Rapid genetic characterization of a novel Enterobacteria phage and determination of its host recognizing genes

Abstract

We isolated a novel Enterobacteria phage IME08 from hospital sewage, then confirmed it was a double-stranded DNA phage by digesting its genetic material with DNase I, RNase A and several restriction endonucleases respectively. BLAST results of random fragments generated by a random PCR cloning method revealed that it belonged to T4-like virus. We subsequently determined the host recognizing genes (g37 and g38) sequence with a PCR-based "genome jumping" protocol based on highly conserved region at 5' terminus of g37 from four other T4-like Bacteriophages (T4, JS98, T2 and K3). These molecular biological methods enabled us to readily characterize the bacteriophage and efficiently determine the sequence of the genes of interest based on very limited conserved sequence information.

以大肠杆菌8099为宿主自医院污水中分离出一株肠杆菌噬菌体IME08，其遗传物质经RNA酶、DNA酶处理证实其为DNA，用限制性内切酶处理该DNA证实其为双链DNA。利用随机引物PCR技术扩增并克隆该噬菌体基因组的随机片段，经测序后同源比对，判断该噬菌体是一株新的T4-like噬菌体。根据4株T4-like噬菌体 (T4，JS98，T2及K3) 宿主识别基因 (g37) 5'端的高度保守序列，采用随机PCR与巢式PCR结合的“基因组跳跃”策略快速克隆出了该噬菌体的宿主识别基因g37和g38。