdown of Plasmids Derived from Bacteriophage Lambda Replicons

Abstract

Transduction with phage derived from a 2-year-old lysate of λ cam105 (λ::Tn9) gave rise to chloramphenicol-resistant (Cm^r) transductants harboring a plasmid (pλCM1) formed from λ cam105 by a Tn9-mediated adjacent deletion to position 36.07 kilobases in the N cistron of λ. The pλCM element can replicate as a plasmid, insert into the bacterial genome, or reproduce lytically as a phage on cells that provide N function. The feasibility of obtaining high titers in encapsidated form and the ease of synchronous introduction into and recovery from bacterial populations make pλCM very suitable for quantitative studies of recombination involving transposable elements. Replicon fusions between pλCM1 and RSF1596 (pMB8::Tn3Δ596) occur by duplication of either IS1 (at low rate in the absence of TnpA activity) or Tn3Δ596 (in the presence of TnpA activity). At 24 or 32°C, the rate of increase of TnpA-mediated fusions per pλCM is about 2% per cell doubling. RSF103 contains the deleted Tn1ΔAp (which lacks intact beta-lactamase and TnpR resolvase coding sequences) adjacent to a streptomycin resistance (Sm^r) determinant. We observed that Tn1ΔAp mediates insertions of external RSF103 sequences into the R388 plasmid. R388::Tn1ΔAp plasmids show transposition immunity in cells lacking TnpR activity. Using the pλCM system, we isolated adjacent transpositions of the RSF103 Sm^r determinant. The resulting pλCM-Sm cosmids contain Sm^r genetic material flanked by direct repeats of Tn1ΔAp, and all are deleted for some RSF103 or pλCM sequences. The pλCM-Sm constructs will fuse into R388 by duplication of a single Tn1ΔAp element. In the presence of tnpR⁺ (but not tnpR) Tn1 or Tn3 elements, all Tn1ΔAp-mediated complex replicons break down completely and rapidly to simple Tn1ΔAp inserts. The equilibrium for resolution is at least 10⁵:1, and resolution is more than 90% complete after 40 min of exposure to a tnpR⁺ cytoplasm. In the absence of TnpR, Rec, and Red activities, Tn1ΔAp-mediated complex replicons yield simple Tn1ΔAp inserts at a lower rate. The presence of intact RSF103 replication determinants between direct Tn1ΔAp repeats appears to accelerate this precise TnpR- and Rec-independent breakdown.