In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn
502 and Tn
512, which are members of the Tn
5053 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. Tn
502 and, under some circumstances, Tn
512 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 (Tn
502 and Tn
512) 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.It is widely recognized that mobile genetic elements contribute to genome plasticity and have been a driving force in the emergence and spread of resistance determinants within and between bacterial species; their impact is ongoing (
10,
51). Significant among these elements are various classes of plasmids, transposons, and integrons which may lack resistance determinants or carry one or multiple determinants. Resistance determinants that have become globally dispersed in environmental and clinically significant bacteria include mercury(II) resistance (
2,
17), evident even in ancient bacteria (
27), and antibiotic resistance, which has increased in dominance since the advent of the antibiotic era (
23,
40).This paper concerns the mercury resistance (
mer) transposons Tn
502 and Tn
512, whose sequence organization and transpositional behavior show that they are new members of a family of elements exemplified by the
mer transposon Tn
5053 (
22). These elements are closely related to those in the Tn
402 family, which contain an integron (
intI) recombination system (
14,
36). Members of the two families differ in the positions of the
mer or
intI determinants (modules) near one end of the transposition (
tni) module. The latter module contains four genes (
tniABQR), and the entire transposon is bounded by 25-bp inverted-repeat termini (IRi and IRt). TniA, TniB, and TniQ are required to form the transpositional cointegrate, which is then resolved by the action of TniR (a serine resolvase) on a resolution (
res) sequence located between
tniR and
tniQ (
22). The transposon in its new location is flanked by 5-bp direct repeats (DRs) (
20,
22). TniA, which contains a D,D(35)E transposase catalytic motif, is thought to function cooperatively with TniB, a putative nucleotide-binding protein, as the active TniAB transposase (
21,
36). Studies of TniA conducted
in vitro show binding to the IRs and to additional 19-bp repeat sequences that make up the complex termini of the transposon (
21). The precise role of TniQ is unknown.An unexpected and unique feature of Tn
5053 and Tn
402 is that they depend on externally coded accessory functions for efficient transposition, namely, a
res site served by a cognate resolvase (
25). As a consequence, these transposons exhibit a strong transpositional bias for some target
res sites (
20,
25,
26) and have aptly been described as “
res site hunters” (
25). One such efficient interaction involves the
res-ParA multimer resolution system of plasmid RP1 (IncPα); other plasmid- or transposon-encoded systems are less efficient or are refractory. Although the role of the external resolvase remains obscure, its capacity to bind to its cognate
res is an essential requirement whereas its catalytic activity is not (
20). For each interaction system, the target sites typically cluster in a single part of
res but not necessarily within the same subregion and, on occasion, can lie in the vicinity of
res. Typically, the transposon is in a single orientation with IRi closest to the resolvase gene. In one study, Tn
402 clustered at two target sites, one within
res and one nearby, and the orientations were different at the two sites (
20).The experimentally observed target preference described above also occurs in natural associations of Tn
5053/Tn
402-like elements and became evident on sequencing class 1 integrons, which were often found positioned close to different
res-resolvase gene regions (
6,
20,
25). Most Tn
402 family elements are comprised of an
intI module that is flanked on the left by IRi and on the right by a 3′ conserved sequence (3′-CS) (
13). In others, a remnant
tni gene cluster may be present instead of the 3′-CS, and IRt occurs at the right flank. The structure of the latter category of integrons strongly indicated that they are defective transposons that were presumably capable of relocation provided that
tni functions were supplied in
trans (
6,
32). The movement of In33 (Tn
2521) from a chromosomal to a plasmid location appears to have been such an in
trans event (
30,
42), and others involving In0 and In2 are demonstrated in this study. In contrast, the integrons that lack the IRt end appear to be nonmobile remnants of Tn
402-like transposons; they belong to several lineages, including those in which the incurred deletions are attributable to acquired insertion sequences (
6). More recently, intact Tn
5053/Tn
402-like transposons and class 1 integrons have increasingly been detected in the
res-
parA region of IncP plasmids (
39), which are arguably the most promiscuous of known plasmids (
50). These various experimental and natural interactions provide insight into the dispersal pathways possible for Tn
5053/Tn
402-like elements.The
res-hunting attribute is a striking feature that is experimentally supported by studies of four family members (namely, Tn
5053 [
22,
25], Tn
402 [
20,
26], and in this study, Tn
502 [
48] and Tn
512). Another facet of the transposition of Tn
502 is explored here. It concerns the observation that loss of the preferred
par target region in RP1 does not abolish transposition of Tn
502 (
48), contrary to the finding with Tn
5053 (
25,
26) and, in this study, Tn
512. The continued, low-frequency transposition of Tn
502 involved at least three dispersed locations (
48); however, nothing is known about the nature of these sites or about the features and requirements of the transposition process. Here we address these issues and uncover the existence of an alternative,
par-independent pathway that is employed by Tn
502 and is available to Tn
512 under some circumstances. The study also provides information on the roles of the TniR and host (RecA) recombination systems in the resolution of transpositional cointegrates and on the ability of the
par-independent transposition pathway to generate plasmid deletions.
相似文献