The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona, United States of America.
PLoS One. 2010 Dec 29;5(12):e15944. doi: 10.1371/journal.pone.0015944.
asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.
革兰氏阴性菌的 asdA 突变体对二氨基庚二酸（DAP）有强制性需求，DAP 是这些生物体细胞壁肽聚糖层的必需成分。在缺乏 DAP 的环境中，即在动物组织中，它们将发生裂解。先前已经利用删除 asdA 基因来开发活减毒重组细菌疫苗的抗生素敏感株。将 Asd(+)质粒引入ΔasdA 突变体会使细菌菌株依赖质粒。这种对 Asd(+)质粒载体的依赖性在细菌菌株和重组质粒之间创建了平衡致死互补。E. ictaluri 是一种肠道革兰氏阴性鱼类病原体，可引起鲶鱼的肠败血症。由于 E. ictaluri 是一种鼻腔/口腔侵袭性细胞内病原体，因此该细菌是开发鲶鱼水产养殖业巴氏/口腔活重组减毒爱德华氏菌疫苗（RAEV）的候选者。作为开发抗生素敏感 RAEV 菌株的第一步，我们对 E. ictaluri 的 asdA 基因进行了表征和删除。E. ictaluri ΔasdA01 突变体表现出对 DAP 生长的绝对需求。E. ictaluri 的 asdA 基因由沙门氏菌的 asdA 基因互补。几种具有不同复制起点的 Asd(+)表达载体被转化为 E. ictaluri ΔasdA01。Asd(+)载体与 pEI1 和 pEI2 E. ictaluri 天然质粒兼容。体内平衡致死系统得到了令人满意的评估。携带 Asd(+)质粒的 E. ictaluri ΔasdA01 合成了重组 GFP、PspA 和 LcrV 蛋白。在这里，我们构建了一个平衡致死系统，这是开发水产养殖业抗生素敏感 RAEV 的第一步。
