Volokhina Elena B, Beckers Frank, Tommassen Jan, Bos Martine P
Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Bacteriol. 2009 Nov;191(22):7074-85. doi: 10.1128/JB.00737-09. Epub 2009 Sep 18.
The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the beta-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.
进化上保守的蛋白质Omp85是革兰氏阴性菌和线粒体中外膜蛋白（OMP）组装所必需的。其大肠杆菌同源物BamA与四种辅助脂蛋白BamB、BamC、BamD和BamE共同发挥作用，一起构成β桶组装机器（Bam）。在此，我们研究了该机器在脑膜炎奈瑟菌中的组成及其组分的功能，脑膜炎奈瑟菌是外膜生物发生研究的模式生物。基因组序列分析揭示了BamC、BamD（先前称为ComL）和BamE的同源物以及第二个BamE同源物Mlp。未发现BamB的同源物。与在大肠杆菌中一样，ComL/BamD对于生存能力和OMP组装似乎是必需的，并且不能被其大肠杆菌同源物替代。BamE不是必需的，但发现它有助于OMP组装的效率并维持外膜完整性。一个bamC突变体仅表现出轻微的OMP组装缺陷，但无法构建bamC bamE双突变体进一步表明了BamC在OMP组装中的功能。一个mlp突变体在OMP组装中未受影响。共纯化分析结果证明了BamC、ComL和BamE与Omp85的关联。半天然凝胶电泳确定RmpM蛋白是Omp85复合物的另一个组分，这在共纯化分析中得到了证实。OMP折叠不需要RmpM，但它能稳定OMP复合物。因此，脑膜炎奈瑟菌中的Bam复合物由Omp85/BamA加上RmpM、BamC、ComL/BamD和BamE组成，其中ComL/BamD和BamE似乎是OMP组装中最重要的辅助组分。
