Sequencing and characterization of three plasmids from Helicobacter pylori strain AL236

Helicobacter pylori, a microaerophilic, spiral-shaped, Gram negative bacterium is a gastrointestinal pathogen. Plasmids from H. pylori are currently being sequenced and characterized in our laboratory to determine their role in H. pylori. A previous study indicated unexpectedly that H. pylori strain AL236 might contain three separate plasmid species. This observation was based on the insertion of the EZ-Tn5™ < R6Kγori/KAN-2> transposon into the total AL236 plasmid DNA, which yielded recombinant plasmids pAL236-2, pAL236-5, and pAL236-11. In this study, all three plasmids were sequenced and characterized.

Utilizing forward and reverse primers specific for the EZ-Tn5™ <R6Kγori/KAN-2> transposon, initial plasmid DNA sequence was obtained. To determine the remaining DNA sequences, unlabeled M13 forward and reverse tailing primers were designed for each recombinant plasmid and utilized to produce PCR products containing M13 primer sites. Sequencing of these PCR products provided the remaining DNA sequence for each plasmid from strain AL236. Results indicated that pAL236-2 is 1448 bp, pAL236-5 is 1216 bp, and pAL236-11 is 3148 bp.

Each plasmid sequence was submitted to BLAST at NCBI, and was reported to have identity with other H. pylori plasmids. Results also indicated the replication proteins of plasmid pAL236-2 and pAL236-5 had identity with H. pylori plasmids that replicate via the rolling-circle mechanism. For plasmid pAL236-11, results indicated the replication protein, RepB, had identity with H. pylori plasmids that replicate via the theta mechanism. Computer analysis of the plasmid sequences was used to determine open reading frames (ORF), promoter, and ribosomal binding sites (RBS).

Phylogenetic trees were constructed through ClustalW2 of known H. pylori plasmid replication proteins. Results showed the existence of three classes of H. pylori replication proteins which included RepA, RepB, and RepH. Both RepA and RepB are involved with theta replication while RepH is involved with rolling-circle replication.