Conjugative Transfer of Promiscuous IncP Plasmids: Interaction of Plasmid-Encoded Products with the Transfer Origin

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-03, Vol.86 (6), p.1771-1775
• Main Authors: Fürste, Jens Peter, Pansegrau, Werner, Ziegelin, Günter, Kröger, Manfred, Lanka, Erich
• Format: Article
• Language: English
• Subjects: Base Sequence; Biochemistry; Biological and medical sciences; Cloning, Molecular; Conjugation, Genetic; DNA; DNA Restriction Enzymes; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA, Recombinant; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gels; Genes; Genetic Vectors; Intergenic DNA; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nucleotide sequences; Open reading frames; Operon; Overproduction; Plasmids; Promoter regions; Promoter Regions, Genetic; Replication
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.6.1771

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of ``relaxosomes'' (plasmid DNA--protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within oriT. The fully functional oriT of RP4 represents an intergenic DNA region of ≈ 350 base paris. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We overproduced gene products and, from analyzing the products of defined deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of oriT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity.