Yakushi T, Tajima T, Matsuyama S, Tokuda H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Japan.
J Bacteriol. 1997 May;179(9):2857-62. doi: 10.1128/jb.179.9.2857-2862.1997.
The major outer membrane lipoprotein (Lpp) of Escherichia coli possesses serine at position 2, which is thought to function as the outer membrane sorting signal, and lysine at the C terminus, through which Lpp covalently associates with peptidoglycan. Arginine (R) is present before the C-terminal lysine in the wild-type Lpp (LppSK). By replacing serine (S) at position 2 with aspartate (D), the putative inner membrane sorting signal, and by deleting lysine (K) at the C terminus, Lpp mutants with a different residue at either position 2 (LppDK) or the C terminus (LppSR) or both (LppDR) were constructed. Expression of LppSR and LppDR little affected the growth of E. coli. In contrast, the number of viable cells immediately decreased when LppDK was expressed. Prolonged expression of LppDK inhibited separation of the inner and outer membranes by sucrose density gradient centrifugation, whereas short-term expression did not. Pulse-labeled LppDK and LppDR were localized in the inner membrane, indicating that the amino acid residue at position 2 functions as a sorting signal for the membrane localization of Lpp. LppDK accumulated in the inner membrane covalently associated with the peptidoglycan and thus prevented the separation of the two membranes. Globomycin, an inhibitor of lipoprotein-specific signal peptidase II, was lethal for E. coli only when Lpp possessed the C-terminal lysine. Taken together, these results indicate that the inner membrane accumulation of Lpp per se is not lethal for E. coli. Instead, a covalent linkage between the inner membrane Lpp having the C-terminal lysine and the peptidoglycan is lethal for E. coli, presumably due to the disruption of the cell surface integrity.
大肠杆菌的主要外膜脂蛋白（Lpp）在第2位含有丝氨酸，该丝氨酸被认为起到外膜分选信号的作用，并且在C末端含有赖氨酸，Lpp通过该赖氨酸与肽聚糖共价结合。在野生型Lpp（LppSK）中，精氨酸（R）存在于C末端赖氨酸之前。通过将第2位的丝氨酸（S）替换为天冬氨酸（D）（假定的内膜分选信号），并删除C末端的赖氨酸（K），构建了在第2位（LppDK）或C末端（LppSR）或两者（LppDR）具有不同残基的Lpp突变体。LppSR和LppDR的表达对大肠杆菌的生长影响很小。相反，当表达LppDK时，活细胞数量立即减少。LppDK的长时间表达通过蔗糖密度梯度离心抑制内膜和外膜的分离，而短期表达则不会。脉冲标记的LppDK和LppDR定位于内膜，表明第2位的氨基酸残基起到Lpp膜定位分选信号的作用。LppDK在内膜中积累并与肽聚糖共价结合，从而阻止了两层膜的分离。球蛋白是脂蛋白特异性信号肽酶II的抑制剂，仅当Lpp具有C末端赖氨酸时对大肠杆菌才具有致死性。综上所述，这些结果表明Lpp本身在内膜中的积累对大肠杆菌并不致命。相反，具有C末端赖氨酸的内膜Lpp与肽聚糖之间的共价连接对大肠杆菌具有致死性，推测这是由于细胞表面完整性的破坏所致。
