Genetic mapping with a thiamine-requiring auxotroph of Escherichia coli K-12 defective in thiamine phosphate pyrophosphorylase.

The pathway of thiamine biosynthesis in Escherichia coli from its pyrimidine and thiazole moieties involves four enzymes (Y. Nose et al., J. Vitaminol. Kyoto 10:105, 1964): hydroxymethylpyrimidine kinase (EC 2.7.1.49), phosphohydroxymethylpyrimidine kinase (EC 2.7.4.7), thiazole kinase (EC 2.7.1.50), and thiamine phosphate pyrophosphorylase (EC 2.5.1.3). A mutant of E. coli W auxotrophic for thiamine (70-23) was shown to be lacking hydroxymethylpyrimidine kinase (Y. Nose et al., J. Vitaminol. Kyoto 10:105, 1964). The genetic determinants specifying information for these four enzymes have not yet been mapped on the chromosome. The thi locus mapped on the chromosome map of E. coli (A. L. Taylor and M. S. Thoman, Genetics 50:659, 1964) has been assumed to be a locus concerned with the synthesis of the thiazole moiety of thiamine. This is based on findings which show that two thiamineless mutants of E. coli K-12 (W4162 and AB1450, obtained from Y. Hirota of Osaka University), with or without other nutritional requirements, are auxotrophic for the thiazole moiety of thiamine as determined by requirement for growth (T. Kawasaki and Y. Nose, unpublished data). A similar finding was recently reported for Salmonella typhimurium (J. L. Parada and M. V. Ortega, J. Bacteriol. 94:707, 1967). As part of a study to locate the genetic determinants of the four enzymes in thiamine synthesis from its pyrimidine and thiazole moieties, mutants which require only thiamine, but not the pyrimidine or thiazole moiety, have been isolated from E. coli K-12 by treatment with N-methyl-N'-nitroN-nitrosoguanidine (E. A. Adelberg, M. Mandel, and G. C. C. Chen, Biochem. Biophys. Res. Commun. 18:788, 1965) and subsequent selection on minimal agar containing 0.01 AM thiamine. Among those mutants obtained, a mutant (KG167) and its Hfr strain (KG1673) were defective in thiamine phosphate pyrophosphorylase (Table 1). The activity of the other three enzymes was comparable with those in a mutant of E. coli W auxotrophic for the thiazole moiety (26-43), which was grown under the same condition of thiamine limitation as used for KG1673. Mapping on the chromosome of thi, which contains the mutation affecting the enzyme described above, was carried out by crossing the Hfr KG1673 with appropriate Fstrains according to procedures reported by M. Ishibashi, Y. Sugino, and Y. Hirota (J. Bacteriol. 87:554, 1964). All strains used for mapping were derivatives of E. coli K-12; strains AB261, W4573, and AB1450 were kindly supplied by Y. Hirota. KG1673 was obtained from KG167 (F+ thilac+) by a threestep procedure. Strain KG1671 (F+ thilac-) was isolated from KG167 by nitrosoguanidine treatment, and then acridine orange treatment (Y. Hirota, Proc. Natl. Acad. Sci. U.S. 46:57, 1960) was used to convert KG1671 to the corresponding Fstrain (KG1672). A cross, KG1672 (Fthimet+ lacj) x AB261 (Hfr thi+ metlac+), and selection of recombinants on minimal eosinemethylene blue (EMB)-lactose-agar (J. Lederberg, Methods Med. Res. 3:5, 1950) supplemented with 0.01 ,M thiamine yielded KB1673, which transfers the chromosome in the same clockwise order (pro ara xyl mal gal lac) as the donor strain AB261. In a preliminary experiment to locate the thi locus, W4573 (Farathi+ xylmalgallacstr-r) was used as a partner in a cross with KG1673 (Hfr ara+ thixyl+ mal+ gal+ lac+ str-s). Recombinants of ara+ str-r, xyl+ str-r, and mal+ str-r) were selected on appropriate EMB-sugaragar (J. Lederberg, Methods Med. Res. 3:5, 1950) containing streptomycin (100 ,g/ml) and were purified twice on the same agar medium. Overnight cultures of the recombinants in minimal medium (B. D. Davis and E. S. Mingioli, J. Bacteriol. 60:17, 1950) supplemented with 0.01 ,uM thiamine were streaked on appropriate EMBsugar-agar and also minimal agar with or without 0.01 tuM thiamine. After overnight incubation at 37 C, unselected markers were scored. The thi locus was found to be linked 4.1, 22, and 3.47% to ara, xyl, and mal genes, respectively.