Geider K, Baldes R, Bellemann P, Metzger M, Schwartz T
Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
Microbiol Res. 1995 Nov;150(4):337-46. doi: 10.1016/S0944-5013(11)80015-7.
The synthetic plasmid pfdC1 with the replication origin of phage fd and fd gene 2 grows autonomously in E. coli cells. DNA sequencing revealed several mutations compared to the fd genome causing reduced expression of viral gene 2 protein, which can be toxic for the host cell. Another adaptation was noticed for E. coli strains with a copy of fd gene 2 on the F-episome and a pfdA-plasmid with a minimal fd replication origin, when maintained at 42 degrees C. The carrier cells adjusted their cellular metabolism to these stress conditions, whereas replication functions of the plasmid or expression of fd gene 2 on the F-episome were not changed. The filamentous bacteriophages tend to reduce their genome size into miniphages, which was also observed for phages with an antibiotic resistance gene. Bacteriophages with a transposon insertion in the viral gene 2 had a tendency to restore the mutated gene by exchange with the functional gene 2 carried in recA-host cells. Mobilization of pfd-plasmids with RP4 transfer functions was reduced due to interference of replication and transfer in the rolling circle mode. The vectors used in these studies can also be applied as cloning vectors, which are compatible with many other plasmid vectors.
具有噬菌体fd复制起点和fd基因2的合成质粒pfdC1在大肠杆菌细胞中自主生长。DNA测序显示,与fd基因组相比存在几个突变,导致病毒基因2蛋白的表达降低,该蛋白可能对宿主细胞有毒。当在42℃下培养时,还注意到在F-附加体上带有一份fd基因2的大肠杆菌菌株和带有最小fd复制起点的pfdA质粒的另一种适应性变化。载体细胞将其细胞代谢调整至这些应激条件,而质粒的复制功能或F-附加体上fd基因2的表达未发生变化。丝状噬菌体倾向于将其基因组大小缩减为微型噬菌体,这在带有抗生素抗性基因的噬菌体中也有观察到。在病毒基因2中插入转座子的噬菌体倾向于通过与recA宿主细胞中携带的功能性基因2交换来恢复突变基因。由于滚环模式下复制和转移的干扰,具有RP4转移功能的pfd质粒的迁移减少。这些研究中使用的载体也可用作克隆载体,与许多其他质粒载体兼容。