Hoang Tung T, Williams Scott, Schweizer Herbert P, Lam Joseph S
Department of Microbiology & Infectious Diseases, University of Calgary Health Sciences Center,Calgary, Alberta,Canada T2N 4N1.
Department of Microbiology, Colorado State University,Fort Collins, CO 80523,USA.
Microbiology (Reading). 1997 Mar;143 ( Pt 3):899-907. doi: 10.1099/00221287-143-3-899.
asd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, the asd gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an Escherichia coli delta asd mutant. The nucleotide sequence of a 2433 bp Smal-Nsil fragment was determined. This fragment contained the C-terminal 47 nucleotides of leuB, encoding 3-isopropylmalate dehydrogenase; asd, encoding aspartate-beta-semialdehyde dehydrogenase (Asd); and orfA, whose product showed similarity to the Asd proteins from Vibrio spp. By subcloning, asd was localized to a 1.24 kb DNA fragment which in an E. coli T7 expression system strongly expressed a 40,000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from P. aeruginosa, E. coli and Haemophilus influenzae revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal delta asd mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that orfA probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the asd gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp P. aeruginosa chromosome.
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必需成分。在缺乏DAP的环境中，例如哺乳动物组织中，它们会发生裂解。这一点以前被用于开发鼠伤寒沙门氏菌疫苗株以及含有asd作为体内选择标记的克隆载体。作为开发铜绿假单胞菌此类系统的第一步，通过从染色体DNA进行PCR扩增、构建小型文库以及互补大肠杆菌天冬氨酸缺陷型突变体的联合方法，克隆了野生型菌株PAO1的asd基因。测定了一个2433 bp的Smal - Nsil片段的核苷酸序列。该片段包含leuB的C末端47个核苷酸，编码3 - 异丙基苹果酸脱氢酶；asd，编码天冬氨酸 - β - 半醛脱氢酶（Asd）；以及orfA，其产物与弧菌属的Asd蛋白具有相似性。通过亚克隆，asd定位于一个1.24 kb的DNA片段，该片段在大肠杆菌T7表达系统中强烈表达一种40,000 Da的蛋白质。从DNA序列推导了氨基酸序列。对来自铜绿假单胞菌、大肠杆菌和流感嗜血杆菌的Asd蛋白进行比较，发现同一性大于63%，证明了革兰氏阴性菌中Asd的保守性质，并确定了含活性位点的共有序列GGNCTVXMLMXXXLGLF为一种可能的特征基序。分离出了染色体天冬氨酸缺陷型突变体。它们对DAP、赖氨酸、蛋氨酸和苏氨酸营养缺陷，并且在没有DAP的情况下裂解。遗传分析表明，orfA可能自然发生了移码，对Asd表型没有贡献。通过脉冲场凝胶电泳（PFGE），asd基因被定位到铜绿假单胞菌5.9 Mbp染色体上坐标1.89至2.15 Mbp之间，即37 - 40分钟处。
