嗜水气单胞菌bamA、bamB、bamD突变株的构建及其对外膜蛋白转运的影响

Construction of Mutant Strain bamA,bamB and bamD of Aeromonas hydrophila and Their Effects on the Outer Membrane Protein Transportation

革兰氏阴性细菌的BAM(β-barrel assembly machinery)系统一般由BamA-E等5个亚基组成,以β-桶状外膜蛋白的结构对细菌的转运和正确折叠中起重要作用。目前对于BAM系统的研究大多集中在大肠杆菌、脑膜炎双球菌等细菌中,而在其他细菌中的相关功能还有待进一步研究。以嗜水气单胞菌为研究对象,首先利用同源重组技术,分别构建完成bamA、bamB、bamD的缺失突变株。尔后,利用SDS-PAGE检测技术对相应突变株差异外膜蛋白进行比较,并对这些差异蛋白进行质谱分析,共鉴定到7个差异蛋白,其中5个为外膜蛋白,2个为位于内膜上的蛋白酶。此外,还通过Western blotting对部分突变株外膜蛋白的表达进行验证。研究结果发现,嗜水气单胞菌BAM系统的突变不仅改变了其外膜蛋白表达,还影响了微生物的蛋白转运进程;且在系统中不同亚基对不同外膜蛋白的转运和表达效果也有所不同,结果提示了嗜水气单胞菌BAM转运系统的不同亚基存在特定外膜蛋白转运的特性。

The BAM（β-barrel assembly machinery）complex of gram-negative bacteria is composed of five subunits of BamA-E,and plays a key role in the transporting and correctly folding of β-barrel outer membrane proteins. The current knowledge of this complex is basedon the several researches on few bacterial species such as Escherichia coli and Neisseria meninyitidis,whereas its function in other bacteriaremains largely not-studied. In this study,we firstly constructed the bamA-,bamB-,and bamD-deletion mutants in Aeromonas hydrophila using homologous recombination method. The altered outer membrane proteins among mutants and wild type control strain were detected by SDSPAGE,and then identified by mass spectrometry. There were 7 differentiated proteins,and 5 of them were outer membrane proteins and rest 2 were proteases in the inner membrane. In addition,the expressions of several selected outer membrane proteins were validated by Westernblotting. Results showed that the mutation of BAM complex of A. hydrophila not only affected their expressions,but also their transportationof outer membrane proteins. The transportation and expression of outer membrane protein varied in the different subunits of the system. Theseresults indicate that the different subunit of BAM transport system in A. hydrophila may have specific characteristics on outer membrane protein transporting and folding.