Isolation of Vitamin B 12 Transport Mutants of Escherichia coli

Abstract

Escherichia coli KBT001, a methionine-vitamin B ₁₂ auxotroph, was found to require a minimum of 20 molecules of vitamin B ₁₂ (CN-B ₁₂ ) per cell for aerobic growth in the absence of methionine. After mutagenesis with N -methyl- N ′-nitro- N -nitrosoguanidine and penicillin selection, two kinds of B ₁₂ transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B ₁₂ to the cell and were unable to grow in the absence of methionine even with CN-B ₁₂ concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B ₁₂ uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B ₁₂ taken up into other cobalamins. In the absence of methionine, some of these strains (e.g., KBT103) were able to grow on media containing 1 ng of CN-B ₁₂ /ml, whereas others (e.g., KBT041) were unable to grow with any of the CN-B ₁₂ concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B ₁₂ but gave a substantial decrease in the secondary rate. Trace amounts of B ₁₂ -binding macromolecules were released from the cells by the osmotic shock, but only from strains such as KBT001 and KBT041 which possessed an active initial phase of CN-B ₁₂ uptake. These results are interpreted as being consistent with the view that the initial CN-B ₁₂ binding site which functions in this transport system is probably bound to the cell membrane.