Hunger M, Schmucker R, Kishan V, Hillen W
Institut für Mikrobiologie und Biochemie, Friedrich-Alexander Universität Erlangen-Nürnberg, F.R.G.
Gene. 1990 Mar 1;87(1):45-51. doi: 10.1016/0378-1119(90)90494-c.
A shuttle plasmid for Acinetobacter calcoaceticus and Escherichia coli has been constructed from a cryptic A. calcoaceticus lwoffi plasmid and pBR322. It is transformed to A. calcoaceticus BD413 by natural competency, yielding about 10(6) transformants per microgram of plasmid DNA. The ApR and TcR genes of pBR322 are functional in A. calcoaceticus. A gene bank was constructed from chromosomal A. calcoaceticus DNA and the shuttle plasmid. Direct transformation to A. calcoaceticus yielded about 95% recombinants, indicating a sixfold enrichment of recombinant plasmids compared to E. coli. One clone complementing a trpE mutation carried a 20-kb insertion and transformed with a 30-fold higher efficiency when compared to the vector. A deletion analysis of the shuttle plasmid indicates that 2.2 kb is necessary for autonomous replication and stable maintenance in A. calcoaceticus. No rearrangements of the DNA or loss of plasmids are found in that organism, even in the absence of selective pressure, when this sequence is present. A further insertional inactivation analysis creating lacZ transcriptional fusions suggests that the origin of replication (ori) is contained within about 1350 bp. Analysis of beta-galactosidase production in A. calcoaceticus indicates that only a weak promoter activity is directed out of one end of this ori. Its sequence contains A + T-rich regions, an 18-bp element with nearly perfect palindromic symmetry and eleven repeats of the consensus sequence, AAAAAATAT, eight of which are clustered within 360 bp. However, no open reading frames or significant homologies to other ori were found.
一种用于醋酸钙不动杆菌和大肠杆菌的穿梭质粒已由一种隐秘的洛菲不动杆菌质粒和pBR322构建而成。它通过自然感受态转化到醋酸钙不动杆菌BD413中，每微克质粒DNA可产生约10⁶个转化体。pBR322的ApR和TcR基因在醋酸钙不动杆菌中具有功能。利用醋酸钙不动杆菌染色体DNA和穿梭质粒构建了一个基因文库。直接转化到醋酸钙不动杆菌中产生了约95%的重组体，这表明与大肠杆菌相比，重组质粒的富集倍数为6倍。一个互补trpE突变的克隆携带一个20 kb的插入片段，与载体相比，其转化效率高30倍。对穿梭质粒的缺失分析表明，2.2 kb对于在醋酸钙不动杆菌中自主复制和稳定维持是必需的。当存在该序列时，即使在没有选择压力的情况下，在该生物体中也未发现DNA重排或质粒丢失。进一步的插入失活分析产生lacZ转录融合，表明复制起点（ori）包含在约1350 bp内。对醋酸钙不动杆菌中β-半乳糖苷酶产生的分析表明，只有一个弱启动子活性从该ori的一端发出。其序列包含富含A + T的区域、一个具有近乎完美回文对称性的18 bp元件以及共有序列AAAAAATAT的11个重复，其中8个聚集在360 bp内。然而，未发现开放阅读框或与其他ori的显著同源性。
