Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751

Abstract

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of T061 (TraC₁) and 746 (TraC₂) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncPβ plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC₂ (RP4) and TraC₄ (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC₃ and TraC₄ and in addition between the TraD proteins of both plasmids. The R751 traC₃ gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.

Key Words:

• bacterial conjugation
• DNA transfer
• evolutionary conservation
• DNA primases