title: An unusual genetic link between vitamin B6 biosynthesis and tRNA pseudouridine modification in Escherichia coli K-12.
abstract: We characterized several unusual phenotypes caused by stable insertion mutations in a gene that is located upstream in the same operon from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. Mutants containing kanamycin resistance (Kmr) cassettes in this upstream gene, which we temporarily designated usg-2, failed to grow on minimal plus glucose medium at 37 and 42 degrees C. However, usg-2::Kmr mutants did form oddly translucent, mucoid colonies at 30 degrees C or below. Microscopic examination revealed that cells from these translucent colonies were spherical and seemed to divide equatorially. Addition of D-alanine restored the shape of the mutant cells to rods and allowed the mutants to grow slowly at 37 degrees C and above. By contrast, addition of the common L-amino acids prevented growth of the usg-2::Kmr mutants, even at 30 degrees C. Furthermore, prolonged incubation of usg-2::Kmr mutants at 37 and 42 degrees C led to the appearance of several classes of temperature-resistant pseudorevertants. Other compounds also supported growth of usg-2::Kmr mutants at 37 and 42 degrees C, including glycolaldehyde and the B6 vitamers pyridoxine and pyridoxal. This observation suggested that usg-2 was pdxB, which had been mapped near hisT. Complementation experiments confirmed that usg-2 is indeed pdxB, and inspection of the pyridoxine biosynthetic pathway suggests explanations for the unusual phenotypes of pdxB::Kmr mutants. Finally, Southern hybridization experiments showed that pdxB and hisT are closely associated in several enterobacterial species. We consider reasons for grouping pdxB and hisT together in the same complex operon and speculate that these two genes play roles in the global regulation of amino acid metabolism.
mesh_terms: D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008040:Genetic Linkage; D019751:Intramolecular Transferases; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D010957:Plasmids; D011560:Pseudouridine / Q000235:genetics / Q000378:metabolism*; D011736:Pyridoxine / Q000096:biosynthesis*; D012343:RNA, Transfer / Q000235:genetics*; D014529:Uridine / Q000031:analogs & derivatives*
chemical_list: D011560:Pseudouridine; D012343:RNA, Transfer; D004262:DNA Restriction Enzymes; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I; D011736:Pyridoxine; D014529:Uridine