Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

Abstract

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previousl shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 + 0.39 ,umol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E.coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreHl), and 19,819 (Urel). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coil, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the KiebsieUla aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes),. whereas this homology was restricted to a

In Vivo Interactome of Helicobacter pylori Urease Revealed by Tandem Affinity Purification

“…These comprised the following interactions: UreA with UreB, UreA with UreH, UreE with UreG, and UreF with UreH; the two latter interactions were confirmed by Y2H and co-immunoprecipitation (47). Phenotypes of insertion mutations in the corresponding urease genes in H. pylori were in agreement with their implication in urease activation (47,48). In addition, our data are consistent with biochemical data obtained for urease of Klebsiella aerogenes.…”

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

“…Indeed, coexpression of the ureI, ureE, ureF, ureG and ureH genes was shown to be required along with structural genes ureA and ureB to yield fully active urease in E. coli (Cussac et al 1992). More recently, Voland and coworkers have shown that H. pylori ureE, ureF, ureG and ureH mutants are severely deficient in urease activity (Voland et al 2003).…”

Genetic Transformation in Helicobacter pylori

“…The gram-negative spiral bacterium Helicobacter pylori, previously named as Campylobacter pylori (6), is an etiological agent of active chronic gastritis and is considered to play an important role in peptic ulcer in human beings (1). A variety of potential pathogenic factors of this microorganism have been so far identified which include urease, extracellular enzymes, hemolysin, and cytotoxin(3), and for example, the genes responsible for the urease activity have been cloned and analyzed in Escherichia coli (2). Unfortunately, lack of extensive studies on the genetic exchange systems in H. pylori has been the bottleneck for unambiguous identification of its pathogenic factor(s).…”