鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析

Isolation and identification of phage SWH-Ab-1 against Acinetobacter baumannii and bioinformatic analysis of its major functional genes

目的分离鲍曼不动杆菌噬菌体,测定其基因组DNA序列,分析预测穿孔素和裂解酶等重要功能基因及其编码蛋白的性质和功能。方法利用双层琼脂平板法从医院污水中分离鲍曼不动杆菌噬菌体,透射电子显微镜观察噬菌体的形态特征;提取噬菌体基因组DNA,采用Illumina Hiseq2000测序仪进行全基因组测序;利用生物信息学方法对噬菌体基因组DNA序列进行功能编码基因定位、同源性分析以及对噬菌体重要功能基因编码蛋白的结构和生物功能等进行预测。结果成功从医院污水中分离获得1株鲍曼不动杆菌噬菌体,命名为噬菌体SWH-Ab-1。噬菌体SWH-Ab-1属长尾噬菌体,对鲍曼不动杆菌具有显著裂解性。噬菌体SWH-Ab-1基因组为全长41 567 bp的双链DNA,G+C%含量为39.42%,包含51个预测功能基因,穿孔素基因和裂解酶基因分别位于噬菌体基因组的ORF02(320~655 bp)和ORF03(642~1 199 bp)开放阅读框,穿孔素基因序列全长336 bp,共编码111个氨基酸,其编码的Hol SA1为疏水性Ⅰ型跨膜蛋白,相对分子质量为11.957 96×103,理论等电点为4.87,可能在脂肪酸代谢、转运载体及信号转导中发挥重要作用。裂解酶基因序列全长558 bp,共编码185个氨基酸,其编码的Lys SA1为亲水性蛋白,相对分子质量为21.010 4×103,理论等电点为9.56,无跨膜区,可能参与氨基酸生物合成、脂肪酸代谢、辅因子生物合成等重要生理过程。两种功能蛋白均不存在信号肽序列,二级结构主要以α-螺旋和无规则卷曲为主。结论噬菌体SWH-Ab-1是一种新分离的长尾裂解性噬菌体,其基因组的ORF02和ORF03开放阅读框是穿孔素和裂解酶的编码基因,编码的Hol SA1和Lys SA1可能组成"二元裂解系统",协同参与噬菌体对宿主菌的裂解过程。

Objective To isolate the Acinetobacter baumannii （A.baumannii） phages, sequence the genome and predict the properties and functions of the major functional genes such as holin and lyase. Methods An A.baumannii phage was isolated from raw sewage of a hospital and identified using double-layer-agar method. The morphology of the phage was observed by electron microscopy. The genomic DNA of the phage was extracted and sequenced using Illumina Hiseq2000 technique. Bioinformatic analysis was used to analyze the nucleotide sequence of the phages, including functional genes location and homology analysis, and to predict the structures and biological functions of the major encoding proteins. Results An A.baumannii phage was successfully isolated and named as SWH-Ab-1. Bacteriophage SWH-Ab-1 belonged to the Siphoviridae, which had significant lytic activity against A.baumannii. The genome of bacteriophage SWH-Ab-1 was a double-strand DNA with a full length of 41 567 bp, in which the G＋C mol% was 39.4% and 51 predicted genes were involved. The holin gene and the lysin gene were located in the ORF02 （320~655 bp） and the ORF03 （642~1 199 bp） of SWH-Ab-1, respectively. HolSA1, consisting of 111 amino acids （336 bp）, was a hydrophobic Ⅰ transmembrane protein, at a molecular mass of 11.957 96×103, with a theoretical isoelectric point of 4.87, which might play an important role in fatty acid metabolism, transport and signal transduction. LysSA1 consisted of 185 amino acids （558 bp）, was a hydrophilic protein without transmembrane regions, its molecular mass was 21.010 4×103, and its theoretical isoelectric point was 9.56, which might function in amino acid biosynthesis, fatty acid metabolism and biosynthesis of cofactors. The secondary structure of the 2 proteins was mainly composed of alpha helixes and random coil, and both of them were lack of signal peptide. Conclusion Bacteriophage SWH-Ab-1 is a new lytic phage, belonging to the Siphoviridae family. The open reading frames ORF02 and ORF03 are the encoding genes of phage SWH-Ab-1 genome. HolSA1 and LysSA1 might form a “two-element lysis system” together in order to lyse the host bacteria.