Kuhn M, Jendrossek D, Fründ C, Steinbüchel A, Schlegel H G
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Federal Republic of Germany.
J Bacteriol. 1988 Feb;170(2):685-92. doi: 10.1128/jb.170.2.685-692.1988.
Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.
在转座子诱变后，分离出了在利用2,3-丁二醇和乙偶姻方面发生改变的嗜糖产碱菌突变体。自杀载体pSUP5011被用于将抗药转座元件Tn5导入嗜糖产碱菌。对利用2,3-丁二醇的亲本菌株CF10141和AS141的卡那霉素抗性接合子进行筛选，以寻找在利用2,3-丁二醇或乙偶姻方面受损的突变体。11个突变体对2,3-丁二醇呈阴性，但对乙偶姻呈阳性；它们无法合成活性发酵型乙醇脱氢酶蛋白（1类）。40个突变体对2,3-丁二醇和乙偶姻均呈阴性（2类）。Tn5-mob也被导入了不利用2,3-丁二醇的亲本菌株H16的链霉素抗性衍生物中。在大约35,000个接合子中，有2个能够在2,3-丁二醇上生长。这两个突变体都组成型地合成发酵型乙醇脱氢酶（3类）。从黏粒文库中克隆了4个第1类和2个第3类突变体基因组DNA的Tn5标记的EcoRI片段。它们被生物素化，并用作探针，在λL47和黏粒基因文库中检测相应的野生型片段。在嗜糖产碱菌中编码发酵型乙醇脱氢酶的基因被克隆，并定位到一个位于11.5-kb EcoRI片段内的2.5-kb SalI片段上。该基因在嗜糖产碱菌JMP222和草酸假单胞菌中进行了异源表达。第3类突变体中Tn5-mob的插入定位在同一个2.5-kb SalI片段上乙醇脱氢酶结构基因附近。
