GENETIC ORGANIZATION OF A SMALL CRYPTIC PLASMID OF HELICOBACTER-PYLORI

A 2.9-kb cryptic plasmid of Helicobacter pylori (Hp), pHe11, was isola ted and the complete nucleotide (nt) sequence was determined. An open reading frame (ORF1) was identified encoding a putative polypeptide of 63709 Da, the existence and correct size of which was confirmed by T7 promoter expression analysis. The ORF1 sequence showed strong amino-a cid sequence identity to a recently identified putative ORF1 protein o f a cryptic Hp plasmid, pHPM180, and significant homologies to putativ e Rep proteins of Campylobacter coli (RepB) and Pediococcus halophilus (RepA), and was therefore designated RepA. A functional role of RepA in replication of pHe11 was demonstrated by the fact that only pHe11 p lasmid derivatives with an intact repA gene were able to autonomously replicate in Hp. Upstream of repA, a 22-bp sequence was recognized whi ch was tandemly repeated four and a half times, a feature typical for many replication origins (ori) and commonly termed a DNA iteron. Analy sis of the repA upstream region by primer extension identified a trans cription start point for the repA mRNA, but did not correspond to know n consensus promoter sequences. Southern hybridizations using pHe11 as a probe under stringent conditions revealed that homologous sequences to pHe11 were present in nearly all plasmid-carrying Hp strains, but not in a plasmid-carrying Helicobacter felis strain, suggesting that t his type of replicon is predominantly found in the Hp species.