Browning Douglas F, Matthews Sophie A, Rossiter Amanda E, Sevastsyanovich Yanina R, Jeeves Mark, Mason Jessica L, Wells Timothy J, Wardius Catherine A, Knowles Timothy J, Cunningham Adam F, Bavro Vassiliy N, Overduin Michael, Henderson Ian R
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
PLoS One. 2013 Dec 23;8(12):e84512. doi: 10.1371/journal.pone.0084512. eCollection 2013.
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.
大肠杆菌的多蛋白β桶组装机器（BAM）负责将含β桶的整合外膜蛋白（OMP）折叠并插入细菌外膜。该复合物的一个重要组成部分是BamA蛋白，它通过其N端的五个多肽转运相关（POTRA）结构域结合未折叠的β桶前体。BamA的C端包含一个β桶结构域，该结构域将BamA tether到外膜，并且也被认为参与OMP插入。在这里，我们使用接头扫描诱变对BamA进行诱变，并证明在我们的实验系统中，所有五个POTRA结构域对于BamA蛋白功能都是必不可少的。此外，我们生成了基于同源性的BamAβ桶模型，并使用插入诱变、缺失分析和免疫荧光测试我们的模型，以识别β链、周质环和细胞外环。我们表明，BamAβ桶的表面暴露环是必不可少的。
