Control Mechanism for Production and Activation of Helicobacter pylori Urease

To define the genes for production of catalytically active H. pylori urease, we camed out study to elucidate the structure of urease gene transcript, to delineate the genetic region which affected the extent of the expression and the activation of urease structural subunits. UreC and ureD were confirmed not to affect the expression of structural genes and active enzyme production, meaning that these genes are not components of the urease gene cluster of H. pylori. p-independent transcriptional stop signal was found in 12 bp down-stream of ureH stop codon. RNA extension test showed that the transcript starts with 267 bp upstream of ureA start codon. Although accessory genes did not affect the extent of the expression of the structural subunits, they were essential for assembling the active urease in E. coli. E. coli transformants of plasmid clones containing ureAB produced catalytically active urease when they are complemented with the plasmid clones of ureIEFGH or coexisted with ureIEFGH, meaning that accessory gene products could be trans-acting as well as cis-acting. The extent of production of urease structural subunits depended on the region of 241 to 57 bp upstream of ureA start codon. E. coli transformant of pBeloBACII clone containing the urease gene cluster, which is maintained with a single copy in host, did not express the urease. Proteins (60, 38, 30, 29, 27, and 24 kDa) that could hold nickel ions were identified in the cell extract of H. pylori. The results in this study will provide the basis to understand the control mechanism for urease gene expression and formation of the active urease.

Physical map of the Helicobacter pylori Chromosome

Helicobacter pylori is a causative agent of type B gastritis and plays a central role in the pathogenesis of gastroduodenal ulcers and gastric cancer. Strategies for the control of H. pylori- induced gastroduodenal diseases based on conventional measures are still of limited utility. Therefore, it seems worthwhile to make a break-through as an alternative strategy by reviewing the host-parasite relationship of H. pylori infection on the basis of genomic structure. In this study, we tried to construct a physical map of H. pylori genome. Chromosomal DNA from a Korean prototype strain, H. pylori 51 was digested with 42 restriction endonucleases to identify restriction patterns suitable for mapping the genome. We identified three enzymes, ApaI, NotI and Sfil, which gave a small number of DNA fragments of higher molecular weight that were well resolved after pulsed-field gel electrophoresis. The H. pylori chromosome contained 7 ApaI fragments ranging from 167 to 311 kb, 7 NotI fragments ranging from 5 to 516 kb and 2 SfiI fragments of 332 and 1,347 kb in size. The genome size of the strain is 1,679 kb. A circular physical map of the H. pylori chromosome was constructed by aligning 3 kinds of restriction fragments by Southern blot analysis of simple ApaI, NotI and SfiI digests or double NotI/ApaI and NotI/SfiI digests with the various probes. When the physical map of H. pylori strain 51 compared with that of strain 26695 of which the cornplete genome sequence was reported, completely different restriction patterns were shown, which suggests the genomic diversity in H. pylori.

Molecular Cloning and Nucleotide Sequence of a Gene Encoding Alcohol Dehydrogenase of Helicobacter pylori

Partially purified H. pylori ADH was used to determine the amino acid sequence of ADH N- terminus. The sequence of the ADH N-terminus was determined as MRVQSKGF. The genomic library of H. pylori that has been prepared by pTZ19U plasmid vector was screened with the deduced oligonucleotide probes to select the plasmid clone containing the entire ADH gene. The clone pTZ19U/ADH-6 was selected and its EcoRI-BamHI fragment (1.3 kb) was subcloned into pBluescript II K/S vector to determine nucleotide sequence. The length of H. pylori ADH gene was 1,044 bp. Ribosomal binding site was found in the upstream of start codon and rho- independent transcriptional stop signal was observed in the downstream of stop codon. The ADH gene encodes a protein of 348 amino acids, of which the predicted molecular size and pI value were 38.6 kDa and 7.1, respectively. ADH activity of E. coli transformant of pBluescript/ADH is 10-times greater compared to that of non-transformants. When H. pylori ADH gene was disrupted by pBluescript/ADH-KM whose internal region of 1.3 kb DNA fragment containing ADH gene was replaced by KM resistance sequence, the strain lost the ADH activity completely, despite the normal growth of the strain. This demonstrates that ADH gene is not essential for the viability of H. pylori.

Characterization and Sequence Analysis of Helicobacter pylori Cryptic Plasmid (pHP489)

The DNA sequence of a plasmid named pHP489 of Helicobacter pylori strain 489 was determined and analyzed to characterize its replication apparatus. The pHP489 plasmid consisted of 1,222 bp and had an overall G+C content of 33.1%. An ORF was predicted to encode the putative protein of 239 amino acid residues (28 kDa). A putative ribosomal binding site and a potential terminator sequence are located upstream and downstream of the ORF, respectively. However, the consensus sequence for a promoter in upstream of ORF was not found. A potential dna A box was found at 317 nt upstream of a start codon and followed by two-57 bp directed repeats and an inverted repeat. The DNA homology was found in the regions of less than 90 bp among pHPK255, pHPM180, and pHel1 of other H. pylori plasmids and Mycoplasma mycoides plasmids. pHP489K that was produced by pHP489 sequence and C. jejuni derived aph(3')-III, was transformed to various H. pylori isolates and were stably maintained in the H. pylori host without the addition of selective antibiotics for the 30-times subcultues. The plasmic vector, in which the ORF region of pHP489 DNA was deleted, could be transformed into H. pylori. However, the plasmid vector, whose the direct repeats region of pHP489 DNA was deleted, failed to be transformed. The direct repeats region of pHP489 DNA was confirmed to be bound with cytosolic factors of H. pylori. These results showed that the direct repeats region of pHP489 DNA is an essential apparatus by which the plasmid could be replicacted in H. pylori. And pHP489 plasmid was supposed to be replicated by host factors rather than plasmic-encoded factors.