Cloning and sequence analysis of plasmid DNA from Helicobactor pylori

The bacterium Helicobacter pylori is recognized as the cause of some types of gastritis and gastric ulcers. Many bacterial species contain plasmid DNA which encodes virulence factors, including antibiotic resistance, adherence factors, degradative enzymes, and toxins. The majority of H. pylori strains contain plasmid DNA, although the size varies from strain to strain. No phenotype has been assigned to the plasmids of H. pylori. The purpose of this study was to characterize the plasmid, pHPM179, isolated from strain HPM179.

Three HindIII fragments (2.2, 1.6, and 1.1 kb) were ligated into the vector pTZ19R and transformed into E. coli DH5$\alpha$F$\backslash sp\prime$ and DH5$\alpha$MCR for sequence determination. The DNA sequence was determined using the dideoxy ribonucleotide chain termination method. This sequence was added to the sequence of the 684 bp HindIII fragment sequenced previously by Chen (Chen, MS thesis 1993, Denton, TX).

The complete DNA sequence was analyzed using DNAsis and DNA inspector IIe computer programs, as well as BLASTP and BLASTN programs provided by the NIH. Three open reading frames (ORFs) were determined: ORF1 could encode a protein of 52 kDa; ORF2 could encode a protein of 10.8 kDa; and ORF3 could encode a protein of 31.9 kDa.

Comparison of the pHPM179 DNA sequence with those in the GenBank revealed matches with other H. pylori sequences. Found were regions of near identity with H. pylori pHPM180, as well as regions highly homologous to another H. pylori plasmid pHPK255. In addition a short stretch of near identity to the chromosomal cagA gene (cytotoxin associated gene) was detected.

The results also indicated that the ORF1 encoded protein was homologous to replication proteins found in nine other plasmids isolated from various organisms, including Campylobacter, Pediococcus, Pseudomonas, Lactococcus, Neisseria, and Klebsiella. Similarly to these other plasmids, pHPM179 contained four 22 bp iterons, i.e. direct repeats that are thought to be the binding site for these replication proteins. Based on this analysis, pHPM179 was predicted to replicate via a theta type mechanism.