Di Girolamo P M, Kadner R J, Bradbeer C
J Bacteriol. 1971 Jun;106(3):751-7. doi: 10.1128/jb.106.3.751-757.1971.
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.
大肠杆菌KBT001是一种甲硫氨酸 - 维生素B12营养缺陷型菌株，发现在缺乏甲硫氨酸的情况下，有氧生长时每个细胞至少需要20个维生素B12（氰钴胺素，CN - B12）分子。在用N - 甲基 - N'-硝基 - N - 亚硝基胍诱变并经青霉素筛选后，从该菌株中分离出两种B12转运突变体。I类突变体，如KBT069，在CN - B12与细胞的初始快速结合方面存在缺陷，即使在CN - B12浓度高达100 ng/ml的情况下，在缺乏甲硫氨酸时也无法生长。II类突变体在CN - B12摄取的初始阶段是完整的，但在次级能量依赖阶段存在缺陷。这些菌株也无法将摄取的CN - B12转化为其他钴胺素。在缺乏甲硫氨酸的情况下，其中一些菌株（如KBT103）能够在含有1 ng/ml CN - B12的培养基上生长，而其他菌株（如KBT041）在所用的任何CN - B12浓度下都无法生长。渗透压休克处理不影响CN - B12的初始摄取速率，但会使次级摄取速率大幅下降。通过渗透压休克从细胞中释放出微量的B12结合大分子，但仅从具有CN - B12摄取活跃初始阶段的菌株如KBT001和KBT041中释放。这些结果被解释为与以下观点一致：在该转运系统中起作用的初始CN - B12结合位点可能与细胞膜结合。
