Isolation of Helicobacter pylori Genes That Modulate Urease Activity

Abstract

Helicobacter pylori urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH3 and CO2. We sought to identify H. pylori genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both H. pylori urease and the NixA nickel transporter. Escherichia coli SE5000 and DH5α transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An H. pylori DNA library was cotransformed into SE5000 (pHP8080) and DH5α (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained hp0548, a gene that potentially encodes a DNA helicase found within the cag pathogenicity island, and hp0511, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in E. coli (pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the flbA (also known asflhA) flagellar biosynthesis/regulatory gene (hp1041), an lcrD homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in E. coli (pHP8080) containing the subcloned flbA gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in E. coli (pHP8080) containing the flbA gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in H. pylori. These results suggest that H. pylori genes may modulate urease activity.

The selective utilization of substrates in vivo by the phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes Ept1p and Cpt1p in yeast

“…1B, white bars). The combined results indicate (i) that the nature of the CDPaminoalcohol substrate of Ept1p does not significantly influence its specificity for the DAG species used as lipid substrate; (ii) that Cpt1p activity affects the substrate use by Ept1p; (iii) that Ept1p plays only a minor role in PC biosynthesis in wildtype yeast in agreement with previous studies [10,29].…”

Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus

“…It includes an inversion of all cagPAI genes except cagA in conjunction with several flanking genes, and a second inversion comprising most of these flanking genes (hp0511-hp0518). Similar rearrangements would also account for earlier observations that the cagA gene is not adjacent to cagB in some strains [11,16]. Interestingly, the corresponding gene locus of Helicobacter acinonychis Sheeba, which is the closest relative to H. pylori known, but probably diverged before acquisition of the cagPAI [17], contains a similar inversion of the same flanking genes and also fragments of a helicase gene that is frequently found downstream of cagA ( Fig.…”

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily

“…When cultured under similar conditions, E. coli transformed with the rocF gene had substantially more specific arginase activity than wild type H. pylori. In contrast, E. coli expressing the H. pylori urease and nickel transporter genes from multicopy to high copy plasmids exhibited 10-fold lower urease activity than that obtained with H. pylori [33]. The higher arginase activity found in E. coli (pBS-rocF) compared to H. pylori correlated well with the greater amount of arginase protein detected in Western blots of E. coli extracts relative to H. pylori extracts.…”

“…coli containing the cloned rocF gene from H. pylori exhibited arginase activity and this activity was markedly higher when heat-activated with cobalt rather than manganese Arginase and urease are both multisubunit, metal cofactor-containing enzymes [11,32]. E. coli can express urease activity when transformed with eight H. pylori genes: the ureABIEFGH genes in the H. pylori urease locus and the nixA nickel transporter [33,34]. Expression of only ureAB (urease structural genes) or ureABIEFGH in E. coli yields little or no urease activity [33,35,36] because nixA is required for nickel transport into the cell, and the accessory proteins UreEFGH are needed for incorporation of nickel into the urease active site [34,37].…”

“…E. coli can express urease activity when transformed with eight H. pylori genes: the ureABIEFGH genes in the H. pylori urease locus and the nixA nickel transporter [33,34]. Expression of only ureAB (urease structural genes) or ureABIEFGH in E. coli yields little or no urease activity [33,35,36] because nixA is required for nickel transport into the cell, and the accessory proteins UreEFGH are needed for incorporation of nickel into the urease active site [34,37]. It was unknown how many H. pylori genes were required for arginase activity in E. coli.…”