Hatten L A, Schweizer H P, Averill N, Wang L, Schryvers A B
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Alberta, Canada.
Gene. 1993 Jul 15;129(1):123-8. doi: 10.1016/0378-1119(93)90707-a.
The asd mutants of Gram- and some Gram+ bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, i.e., mammalian tissues, they will undergo lysis. This has previously been exploited to develop vaccine strains of Salmonella typhimurium and Streptococcus mutans. As a first step for the development of a biosafe Neisseria meningitidis laboratory strain, we have cloned the asd from wild-type strain B16B6 by complementation of an Escherichia coli asd mutant. By subcloning and insertion mutagenesis, the N. meningitidis asd was localized to a 1.5-kb DNA fragment. In a T7 RNA polymerase-T7 promoter expression system, a 38-kDa protein was strongly expressed from this DNA fragment. The N-terminal amino acid (aa) sequence was deduced from the nucleotide sequence, which was determined with the help of an in-frame Asd'::'LacZ protein fusion. A comparison of the N-terminal aa of the Asd proteins from N.meningitidis and E. coli revealed 70% identity, suggesting that the Asd protein may be highly conserved among Gram- bacteria.
革兰氏阴性菌和一些革兰氏阳性菌的天冬氨酸缺陷型突变体对二氨基庚二酸（DAP）有绝对需求，DAP是这些生物体细胞壁的一种必需成分。在缺乏DAP的环境中，即哺乳动物组织中，它们会发生裂解。此前已利用这一点开发了鼠伤寒沙门氏菌和变形链球菌的疫苗株。作为开发生物安全的脑膜炎奈瑟菌实验室菌株的第一步，我们通过补充大肠杆菌天冬氨酸缺陷型突变体，从野生型菌株B16B6中克隆了天冬氨酸缺陷型基因（asd）。通过亚克隆和插入诱变，将脑膜炎奈瑟菌的asd定位到一个1.5kb的DNA片段上。在T7 RNA聚合酶-T7启动子表达系统中，该DNA片段强烈表达一种38kDa的蛋白质。从核苷酸序列推导N端氨基酸（aa）序列，该序列借助框内Asd'::'LacZ蛋白融合来确定。对脑膜炎奈瑟菌和大肠杆菌Asd蛋白的N端氨基酸进行比较，发现同一性为70%，这表明Asd蛋白在革兰氏阴性菌中可能高度保守。
