title: Helicobacter pylori glutamine synthetase lacks features associated with transcriptional and posttranslational regulation.
abstract: Helicobacter pylori urease, produced in abundance, is indispensable for 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 protein by glutamine synthetase (EC 6.3.1.2), which catalyzes the reaction: NH3 + glutamate + ATP-->glutamine + ADP + Pi. We hypothesized that glutamine synthetase plays an unusually critical role in nitrogen assimilation by H. pylori. We developed a phenotypic screen to isolate genes that contribute to the synthesis of a catalytically active urease. Escherichia coli SE5000 transformed with plasmid pHP808 containing the entire H. pylori urease gene cluster was cotransformed with a pBluescript plasmid library of the H. pylori ATCC 43504 genome. A weakly urease-positive 9.4-kb clone, pUEF728, was subjected to nucleotide sequencing. Among other genes, the gene for glutamine synthetase was identified. The complete 1,443-bp glnA gene predicts a polypeptide of 481 amino acid residues with a molecular weight of 54,317; this was supported by maxicell analysis of cloned glnA expressed in E. coli. The top 10 homologs were all bacterial glutamine synthetases, including Salmonella 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 (residues 405 to 410). Since the Tyr (Y) residue is the target of adenylation and since the H. pylori glutamine synthetase lacks that residue in four strains examined, we conclude that no adenylation occurs within this motif. Cloned H. pylori glnA complemented a glnA mutation in E. coli, and GlnA enzyme activity could be measured spectrophotometrically. In an attempt to produce a GlnA-deficient mutant of H. pylori, a kanamycin resistance cassette was cloned into the Tth111I site of H. pylori glnA. By using the standard technique of allelic exchange mutagenesis, no verifiable glutamine synthetase double-crossover mutant of strain UMAB41 could be isolated, suggesting that the mutation is lethal. We conclude that glutamine synthetase is critical for nitrogen assimilation in H. pylori and is active under all physiologic conditions.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Garner
authors.forename: J
authors.initials: J
authors.lastname: Fulkerson
authors.forename: H L
authors.initials: HL
authors.lastname: Mobley
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014510:Urease / Q000096:biosynthesis
chemical_list: D014510:Urease; D005974:Glutamate-Ammonia Ligase