Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori.
The aroB gene from Helicobacter pylori strain P1 was cloned and further characterized by sequence analysis and by functional complementation of the aroB mutation in Escherichia coli. The aroB gene encodes the enzyme 3-dehydroquinate synthase which catalyzes one of the early steps in the shikimate pathway. This pathway, which creates aromatic molecules from sugar precursors, is present in prokaryotes, fungi and plants but is absent from mammalian cells. The predicted amino acid sequence of the H. pylori aroB gene product showed significant homology (30-40% identity and 50-60% similarity) to 3-dehydroquinate synthases from various other prokaryotes and eukaryotes. The single gene on a plasmid was biologically active in E. coli. It suppressed the specific phenotype of aroB mutants by restoring the shikimate pathway-dependent synthesis of aromatic amino acids and the production of the siderophore enterobactin. Two other reading frames were found adjacent to the aroB gene. The first, designated as orf1, had no significant homology to proteins and genes present in databases, whereas the second was found to share a significant degree of homology with the tgt gene encoding tRNA-guanine transglycosylase from a variety of other bacteria (40-50% identity and 60-70% similarity). The function of the tgt gene was confirmed by heterologous complementation. The gene on a plasmid was shown to complement the queuosine biosynthesis defect in a genetically defined tgt- strain of E. coli. The presence of the aroB gene and the putative tgt homologue in unrelated H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers.
Bereswill
S
S
Institut für Medizinische Mikrobiologie, Abteilung Mikrobiologie und Hygiene, Freiburg, Germany. bereswil@sun1.ukl.uni-freiburg.de
Fassbinder
F
F
Völzing
C
C
Haas
R
R
Reuter
K
K
Ficner
R
R
Kist
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010430:Pentosyltransferases / Q000235:genetics*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012765:Shikimic Acid / Q000378:metabolism
D004269:DNA, Bacterial; D012765:Shikimic Acid; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases