Costello G M, Vipond R, MacIntyre S
School of Animal and Microbial Sciences, University of Reading, UK.
J Bacteriol. 1996 Mar;178(6):1623-30. doi: 10.1128/jb.178.6.1623-1630.1996.
Two homologs of the outer membrane protein OmpA were identified in Aeromonas salmonicida by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and amino-terminal sequence analyses. An A. salmonicida genomic DNA library was constructed by using lambda GEM-11 and recombinant phage carrying both genes ompAI and ompAII) selected by immunoscreening. A 5.0-kb BamHI fragment containing the two genes in tandem was subcloned in pBluescript and used for further subcloning and sequencing of the genes. The encoded proteins (Mr = 33,564 and 32,536 for mature OmpAI and OmpAII, respectively) had only 64% identity with each other and otherwise had the highest level of homology to OmpA proteins from the members of the family Enterobacteriaceae. Based on the Escherichia coli OmpA model, an eight-stranded amphipathic beta-barrel model for the membrane assembly of the N-terminal half of OmpAI and OmpAII was predicted. Most variation between the two proteins was localized to the predicted surface loops and periplasmic turns, while the transmembrane strands and C-terminals domains were highly conserved. Expression of ompAI and ompAII separately in E. coli indicated that both genes could be independently transcribed from their own promoters and that both gene products were assembled into the E. coli outer membrane. A survey of different Aeromonas spp. by PCR revealed that possession of two tandem ompA genes was widespread among this genus. This is the first report of any bacterial species possessing two genes for homologs of this major outer membrane protein.
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、免疫印迹和氨基末端序列分析,在杀鲑气单胞菌中鉴定出两种外膜蛋白OmpA的同源物。利用λGEM-11构建了杀鲑气单胞菌基因组DNA文库,并通过免疫筛选选择携带ompAI和ompAII两个基因的重组噬菌体。将包含这两个串联基因的5.0 kb BamHI片段亚克隆到pBluescript中,并用于基因的进一步亚克隆和测序。编码的蛋白质(成熟的OmpAI和OmpAII的分子量分别为33,564和32,536)彼此之间只有64%的同一性,并且与肠杆菌科成员的OmpA蛋白具有最高水平的同源性。基于大肠杆菌OmpA模型,预测了OmpAI和OmpAII N端一半在膜组装中的八链两亲性β桶模型。两种蛋白质之间的大多数差异定位于预测的表面环和周质转角,而跨膜链和C端结构域高度保守。ompAI和ompAII在大肠杆菌中的单独表达表明,这两个基因都可以从它们自己的启动子独立转录,并且两个基因产物都组装到大肠杆菌外膜中。通过PCR对不同气单胞菌属进行的调查显示,拥有两个串联的ompA基因在该属中很普遍。这是关于任何细菌物种拥有该主要外膜蛋白两种同源基因报道的首例。