Kretzschmar Utta, Schobert Max, Görisch Helmut
Fachgebiet Technische Biochemie, Institut für Biotechnologie der Technischen Universität Berlin, Seestraße 13, D-13353 Berlin, Germany1.
Microbiology (Reading). 2001 Oct;147(Pt 10):2671-2677. doi: 10.1099/00221287-147-10-2671.
Pseudomonas aeruginosa ATCC 17933 uses a pyrroloquinoline quinone-dependent ethanol oxidation system. Two mutants of P. aeruginosa, unable to grow on ethanol and showing no acetyl-CoA synthetase (ACS) activity under standard test conditions, were complemented by cosmid pTB3018. Subcloning led to the isolation of a gene which encodes a protein with high similarity to acetyl-CoA synthetases. Interruption of the putative acsA gene by a kanamycin-resistance cassette resulted in a mutant also unable to grow on ethanol and with very low residual acetyl-CoA-forming activity. Complementation by the wild-type allele of the acsA gene restored growth and led to the expression of ACS activity in excess of that of wild-type cells. In wild-type P. aeruginosa, ACS activity was induced upon growth on ethanol, 2,3-butanediol, malonate and acetate. The wild-type and mutants defective in ACS activity showed an active acetate kinase (ACK) under the growth conditions used; however, phosphotransacetylase (PTA) could not be detected. The data indicate that P. aeruginosa requires active acsA gene product for growth on ethanol.
铜绿假单胞菌ATCC 17933利用一种依赖吡咯喹啉醌的乙醇氧化系统。铜绿假单胞菌的两个突变体在乙醇上无法生长，并且在标准测试条件下没有乙酰辅酶A合成酶（ACS）活性，它们通过黏粒pTB3018得到了互补。亚克隆导致分离出一个基因，该基因编码一种与乙酰辅酶A合成酶高度相似的蛋白质。用卡那霉素抗性盒中断假定的acsA基因导致一个突变体也无法在乙醇上生长，并且具有非常低的残余乙酰辅酶A形成活性。用acsA基因的野生型等位基因进行互补恢复了生长，并导致ACS活性的表达超过野生型细胞。在野生型铜绿假单胞菌中，在乙醇、2,3 - 丁二醇、丙二酸和乙酸上生长时，ACS活性被诱导。在所用的生长条件下，野生型和ACS活性有缺陷的突变体都显示出有活性的乙酸激酶（ACK）；然而，未检测到磷酸转乙酰酶（PTA）。数据表明，铜绿假单胞菌在乙醇上生长需要有活性的acsA基因产物。
