Tn502 and Tn512 are res site hunters that provide evidence of resolvase-independent transposition to random sites

Abstract

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.

FIG. 1.

Preferential insertion of Tn502, Tn512, In0, and In2 in the res region of pUB307 (par⁺) and random insertion of Tn502 in the par-deleted derivative pUB1601. (A) Simplified map of pUB1601 linearized at the unique EcoRI site. The insertion sites of Tn502 are as follows, relative to the DNA sequence of RP1 (GenBank accession no. BN000925), from left to right: Ω2 (nt 2306), tnpA::Ω11 (nt 10325), Ω6 (nt 11934), tetR::Ω4 (nt 13831), tetA::Ω8 (nt 14239), tetA::Ω3 (nt 14414), tetA::Ω7 (nt 15209), traA::Ω9 (nt 39694) (pVS1717), traC2::Ω1 (nt 41924), kfrA::Ω12 (nt 55630), kfrA::Ω10 (nt 55626), and kfrA::Ω5 (nt 55816). Open and filled symbols indicate the IRi-mer-tni-IRt and IRt-tni-mer-IRi orientations, respectively. (B) Sequence of part of the par-res region that is situated between the Tra2 region and the Km^r determinant in RP1 and pUB307. The parCBA and parDE operons are separated by the res region, which contains subsites resI, resII, and resIII. Only the resII subsite is shown (shaded sequence), and the contained DraI cleavage site appears in boldface. Symbols representing insertion sites are as follows: open triangle, Tn502; gray-shaded triangle, Tn502ΔtniQ; filled triangle, Tn512; filled arrow, In0; and open arrow, In2. Numbers adjacent to symbols represent the number of insertion mutations at each site. The most common site of the wild-type elements, relative to the DNA sequence of RP1, is at nt 35017. The Tn502 insertions in parE and at nt 35030 were aberrant insertions from 50 tested that were not in the preferred region (see the text). The insertions of Tn502ΔtniQ in parC and upstream of parD were unselected. Insertions are in the IRi-mer-tni-IRt (Tn) or IRi-intI-Δtni-IRt orientation, except two indicated by a filled, curved arrow; these are in the IRt-tni-mer-IRi orientation.

FIG. 2.

Model for cointegrate resolution during transposition. (A) The scheme represents an unresolved cointegrate such as that formed in RecA⁺ and RecA⁻ hosts between an In2⁺ plasmid (pACYC::In2) and pUB307. The duplicated In2 element (white boxes) has disrupted the resII subsite, separating it from the flanking resI and resIII subsites (black boxes). These subsites are the binding sites for the ParA resolvase protein encoded by pUB307 (8). (B) The ability of ParA to form a res-ParA nucleoprotein complex facilitates synapsis of the two In2 elements and, in a RecA⁺ host, enables RecA-dependent recombination. (C) RecA-dependent recombination between the synapsed In2 elements resolves the cointegrate. If the cointegrate had contained copies of a functional transposon, such as Tn502, resolution could have occurred either via the transposon-encoded site-specific resolution system (res-TniR) or via the homology-dependent RecA pathway.

FIG. 3.

Map positions of Tn502-mediated deletions in pUB307parE::Tn502 and pUB1601traA::Tn502. The upper portion is a simplified map of RP1 (and pUB307) showing the relative locations of par genes, IS21, aph, and flanking trb and tra genes. A PstI fragment (represented by the broken line) is absent in pUB1601. The different orientations of Tn502 in pUB1601traA::Tn502 (pVS1717) and pUB307parE::Tn502 (pVS1716) are indicated, as is the 5-bp DR sequence that flanks each insertion. The lower portion shows the extent of deletions in two mutants of pUB1601traA::Tn502 and three mutants of pUB307parE::Tn502. The 5-bp DR sequences that flank Tn502 in each mutant are indicated in parentheses; the associated numbers indicate the location of Tn502 in each mutant relative to the nucleotide sequence of RP1 (GenBank accession no. BN000925).

FIG. 4.

Deletions of the backbone of natural IncPβ plasmids. The map depicts the gene organization of pBP136 in the region between trbP (part of Tra2) and traC (part of Tra1). This region is thought to represent the ancestral IncPβ backbone and contains the res-parA preferred target region for Tn5053/Tn402-like elements; it includes repeat sequences, indicated by the vertical lines. The location of a Tn5053/Tn402-like element in pB3 is indicated by the triangle. The bars below the map show the extent of the IncPβ backbone that has been deleted and replaced with an inserted Tn5053/Tn402-like element in the plasmids listed. The inserts are all flanked by IRi and IRt.