Ow D W, Ausubel F M
Mol Gen Genet. 1980;180(1):165-75. doi: 10.1007/BF00267366.
We demonstrate the use of bacteriophage P4 as a molecular cloning vector in Klebsiella pneumoniae. A hybrid P4 phage, constructed in vitro, that contains a K. pneumoniae hisDG DNA fragment can be propagated either as a lytic viable specialized transducing phage or as an autonomous, self-replicating plasmid. Hybrid P4 genomes existing as plasmids can be readily converted into non-defective P4-hybrid phage particles by superinfection with helper phage P2. Infection of a K. pneumoniae hisD non-P2 lysogen with P4-hisD hybrid phage results in approximately 90% of the infected cells becoming stably transduced to HisD+. Because P4 interferes with P2 growth, high titre stocks of P4 hybrid phages are relatively free (less than or equal to 10(-6) of P2 contamination. The hisG gene product was detected in ultraviolet light irradiated host cells infected by the P4-hisDG hybrid phage. A mutant of P4 (P4sid1) that directs the packaging of P4 DNA into P2 sized capsids should permit the construction of hybrid phages carrying 26 kilobase inserts.
我们展示了噬菌体P4作为肺炎克雷伯菌分子克隆载体的应用。一种在体外构建的含有肺炎克雷伯菌hisDG DNA片段的杂交P4噬菌体，可以作为裂解性有活力的特异性转导噬菌体或作为自主的自我复制质粒进行增殖。以质粒形式存在的杂交P4基因组可以通过辅助噬菌体P2的超感染轻易地转化为无缺陷的P4杂交噬菌体颗粒。用P4-hisD杂交噬菌体感染肺炎克雷伯菌hisD非P2溶原菌，约90%的受感染细胞会稳定转导为HisD+。由于P4会干扰P2的生长，P4杂交噬菌体的高滴度原液相对不含（小于或等于10^(-6)）P2污染。在受P4-hisDG杂交噬菌体感染的紫外线照射宿主细胞中检测到了hisG基因产物。一种将P4 DNA包装到P2大小衣壳中的P4突变体（P4sid1）应允许构建携带26千碱基插入片段的杂交噬菌体。
