两株侵染寡养单胞菌噬菌体的生物学特性及全基因组分析

Biological characteristics and genome sequences of two bacteriophage strains infecting Stenotrophomonas indicatrix

摘要:

目的 寡养单胞菌(Stenotrophomonas)对植物和水体藻类具有促生效果，但目前关于寡养单胞菌噬菌体的相关研究报道较少。本研究从水体中分离侵染寡养单胞菌的噬菌体，并对其生物学及基因组特征进行研究，有助于加强对寡养单胞菌及其噬菌体生态功能的理解。方法 以蓝藻附生细菌指示器寡养单胞菌(S. indicatrix) EB12为宿主，采用双层平板法从湖水中分离噬菌体，并对其生物学特性、全基因组序列特征、基因功能注释、系统进化及蛋白质结构进行分析。结果 透射电镜显示，分离获得的2株噬菌体Ste-X和Ste-D均为短尾噬菌体；其潜伏期均为20 min；最佳感染复数分别为1 000和10；裂解量分别为28.8 PFU/cell和131.1 PFU/cell；温度耐受范围分别为20-60 ℃和20-70 ℃；pH耐受范围分别为pH 4.0-13.0和pH 3.0-12.0；在紫外照射下，Ste-X和Ste-D分别于60 min和120 min后失活，且宿主范围较窄；2株噬菌体的基因组长度均为39 429 bp，G+C含量分别为57.80%和57.85%，均含有65个开放阅读框(open reading frame, ORF)，2株噬菌体仅含3个差异碱基，但与其他噬菌体的同源性较低，均为新的短尾噬菌体。对差异碱基所在的开放阅读框ORF34和ORF52所编码的蛋白质结构进行预测分析，结果显示，ORF34编码的蛋白质均为酸性、稳定的亲水蛋白，均含有ChtBD3和PHA00661这2个保守结构域，其空间结构以无规则卷曲为主；ORF52编码的蛋白质均为碱性、不稳定的亲水蛋白，无保守结构域，空间结构以α螺旋为主。结论 从水体中分离得到2株全基因组高度相似但生物学特性明显不同的新型短尾噬菌体。

Abstract:

Objective Stenotrophomonas can promote the growth of plants and aquatic algae. However, limited studies have focused on the bacteriophages infecting Stenotrophomonas. In this study, we isolated bacteriophages infecting Stenotrophomonas from water and studied their biological and genomic characteristics, aiming to improve our understanding of the ecological functions of Stenotrophomonas and its bacteriophages.Methods The double-layer plate method was employed to isolate bacteriophages infecting S. indicatrix EB12 from lake water. The biological characteristics, whole genome sequence characteristics, gene functions, phylogenetic relationship, and protein structures of the isolates were analyzed.Results Transmission electron microscopy showed that two isolates Ste-X and Ste-D were short-tailed bacteriophages. The incubation period of both strains was 20 min. Ste-X and Ste-D showed the optimal multiplicities of infection being 1 000 and 10, burst sizes of 28.8 PFU/cell and 131.1 PFU/cell, temperature tolerance ranges of 20-60 °C and 20-70 °C, and pH tolerance ranges of pH 4.0-13.0 and pH 3.0-12.0, respectively. Ste-X and Ste-D were deactivated by UV irradiation for 60 min and 120 min, respectively. Their host ranges were narrow. The genomes of both strains showed the lengths of 39 429 bp, with the G+C content of 57.80% and 57.85%, respectively, and both of them contained 65 open reading frames (ORFs). The genomes of the two strains contained only three different bases. However, their homology with other bacteriophages was low, which suggested that the two strains were novel short-tailed bacteriophages. The structures of the proteins encoded by ORF34 and ORF52 were predicted. Result showed thant the proteins encoded by ORF34 were acidic, stable, and hydrophilic, containing two conserved domains, ChtBD3 and PHA00661, with the three-dimensional structures dominated by random coils. The proteins encoded by ORF52 were alkaline, unstable, hydrophilic, with no conserved domain and the three-dimensional structures dominated by α-helixes.Conclusion Two novel strains of short-tailed bacteriophages with high genome similarity but distinct biological characteristics were isolated from water.