Helicobacter pylori ABC transporter: Effect of allelic exchange mutagenesis on urease activity

Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity, Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H, pylori retain significant urease activity, suggesting the presence of alternate nickel transporters, Analysis of the nucleotide sequence of the H, pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli, Based on this sequence, a 378-bp DNA fragment was PCR amplified from H, pylori genomic DNA and used as a probe to identify an H, pylori lambda ZAPII genomic library clone that carried these sequences, Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively, The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E, coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases, To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H, pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mu mol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mu mol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mu mol of NH3/min/mg of protein in the parent [P < 0.0001]), Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abe genes, We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.