Stringent and Relaxed Recognition of oriT by Related Systems for Plasmid Mobilization: Implications for Horizontal Gene Transfer

Abstract

The plasmids R1162 and pSC101 have origins of conjugative transfer (oriTs) and corresponding relaxases that are closely related. The oriTs are made up of a highly conserved core, where DNA is cleaved by the relaxase prior to transfer, and an inverted repeat that differs in size and sequence. We show that in each case the seven base pairs adjacent to the core and within one arm of the inverted repeat are sufficient to determine specificity. Within this DNA there are three AT base pairs located 4 bp from the core. Mutations in the AT base pairs suggest that the relaxase makes essential contacts at these locations to the minor groove of the DNA. The remaining four bases are different for each oriT and are both necessary and sufficient for stringent recognition of oriT by the pSC101 mobilization proteins. In contrast, the R1162 mobilization proteins have a much more relaxed requirement for the base sequence of this specificity region. As a result, the R1162 mobilization proteins can initiate transfer from a variety of sites, including those derived from the chromosome. The R1162 mobilization proteins could therefore contribute to the horizontal gene transfer of DNA from diverse sources.The plasmids R1162 and pSC101 have closely related systems for conjugative mobilization, although they are otherwise unrelated. These mobilization (Mob) systems belong to a large family, with examples found in plasmids from both gram-negative and gram-positive bacteria (12). Within this family, the general structures of the origins of conjugative transfer (oriTs) are similar and are characterized by a highly conserved region called the core and an adjacent, inverted repeat that varies in both size and sequence (3). These features are shown for the R1162 and pSC101 oriTs in Fig. 1A. We recently suggested that the oriTs of this family were generated by the duplication and inversion of the core and adjacent DNA on several separate occasions (3). The resulting second copy of the core was then lost by deletion, although remnants have remained in several cases, including the oriT of pSC101. The properties of the relaxases indicated that this duplication and inversion was necessary in the evolution of a system for the efficient transfer of circular DNA. The cognate relaxase binds double-stranded DNA, initiates strand separation within the core, and cleaves one of these strands at the nick site. The cleavage takes place by a transesterification that results in transient covalent linkage of the protein to the 5Ј end of the DNA strand (8,22,23). The relaxase and the attached strand, unwound from its complement, are then transferred into a recipient cell by type IV secretion (11). Duplication and inversion of DNA next to the core generates an inverted repeat, which re-creates the binding site for the attached relaxase. This allows the subsequent reversal of the transesterification to regenerate a circular molecule of DNA. The outer arm of the inverted repeat is not required for relaxase-induced nicking of supercoiled DNA at the beginning of a ro...

The R1162 Mob Proteins Can Promote Conjugative Transfer from Cryptic Origins in the Bacterial Chromosome

“…We previously showed that one of these sites (bp 1306755 to 1306773, Fig. 2) is active for the initiation of plasmid DNA transfer (21). This DNA is between the genes hupB (HU protein ␤ subunit) and ppiD (peptidyl-prolyl cis-trans isomerase) and is unlikely to be part of an integrated plasmid.…”

“…2). The activity of the origin (the proportion of white transconjugant colonies; see Materials and Methods) was 0.75%, indicating that this origin is ϳ15 times more active than the one in Erwinia (21).…”

“…The initiating activity of a cryptic oriT on a plasmid was tested as described previously (21). In brief, the test plasmid (pUT2045, Fig.…”

“…We previously identified one such site in the chromosome of Erwinia carotovora subsp. atroseptica (now Pectobacterium atrosepticum) (21). In addition, by testing libraries of oriTs with one or more mutations for relaxase-induced nicking, we found that a large population of DNAs with different sequences was potentially active (21).…”

“…Although the mechanism of transfer is similar for the two plasmids, the oriTs are structurally very different. In prior studies we have determined that the R1162 oriT is small, structurally simple, and able to accommodate base changes at different positions without a complete loss of function (5,21). As a result of this relaxed specificity, the R1162 mobilization (Mob) proteins can activate the related but different oriT of another plasmid, pSC101 (28).…”