Nishijima M, Bulawa C E, Raetz C R
J Bacteriol. 1981 Jan;145(1):113-21. doi: 10.1128/jb.145.1.113-121.1981.
A conditionally lethal mutant of Escherichia coli lacking phosphatidylglycerol in vivo at 42 degrees C has been previously isolated by two-stage mutagenesis (M. Nishijima and C. R. H. Raetz, J. Biol. Chem. 254:7837-7844, 1979). In the first step (designated pgsA444) the phosphatidylglycerophosphate synthetase is partially inactivated, but the resulting strain continues to make about two-thirds of the normal level of phosphatidylglycerol and is not temperature sensitive. The second lesion, termed pgsB1, causes temperature-sensitive growth and phosphatidylglycerol synthesis in strains harboring pgsA444. The pgsA locus appears to be the structural gene for the synthetase and maps near min 42. In the present study we mapped the pgsB1 mutation and characterized its interaction with pgsA444 by genetic and biochemical methods. Unexpectedly, pgsB1 was not a second lesion in the pgsA structural gene, but rather mapped at a distinct site near minute 4. P1 vir-mediated contransduction suggested the gene order pantonA-dapD-pgsB-dnaE (clockwise). Independent evidence for the genetic mapping was provided by the identification of two hybrid ColE1 plasmids (pLC26-43 and pLC34-20. L. Clarke and J. Carbon, Cell 9:91-99, 1976) which both carry pgsB+ and dnaE+. Introduction of either the pgsA+ or the pgsB+ gene (via episomes, hybrid plasmids or P1 vir transduction) suppressed the temperature sensitivity of the double mutant (pgsA444 pgsB1) and restored normal levels of phosphatidylglycerol at 42 degrees C. In addition, strains with the pgsA+ pgsB1 genotype produced a novel lipid (X) at all temperatures, whereas the double mutant (pgsA444 pgsB1) contained two unusual lipids (X and Y) after 3 h at 42 degrees C. Both X and Y are precursors of lipopolysaccharide, and introduction of pgsB+ into the double mutant caused the disappearance of X and Y. Although the biochemical basis of the pgsB1 lesion is unknown, its existence suggests a previously unrecognized link between lipopolysaccharide and phosphatidylglycerol syntheses in E. coli.
通过两阶段诱变，先前已分离出一种大肠杆菌的条件致死突变体，该突变体在42℃时体内缺乏磷脂酰甘油（M. Nishijima和C. R. H. Raetz，《生物化学杂志》254:7837 - 7844，1979）。在第一步（命名为pgsA444）中，磷脂酰甘油磷酸合成酶部分失活，但所得菌株继续产生约正常水平三分之二的磷脂酰甘油，并且对温度不敏感。第二个损伤，称为pgsB1，导致携带pgsA444的菌株出现温度敏感生长和磷脂酰甘油合成。pgsA位点似乎是合成酶的结构基因，定位在约42分钟处。在本研究中，我们通过遗传和生化方法对pgsB1突变进行了定位，并表征了其与pgsA444的相互作用。出乎意料的是，pgsB1不是pgsA结构基因中的第二个损伤，而是定位在约4分钟处的一个不同位点。P1噬菌体介导的共转导表明基因顺序为pantonA - dapD - pgsB - dnaE（顺时针）。通过鉴定两个携带pgsB⁺和dnaE⁺的杂种ColE1质粒（pLC26 - 43和pLC34 - 20。L. Clarke和J. Carbon，《细胞》9:91 - 99，1976），为遗传定位提供了独立证据。引入pgsA⁺或pgsB⁺基因（通过附加体、杂种质粒或P1噬菌体转导）可抑制双突变体（pgsA444 pgsB1）的温度敏感性，并在42℃时恢复磷脂酰甘油的正常水平。此外，具有pgsA⁺ pgsB1基因型的菌株在所有温度下都产生一种新的脂质（X），而双突变体（pgsA444 pgsB1）在42℃处理3小时后含有两种异常脂质（X和Y）。X和Y都是脂多糖的前体，将pgsB⁺引入双突变体导致X和Y消失。尽管pgsB1损伤的生化基础尚不清楚，但其存在表明大肠杆菌中脂多糖和磷脂酰甘油合成之间存在先前未被认识的联系。
