ISOLATION OF VITAMIN-B12 TRANSPORT MUTANTS OF ESCHERICHIA-COLI
ISOLATION OF VITAMIN-B12 TRANSPORT MUTANTS OF ESCHERICHIA-COLI
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DOI：
10.1128/jb.106.3.751-757.1971
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发表时间：
1971-01-01
影响因子：
3.2
通讯作者：
BRADBEER, C
中科院分区：
文献类型：
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作者：
DIGIROLA.PM;KADNER, RJ;BRADBEER, C
大肠杆菌KBT 001是一种蛋氨酸-维生素B12营养缺陷型菌，在不含蛋氨酸的条件下，每细胞需至少20个维生素B12分子（CN-B12）才能有氧生长。经N-甲基-N′-硝基-N-亚硝基胍诱变和青霉素筛选，从该菌株中分离到两种B12转运突变株。第一类突变体，如KBT 069，在CN-B-12与细胞的初始快速结合方面存在缺陷，即使在CN-B-12浓度高达100纳克/毫升的情况下，在没有甲硫氨酸的情况下也无法生长。II类突变体具有完整的CN-B12摄取的初始阶段，但在次级能量依赖阶段有缺陷。这些菌株也不能将摄取的CN-B 12转化为其他钴。在不存在甲硫氨酸的情况下，这些菌株中的一些（例如，KBT 103）能够在含有1 ng/ml氯化萘-B 12的培养基上生长，而其他菌株（例如，KBT 041）在所使用的任何CN-B 12浓度下均不能生长。渗透冲击处理不影响CN-B 12的初始摄取率，但使次级摄取率大幅下降。通过渗透压休克，从细胞中释放出痕量的B12结合大分子，但仅来自具有CN-B12摄取活跃初始相的菌株，如KBT 001和KBT 041。这些结果被解释为与在该转运系统中起作用的CN-B 12的初始结合位点可能与细胞膜结合的观点一致。
Escherichia coliKBT001, a methionine-vitamin B12auxotroph, 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 withN-methyl-N′-nitro-N-nitrosoguanidine and penicillin selection, two kinds of B12transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B12to the cell and were unable to grow in the absence of methionine even with CN-B12concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B12uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B12taken 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-B12concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B12but 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-B12uptake. These results are interpreted as being consistent with the view that the initial CN-B12binding site which functions in this transport system is probably bound to the cell membrane.