Polissi A, Georgopoulos C
Départment de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Mol Microbiol. 1996 Jun;20(6):1221-33. doi: 10.1111/j.1365-2958.1996.tb02642.x.
The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5 degrees C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low-copy-number plasmid vector it was able to suppress the temperature-sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP-binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP-binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single-amino-acid substitutions in non-conserved residues and were able to support cell growth at 30 degrees C but not at 43 degrees C. The remaining two mutations behaved as recessive lethals, and resulted in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that N-acetyl [3H]-glucosamine, a precursor of lipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. A possible role for MsbA as a translocator of lipopolysaccharides or its precursors is discussed.
htrB基因的发现是因为其插入失活会干扰大肠杆菌在32.5摄氏度以上温度下的生长和生存能力，这是磷脂积累的结果。msbA基因最初被发现是因为当它克隆在低拷贝数质粒载体上时，能够抑制htrB缺失突变体的温度敏感生长表型以及磷脂的积累。msbA基因产物属于ABC转运蛋白超家族，这是一个普遍保守的蛋白质家族，其特征是具有高度保守的ATP结合结构域。msbA基因在所有温度下对细菌的生存能力都是必需的。为了了解MsbA蛋白的生理作用，我们使用随机PCR诱变对ATP结合结构域进行了突变。分离并鉴定了六个独立的突变体。其中四个突变导致非保守残基的单氨基酸替换，能够在30摄氏度下支持细胞生长，但在43摄氏度下不能。其余两个突变表现为隐性致死，导致ATP结合结构域两个高度保守区域之一的沃克基序B中的单氨基酸替换。尽管这两种突变蛋白都不能支持大肠杆菌生长，但它们在体外都保留了结合ATP的能力。此外，我们提供证据表明，脂多糖的前体N-乙酰[3H]-葡萄糖胺在htrB缺失或msbA条件致死菌株的内膜中在非允许温度下积累。通过用含有野生型msbA基因的质粒转化，前体向外膜的转运得以恢复。本文讨论了MsbA作为脂多糖或其前体转运体的可能作用。
