Single-step PCR amplification and enzyme restriction analysis of the entire Helicobacter pylori cytotoxin vacA gene for genetic variability studies.
ABSTRACT To monitor changes along the entire Helicobacter pylori vac A gene we carried out full-length single-step PCR amplification in 21 gastritis and gastric cancer isolates. HindIII restriction analysis led us to detect a > 400-bp internal insertion in vacA subsequently shown to be a direct 451-bp gene duplication. We found HindIII profiles for 16 genes that allowed their grouping into two restriction patterns that were related to theoretical profiles for previously sequenced Western genes. Comparisons with theoretical HindIII patterns for Japanese isolates appear suggestive of geographical H. pylori clonality. Full-length single-step PCR amplification seems suitable for quick restriction pattern assignment and detection of gene size changes.
Article: Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates.[show abstract] [hide abstract]
ABSTRACT: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.BMC Microbiology 01/2007; 7:26. · 3.04 Impact Factor