HELICOBACTER-PYLORI GLUTAMINE-SYNTHETASE LACKS FEATURES ASSOCIATED WITH TRANSCRIPTIONAL AND POSTTRANSLATIONAL REGULATION

Helicobacter pylori urease, produced in abundance, is indispensable fo r the survival of H. pylori in animal hosts. Urea is hydrolyzed by the enzyme, resulting in the liberation of excess ammonia, some of which neutralizes gastric acid, The remaining ammonia is assimilated into pr otein by glutamine synthetase (EC 6.3.1.2), which catalyzes the reacti on: NH3 + glutamate + ATP-->glutamine + ADP + P-i, We hypothesized tha t glutamine synthetase plays an unusually critical role in nitrogen as similation by H. pylori, We developed a phenotypic screen to isolate g enes that contribute to the synthesis of a catalytically active urease , Escherichia coli SE5000 transformed with plasmid pHP808 containing t he entire H. pylori urease gene cluster was cotransformed with a pBlue script plasmid library of the H. pylori ATCC 43504 genome. A weakly ur ease-positive 9.4-kb clone, pUEF728, was subjected to nucleotide seque ncing, Among other genes, the gene for glutamine synthetase was identi fied. The complete 1,443-bp glnA gene predicts a polypeptide of 481 am ino acid residues with a molecular weight of 54,317; this was supporte d by maxicell analysis of cloned glnA expressed in E. coli, The top 10 homologs were all bacterial glutamine synthetases, including Salmonel la typhimurium glnA, The ATP-binding motif GDNGSG (residues 272 to 277 ) of H. pylori GlnA exactly matched and aligned with the sequence in 8 of the 10 homologs, The adenylation site found in the top 10 homologs (consensus sequence, NLYDLP) is replaced in H. pylori by NLFKLT (resi dues 405 to 410), Since the Tyr (Y) residue is the target of adenylati on and since the H. pylori glutamine synthetase lacks that residue in four strains examined, we conclude that no adenylation occurs within t his motif, Cloned H. pylori glnA complemented a glnA mutation in E. co li, and GlnA enzyme activity could be measured spectrophotometrically. In an attempt to produce a GlnA-deficient mutant of H. pylori, a kana mycin resistance cassette was cloned into the Tth111I site of H. pylor i glnA, By using the standard technique of allelic exchange mutagenesi s, no verifiable glutamine synthetase double crossover mutant of strai n UMAB41 could be isolated, suggesting that the mutation is lethal. We conclude that glutamine synthetase is critical for nitrogen assimilat ion in H. pylori and is active under all physiologic conditions.