Stancek Martin, Isaksson Leif A, Rydén-Aulin Monica
Department of Microbiology, Stockholm University, S-106 91 Stockholm, Sweden.
Microbiology (Reading). 2003 Sep;149(Pt 9):2427-2433. doi: 10.1099/mic.0.26337-0.
Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 degrees C but the level is still sufficient to support normal growth. At 42 degrees C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 degrees C, while a large decrease leads to a more pleiotropic phenotype.
温度敏感型大肠杆菌突变体72c的分离此前已有报道。该突变等位基因被命名为fusB，会导致多效性表型，除温度敏感性外，其最显著的特征是无法在合成培养基上生长以及对三甲氧苄氨嘧啶超敏感，三甲氧苄氨嘧啶是一种抑制C1代谢的抗生素。这项研究表明，fusB突变是编码烟酸单核苷酸腺苷酸转移酶的nadD基因中的一个移码突变。移码导致最后10个氨基酸发生变化，并额外添加了17个氨基酸。这种病变被重新命名为nadD72，在允许温度下导致极少的NAD+和NADPH合成，在非允许温度下基本不合成。作为对比，在nadD基因中分离出了一个新突变，其ATP结合位点发生了氨基酸变化。它在30℃时NAD+合成减少，但水平仍足以支持正常生长。在42℃时，NAD+合成进一步减少，这导致在基本培养基上出现温度敏感性。这个突变被命名为nadD74。因此，NAD+水平的小幅下降会影响在42℃时在基本培养基上的生长能力，而大幅下降则会导致更具多效性的表型。
