CLONING AND FUNCTIONAL-CHARACTERIZATION OF THE GENES ENCODING 3-DEHYDROQUINATE SYNTHASE (AROB) AND TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE (TGT) FROM HELICOBACTER-PYLORI
S. Bereswill et al., CLONING AND FUNCTIONAL-CHARACTERIZATION OF THE GENES ENCODING 3-DEHYDROQUINATE SYNTHASE (AROB) AND TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE (TGT) FROM HELICOBACTER-PYLORI, Medical microbiology and immunology, 186(2-3), 1997, pp. 125-134
The aroB gene from Helicobacter pylori strain P1 was cloned and furthe
r characterized by sequence analysis and by functional complementation
of the aroB mutation in Escherichia coli. The aroB gene encodes the e
nzyme 3-dehydroquinate synthase which catalyzes one of the early steps
in the shikimate pathway. This pathway, which creates aromatic molecu
les 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 v
arious other prokaryotes and eukaryotes. The single gene on a plasmid
was biologically active in E. coli. It suppressed the specific phenoty
pe of aroB mutants by restoring the shikimate pathway-dependent synthe
sis of aromatic amino acids and the production of the siderophore ente
robactin. Two other reading frames were found adjacent to the aroB gen
e. The first, designated as orf1, had no significant homology to prote
ins and genes present in databases, whereas the second was found to sh
are a significant degree of homology with the tgt gene encoding tRNA-g
uanine transglycosylase from a variety of other bacteria (40-50% ident
ity and 60-70% similarity). The function of the tgt gene was confirmed
by heterologous complementation. The gene on a plasmid was shown to c
omplement the queuosine biosynthesis defect in a genetically defined t
gt(-) strain of E. coli. The presence of the aroB gene and the putativ
e tgt homologue in unrelated H. pylori strains was confirmed by Southe
rn blot hybridization and by polymerase chain reaction with specific p
rimers.