阴沟肠杆菌Tn5－nifA重组质粒的构建及其nifA的表达分析

Study on Characterization of a Tn5-nifA Plasmid and on Expression of nifA in Enterobacter cloacae

构建的重组质粒ＰＢＦ１０１带有阴沟肠杆菌（Ｅｎｔｅｒｏｂａｃｔｅｒｃｌｏａｃａｅ）Ｅ２６固氮基因ｎｉｆＡ片段，该质粒具有广泛宿主范围接合转移特性和转座子Ｔｎ５的转座作用。验证了Ｅ２６ｎｉｆＡ产物能激活肺炎克氏杆菌（Ｋｌｅｂｓｉｅｌｌａｐｎｅｕｍｏｎｉａｅ）ｎｉｆＨ启动子的表达，解除氨对固氮酶合成的阻遏作用。发现当ＰＢＦ１０１引入自生固氮催娩克氏杆菌（Ｋ．ｏｘｙｔｏｃａ）ＮＧ１３后，可观察到固氮酶的组成型生成，同时在３９℃高温条件下的细菌仍能检测到部分固氮活性。

The plasmid pBF101 with a broadhOSt range was constructed by insertionof 2. 1 kb DNA fragment containingEnterobacter cloacae nifA gene into Sal Isite of a mobilizable vector pSZ21(Fig. 2). NifA expression was undercontrol of Tc promoter. The plasmidpBF101 can be mobilized with highfrequency from the donor E. coli S171 by mating and be widely applicableas transposon carrier for transposon insertion into the recipient genome(Table 1).We investigated the effect of E.cloacae NifA on the m f transcription inKlebsiella pneumoniae and on the activation of the K. oxytoca nif genes bymonitoring β-galactosidase synthesisand assaying acetylene-reduction (Tables 2, 3). The evidence presentedhere demonstrates that the mechanismof regulation in E. cloacae and K. oxy-toca is similar to that in K. pneumoniae.