Abstract
Escherichia coli KBT001, a methionine-vitamin B12 auxotroph, was found to require a minimum of 20 molecules of vitamin B12 (CN-B12) 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 B12 transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B12 to the cell and were unable to grow in the absence of methionine even with CN-B12 concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B12 uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B12 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-B12/ml, whereas others (e.g., KBT041) were unable to grow with any of the CN-B12 concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B12 but gave a substantial decrease in the secondary rate. Trace amounts of B12-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-B12 uptake. These results are interpreted as being consistent with the view that the initial CN-B12 binding site which functions in this transport system is probably bound to the cell membrane.
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