Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4

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Abstract
The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tral region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectio nally from oriT with the leader operon heading the DNA to be transferred. The tral gene of the relaxase operon includes within its 3′ terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes tral and traK exhibit greater sequence divergence than the other genes of the corres ponding operons. This is conceivable, because tral and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the pro duct of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.