Functional analysis of two putative chromosomal replication origins from Pseudomonas aeruginosa

Abstract

Two autonomously replicating elements previously isolated from Pseudomonas aeruginosa were characterized in vitro for pre-priming complex formation using combinations of replication proteins from P. aeruginosa and Escherichia coli. The results of these studies showed that the P. aeruginosa DnaA and DnaB proteins could form a pre-priming complex on plasmid templates containing either of the two autonomously replicating elements of P. aeruginosa, pYJ50 (containing oriCI), and pYJ52 (containing oriCII), or the E. coli chromosomal origin (plasmid pYJ2). The E. coli DnaA, DnaB, and DnaC proteins were also able to form a pre-priming complex on pYJ2, pYJ50, and pYJ52. Neither pYJ50 nor pYJ52 could be established in E. coli, suggesting a block in steps subsequent to the formation of the pre-priming complex. Similarly, pYJ2 could not be established in P. aeruginosa. Since pYJ50 and pYJ52 could be established in P. aeruginosa and both putative origins form a pre-priming complex in vitro, attempts were made to delete each of these two putative origins. The results indicate that the oriCI sequence is essential for cell viability under typical laboratory growth conditions but that oriCII is not.