Mechanisms of Strand Replacement Synthesis for Plasmid DNA Transferred by Conjugation

摘要: It has been known for some time that only one of the two plasmid DNA strands is transferred during conjugation (9). As a result, complementary strand must be synthesized prior to establishment in new host. In donor cell, departing also replaced, although this not essential transfer (17). The mechanisms used replacement synthesis are understood. particular, it what extent components mechanism vegetative replication plasmid, or other plasmid-encoded proteins, recruited process. IncQ R1162, which efficiently comobilized conjugative IncP-1 plasmids, such as RK2 and R751, particularly suitable investigating question. R1162 encodes proteins required several steps initiating its own replication, rather than relying on host counterparts, feature no doubt contributes broad range. This independence could extended important process conjugation. Iterons within origin (oriV) target DNA-binding protein, encoded by locally distorts helix allows entry helicase, (16, 31). helicase activates oriL oriR, opposite-facing sites where initiated. These recognized third primase (30). both free protein C-terminal domain relaxase, (32). relaxase cleaves at (oriT), with becoming covalently linked 5′ end DNA. cleaved unwound from complement, intermediate transported into cell type IV secretory machine. Displacement trailing 3′ forms circular, single-stranded molecule, then synthesized. linkage suggests involved transfer. However, when mob genes cloned vector, pBR322, resulting molecule mobilizable even deleted (4). Thus, absolutely transfer, perhaps because can replaced priming systems active vector. Evidence system nevertheless was obtained under conditions mobilization inefficient. case, frequency increased presence cognate initiation (12). Assessing contribution difficult due ongoing cell. We recently developed first introduced electroporation potential cells, containing complete set but immediately mating these cells recipients encoding λ integrase (29). Since contains att site, captured after integration attB. Furthermore, parental daughter marked different restriction sites, using mismatched oligonucleotide electroporation. found appropriately oriented site regeneration round repaired molecules able participate subsequent rounds transfer. In experiments described here, we ask if similarly step recipient. Although newly created transconjugant do themselves provide source R1162-encoded specific incoming still nonetheless, domain. Priming initiated neighboring oriR released recircularization. addition, independently transport machine levels sufficient detection (21), entering Our results show Escherichia coli there robust cellular strand. Salmonella, less active, deployed initiate synthesis. parallel, sense, those larger, self-transmissible encode dispensable E. coli.