HELICOBACTER-PYLORI NICKEL-TRANSPORT GENE NIXA - SYNTHESIS OF CATALYTICALLY ACTIVE UREASE IN ESCHERICHIA-COLI INDEPENDENT OF GROWTH-CONDITIONS

Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastriti s and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structura l subunits are overexpressed, exogenous NICI2 is added, and the host s train is grown in medium that does not chelate free Ni2+. As wild-type H. pylori does not require such conditions for very high levels of ur ease expression, we reasoned that additional genes were required to ac cumulate the metal ion. To isolate such genes, E. coli SE5000 (pHP808) , which carries the H. pylori urease gene cluster, was complemented wi th a lambda, ZAP-derived plasmid library of the H. pylori chromosome, One of 1000 ampicillin-resistant clones, plated onto urea segregation agar, produced detectable urease, Urease activity of this co-transform ant, grown in Luria broth containing 1 mu M NiCl2, was 36 mu mol NH3 m in(-1) mg(-1) protein. Urease-enhancing activity, which is not directl y linked to the urease gene cluster, was localized by subcloning and n ucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34 317 Da that displayed characteristics o f an integral membrane protein. In vitro transcription-translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. A n in-frame Bal31 deletion within nixA abolished urease-enhancing activ ity. At 50 nM NICl2, E. coli containing the nixA clone transported 125 0 +/- 460 pmol Ni2+ min(-1) 10(-8) cells, whereas the vector control t ransported only 140 +/- 85 pmol Ni2+ min(-1) 10(8) cells, i.e, signifi cantly less (P = 0.01). We conclude that NixA confers upon E. coil a h igh-affinity nickel-transport system (K-T = 11.3 +/- 2.4nM; V-max = 17 50 +/- 220 pmol Ni2+ min(-1) 10(-8) cells) and is necessary for expres sion of catalytically active urease, regardless of growth conditions.