Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.
Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate. When wild-type H. pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation. Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H. pylori despite evidence that insertional mutagenesis had occurred. The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies. The data imply that the H. pylori dapE mutant may be useful as a conditionally lethal vaccine.
Karita
M
M
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605, USA.
Etterbeek
M L
ML
Forsyth
M H
MH
Tummuru
M K
MK
Blaser
M J
MJ
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003960:Diaminopimelic Acid / Q000378:metabolism; D005798:Genes, Bacterial; D005804:Genes, Lethal; D016480:Helicobacter pylori / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D009876:Operon; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000581:Amidohydrolases; D001426:Bacterial Proteins; D003960:Diaminopimelic Acid; C108512:glucose-inhibited division protein A, bacteria; C054685:N-succinyl-l,l-diaminopimelic acid desuccinylase