Analysis The Genome Of Thermus Phage TSP4 And Its Helicase Gene Expression

Phages are viruses which infect bacteria, archaea and fungi, they are the most abundant and diverse form of life on earth and may exert major influence over the microbial world.As a model organism of thermophiles, researches on Thermus phages have obtained more attention. Up to date, four families of Thermus bacteriophages: Myoviridae, Siphoviridae, Tectiviridae, Inoviridae were isolated, and the genomes of five Thermus bacteriophages have been sequenced.In this research, a novel Thermus bacteriophage named TSP4 was isolated from Tengchong hot springs. TSP4 was a typical Siphoviridae phage with a polyhedral head (67 nm in diameter), a long flexible tail with 837 nm (10 nm in length). Tail-plate and tail collar (shrink at the neck) were observed.The genome of TSP4 was 83142 bp. The C+G content of TSP4 genome was 56.0%.37 inverted repeats,18 tandem repeats,150 promoters,36 regions with significantly higher G+C content islands were found. Analysis of the frequent codon usage showed that TSP4 and its host bacteria have an obvious codon usage bias with different characterization.TSP4 has 108 hypothesis genes. They were divided into two gene clusters, one with counterclockwise, and another, with clockwise. DNA metabolism, replication, recombination and transcription-related proteins mainly distributed on counterclockwise cluster, while phage structure and capsid proteins were lined on clockwise cluster. The genomic characterization of TSP4 has high homology with Siphoviruses P23-45 and P74-26, which were isolated from Kamchatka, Russia.The phylogenetic evolution and motif of ORF 4,7,11,26,39 and 42 were analyzed, and some protein three-dimensional structures were predicted. Finally, the Helicase DnaB gene of TSP4 was cloned and expressed in E. coli successfully. And the Helicase activity was determined by 32P labled ssDNA and AFM (atomic force microscopy).Based on the research of Thermus phage diversity in Tengchong hot springs, cloning and expression of the Helicase DnaB gene of TSP4 would help to understand the law of phage evolution, mechanisms of horizontal gene transfer between species, and also would be beneficial to develop the unique genetic resources from thermophilic phages.