Giladi M, Champion C I, Haake D A, Blanco D R, Miller J F, Miller J N, Lovett M A
Department of Medicine, UCLA School of Medicine 90024.
J Bacteriol. 1993 Jul;175(13):4129-36. doi: 10.1128/jb.175.13.4129-4136.1993.
We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spanning sequences or signal peptides. This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkaline phosphatase (AP) fusion proteins from inner membrane AP fusion proteins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the "blue halo" assay first described by Strauch and Beckwith (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988). The lipoprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces periplasmic proteolytic degradation of AP fusion proteins. pMG AP fusions containing cleavable signal peptides, including the E. coli periplasmic protein beta-lactamase, the E. coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos. In contrast, inner membrane AP fusions derived from E. coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T. pallidum AP recombinant which does not contain a signal peptide, resulted in blue colonies without blue halos. Lipoprotein-AP fusions, including the Borrelia burgdorferi OspA and T. pallidum Tp 75 and TmpA showed halo formation, although there was significantly less halo formation than that produced by either periplasmic or outer membrane AP fusions. In addition, we applied this approach to screen recombinants constructed from a 9.0-kb plasmid isolated from the B31 virulent strain of B. burgdorferi. One of the blue halo colonies identified produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site. The pMG/KS330r- cloning and screening approach can identify genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.
我们最近报道了一种称为pMG的phoA表达载体，它与TnphoA一样，可用于鉴定编码跨膜序列或信号肽的基因。通过将pMG重组体转化到大肠杆菌KS330中，该克隆系统已被改进，以促进区分外膜和周质碱性磷酸酶（AP）融合蛋白与内膜AP融合蛋白，KS330是Strauch和Beckwith首次描述的用于“蓝色晕圈”检测的菌株（《美国国家科学院院刊》85:1576 - 1580, 1988）。KS330的脂蛋白突变lpp - 5508导致外膜对大分子有渗漏，其degP4突变极大地减少了周质中AP融合蛋白的蛋白水解降解。含有可裂解信号肽的pMG AP融合蛋白，包括大肠杆菌周质蛋白β - 内酰胺酶、大肠杆菌和沙眼衣原体的外膜蛋白OmpA和MOMP，以及梅毒螺旋体AP重组体Tp 9，扩散穿过KS330渗漏的外膜，产生带有蓝色晕圈的蓝色菌落。相比之下，源自大肠杆菌蛋白的内膜AP融合蛋白，包括前导肽酶、SecY和四环素抗性基因产物，以及不含信号肽的梅毒螺旋体AP重组体Tp 70，产生没有蓝色晕圈的蓝色菌落。脂蛋白 - AP融合蛋白，包括伯氏疏螺旋体OspA和梅毒螺旋体Tp 75和TmpA，显示出晕圈形成，尽管晕圈形成明显少于周质或外膜AP融合蛋白产生的晕圈。此外，我们应用这种方法筛选了由从伯氏疏螺旋体B31毒力菌株分离的9.0 - kb质粒构建的重组体。鉴定出的一个蓝色晕圈菌落产生了一种AP融合蛋白，该蛋白含有一个带有前导肽酶I裂解识别位点的信号肽。pMG/KS330r - 克隆和筛选方法可以鉴定编码具有可裂解信号肽的蛋白质的基因，因此可作为鉴定编码潜在毒力因子基因的第一步。
