Wong K R, Buckley J T
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Mol Microbiol. 1993 Sep;9(5):955-63. doi: 10.1111/j.1365-2958.1993.tb01225.x.
Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas beta-lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.
气单胞菌溶血素是嗜水气单胞菌分泌的一种形成通道的蛋白质。为了确定气单胞菌溶血素的区域是否能指导另一种蛋白质的分泌，将aerA的部分片段与大肠杆菌碱性磷酸酶基因phoA融合，并克隆到大肠杆菌、杀鲑气单胞菌和嗜水气单胞菌中。我们惊讶地发现，两种气单胞菌属细菌对该酶的分泌均独立于与其融合的气单胞菌溶血素片段。最小的融合产物仅包含气单胞菌溶血素的信号序列和两个氨基酸。最大的融合产物包含气单胞菌溶血素分子的90%以上。在含有葡萄糖的LB培养基中生长的大肠杆菌和杀鲑气单胞菌的周质中以及经休克处理的细胞中发现了融合蛋白。在这些条件下，杀鲑气单胞菌自身分泌气单胞菌溶血素。相反，当含有任何融合基因的杀鲑气单胞菌在不含葡萄糖的LB培养基中生长时，大部分碱性磷酸酶活性位于细胞外，而β-内酰胺酶仍保留在其正常的周质位置。嗜水气单胞菌也得到了类似的结果。杀鲑气单胞菌中该酶位置的变化似乎与生长培养基的pH值有关。杀鲑气单胞菌和嗜水气单胞菌也分泌天然的大肠杆菌碱性磷酸酶，但一般分泌途径发生突变的嗜水气单胞菌菌株无法释放该酶。我们得出结论，气单胞菌分泌系统能够将大肠杆菌酶识别为细胞外蛋白并将其引导至细胞外。
