Thiamine uptake in Escherichia coli: II. The isolation and properties of a mutant of Escherichia coli defective in thiamine uptake

Abstract

A mutant (KG 900) of Escherichia coli, which requires a high concentration of thiamine for growth, has been isolated from a strain KG 33, which is a mutant of E. coli K12, auxotrophic for the thiazole moiety of thiamine and requires a low concentration of either thiamine or thiamine thiazole for growth. The isolation procedure includes mutation by nitrosoguanidine treatment, subsequent growth at a high concentration of thiamine, and penicillin screening during cultivation of the cells at a low thiamine concentration. Thiamine concentration required for a half maximal growth of KG 900 was approximately 150-fold higher than that of the parent KG 33. Thiamine thiazole requirement for growth of KG 900 is only 2-fold that of KG 33. Uptake of 14C-thiamine by cells of KG 900 is negligible when incubated with 0.2, 1.0, and 5.0 μ pm 14C-thiamine, respectively, for 30 min at 37 ° in the presence of 0.4% glucose, whereas the parent cells take up 14C-thiamine readily from the external medium. The rates of exit of 14C-thiamine taken up by cells of both strains were almost identical. With KG 900 the initial rate and total extent of exchange at 0 ° between a high intracellular thiamine concentration and a low level of extracellular 14C-thiamine were approximately a half as great as those observed with KG 33. Thiamine kinase activity in the sonic membrane fraction, and the activities of four soluble enzymes involved in thiamine synthesis from the pyrimidine and thiazole moieties were higher in preparations from KG 900 than those from the parent strain, KG 33. The results strongly suggest that the defect manifested by strain KG 900 is in the passage of thiamine through the cell membrane. A model for the system of thiamine uptake in E. coli is presented, which assumes that a “carrier” specific for thiamine functions in transporting thiamine across the cell membrane and then thiamine kinase (ATP-thiamine pyrophosphotransferase, EC 2.7.6.2) in the membrane facilitates accumulation of thiamine as thiamine pyrophosphate in the cytoplasm.