A triple mutant of Escherichia coli lacking secondary acyl chains on lipid A
A triple mutant of Escherichia coli lacking secondary acyl chains on lipid A
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DOI：
10.1074/jbc.m200409200
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发表时间：
2002-04-19
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
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作者：
Vorachek-Warren, MK;Ramirez, S;Raetz, CRH
在大肠杆菌K12 W3110中产生了编码脂质A生物合成的酰基载体蛋白依赖的晚期酰基转移酶lpxL（htrB）、lpxM（msbB）和lpxP的三个基因的所有可能的插入突变组合。lpxM缺陷突变体合成五酰化脂质A分子并正常生长。缺乏lpxP的菌株在12℃的温度下不能将棕榈油酸酯纳入其脂质A中，但能产生正常数量的六酰化脂质A，并能存活。尽管lpxL突变体和lpxL lpxM双突变体在所有温度下的最低培养基上生长缓慢，但它们在32℃以上的营养液上不能生长。这些突变体保留了合成一些五酰化和六酰化脂质A分子的能力，因为在30℃的温度下lpxP的诱导有限，而在32℃以上则没有。MKV15是一种大肠杆菌lpxL、lpxM、lpxP三重突变体，同样在所有温度的最低培养基上生长缓慢，但在任何温度的营养液上都不能生长。MKV15合成一种脂质a分子，只含有4个伯(R)-3-羟基肉豆蔻酰基链。MKV15的外膜定位和脂质A含量、甘油磷脂和膜蛋白组成几乎正常。然而，与野生型相比，MKV15的四酰化脂质A输出到外膜的速率降低了。根据抗生素过敏判断，MKV15外膜的完整性受到损害，MKV15在离心后发生裂解。MKV15可能被证明是一种有用的宿主菌株，可以表达来自其他革兰氏阴性菌的晚期酰基转移酶基因，促进活细胞中脂质a结构的重组和新型疫苗的设计。
All possible combinations of insertion mutations in the three genes encoding the acyl carrier protein-dependent late acyltransferases of lipid A biosynthesis, designated lpxL(htrB), lpxM(msbB), and lpxP, were generated in Escherichia coli K12 W3110. Mutants defective in lpxM synthesize penta-acylated lipid A molecules and grow normally. Strains lacking lpxP fail to incorporate palmitoleate into their lipid A at 12 degreesC but make normal amounts of hexa-acylated lipid A and are viable. Although lpxL mutants and lpxL lpxM double mutants grow slowly on minimal medium at all temperatures, they do not grow on nutrient broth above 32 degreesC. Such mutants retain the ability to synthesize some penta- and hexa-acylated lipid A molecules because of limited induction of lpxP at 30 degreesC but not above 32 degreesC. MKV15, an E. coli lpxL lpxM lpxP triple mutant, likewise grows slowly on minimal medium at all temperatures but not on nutrient broth at any temperature. MKV15 synthesizes a lipid A molecule containing only the four primary (R)-3-hydroxymyristoyl chains. The outer membrane localization and content of lipid A are nearly normal in MKV15, as is the glycerophospholipid and membrane protein composition. However, the rate at which the tetra-acylated lipid A of MKV15 is exported to the outer membrane is reduced compared with wild type. The integrity of the outer membrane of MKV15 is compromised, as judged by antibiotic hypersensitivity, and MKV15 undergoes lysis following centrifugation. MKV15 may prove useful as a host strain for expressing late acyltransferase genes from other Gram-negative bacteria, facilitating the re-engineering of lipid A structure in living cells and the design of novel vaccines.