Lathrop J T, Wei B Y, Touchie G A, Kadner R J
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908, USA.
J Bacteriol. 1995 Dec;177(23):6810-9. doi: 10.1128/jb.177.23.6810-6819.1995.
The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.
大肠杆菌的btuB基因编码维生素B12、大肠杆菌素、大肠杆菌素A和噬菌体BF23的外膜转运蛋白。构建了一系列由二肽序列插入以及框内重叠缺失和重复导致的BtuB突变形式。分析了以单拷贝和多拷贝形式表达变异基因的菌株的BtuB功能、外膜中BtuB多肽的水平以及外膜通透性屏障的变化。大多数二肽插入具有正常的转运功能且能在膜中组装。在跨越第5位和第514位残基的27个缺失中，只有2个具有转运功能，其余大多数不能稳定地插入膜中。大多数重复（21个中的19个）保留了转运功能并插入到外膜中，尽管有些会被蛋白酶水解。即使是含有多达340个重复氨基末端残基的长重复序列也保留了功能，这表明BtuB膜插入的序列要求具有相当大的可塑性。许多缺失和重复蛋白的表达使细胞对通常被外膜排除的结构不相关抑制剂的敏感性增加。这些结果可能与构成门控通道的BtuB细胞外环或跨膜段的突变破坏一致，但BtuB全长的改变都会影响膜通透性特性这一发现表明，改变后的蛋白质可能会扰乱外膜结构本身。
