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.