Genetic organization of a small cryptic plasmid of Helicobacter pylori

Abstract A 2.9-kb cryptic plasmid of Helicobacter pylori (Hp) , pHell, was isolated and the complete nucleotide (nt) sequence was determined. An open reading frame (ORF1) was identified encoding a putative polypeptide of 63 709 Da, the existence and correct size of which was confirmed by T7 promoter expression analysis. The ORF1 sequence showed strong amino-acid sequence identity to a recently identified putative ORF1 protein of a cryptic Hp plasmid, pHPM180, and significant homologies to putative Rep proteins of Campylobacter coli (RepB) and Pediococcus halophilus (RepA), and was therefore designated RepA. A functional role of RepA in replication of pHell was demonstrated by the fact that only pHell plasmid derivatives with an intact repA gene were able to autonomously replicate in Hp . Upstream of repA , a 22-bp sequence was recognized which was tandemly repeated four and a half times, a feature typical for many replication origins ( ori ) and commonly termed a DNA iteron. Analysis of the repA upstream region by primer extension identified a transcription start point for the repA mRNA, but did not correspond to known consensus promoter sequences. Southern hybridizations using pHell as a probe under stringent conditions revealed that homologous sequences to pHell were present in nearly all plasmid-carrying Hp strains, but not in a plasmid-carrying Helicobacter felis strain, suggesting that this type of replicon is predominantly found in the Hp species.