Mutational and Topological Analysis of the <i>Escherichia coli</i> BamA Protein

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.大肠杆菌（Escherichia coli）的多蛋白β桶组装机器（β-barrel assembly machine，BAM）负责将含有β桶结构的整合型外膜蛋白（integral outer membrane proteins，OMPs）折叠并插入细菌外膜之中。该复合物的必需组分为BamA蛋白，其N端包含五个多肽转运相关结构域（polypeptide transport-associated domains，POTRA），可结合未折叠的β桶前体蛋白。BamA的C端含有一个β桶结构域，该结构域将BamA锚定在外膜上，同时也被认为参与了OMP的插入过程。本研究采用接头扫描诱变技术对BamA进行诱变实验，结果表明在本实验体系中，五个POTRA结构域均为BamA蛋白发挥功能所必需。此外，我们构建了BamA β桶结构域的同源建模模型，并通过插入诱变、缺失分析与免疫荧光实验对该模型进行验证，以此鉴定出β链、周质转角以及胞外环。我们证实，BamA β桶结构域的表面暴露环为其功能所必需。