新型聚球藻噬藻体Yong-L2-223的分离及基因组分析

Isolation and genome analysis of a novel Synechococcus cyanophage Yong-L2-223

摘要:

【目的】噬藻体(cyanophages)是特异性侵染蓝藻(cyanobacteria)的病毒，广泛分布于各类水体中，在调节蓝藻种群动态和密度、推动生物地球水生生态系统循环中起着重要作用。本研究的目的在于分离、鉴定噬藻体。【方法】本研究以海洋聚球藻(Synechococcus sp.) PCC 7002为指示宿主，从淡水水样中分离培养一株新型噬藻体Yong-L2-223，对其进行了宿主范围实验、全基因组测序、基因功能注释和系统进化分析。【结果】针对31株供试蓝藻的宿主范围实验，结果除指示藻PCC 7002 [属于聚球藻目(Synechococcales)]外，Yong-L2-223能够感染2株淡水蓝藻，分别是来源于滇池的绿色微囊藻(Microcystis viridis) FACHB-1342 [属于色球藻目(Chroococcales)]和水华束丝藻(Aphanizomenon flos-aquae) FACHB-1209 [属于念珠藻目(Nostocales)]。既可在高盐条件下感染海洋蓝藻，又可在低盐条件下感染淡水蓝藻，Yong-L2-223具有广盐性。透射电镜观察表明，Yong-L2-223呈肌尾病毒样(myovirus-like)，头部直径约60 nm，尾部长约136 nm。Yong-L2-223的双链DNA基因组全长为65 725 bp，G+C含量为58.6%，含有100个开放阅读框(open reading frame, ORFs)。Yong-L2-223基因组中含有编码Cas4、基因转移因子(gene transfer factor, GTA)的基因，含有辅助代谢基因(auxiliary metabolic genes, AMGs)，含有pre-Q0生物合成基因簇(gene cluster)。这些基因的编码产物可能有助于该噬藻体对宿主的适应、感染，从而实现跨3个目(order)感染蓝藻。将Yong-L2-223基因组与现有数据库中所有病毒基因组进行序列配对比较(pairwise sequence comparison, PASC)，结果最高PASC值仅为3.78%，远远低于国际病毒分类委员会(International Committee on Taxonomy of Viruses, ICTV)界定属的边界值(70%)。在基于全基因组的蛋白谱系统进化树中，Yong-L2-223形成一个独立的分支(branch)，与其他病毒间进化距离远。【结论】 Yong-L2-223在有尾纲中代表一个新的未知的属。本研究首次以海洋蓝藻为指示藻从淡水中分离获得噬藻体，且该噬藻体序列新颖，拓宽了对噬藻体的认知，丰富了噬藻体库、噬藻体基因库，为噬藻体资源开发奠定了基础。

Abstract:

[Objective] Cyanophages, the viruses specifically infecting cyanobacteria, are ubiquitous in water environments. They play a role in regulating the population dynamics and density of cyanobacteria and promote the biogeochemical cycling of the aquatic ecosystem. This study aims to isolate and identify a cyanophage. [Methods] A novel cyanophage Yong-L2-223 was isolated from fresh water samples with marine Synechococcus sp. PCC 7002 as the indicator host. The host range, genome sequence, open reading frames (ORFs), and phylogenetic relationship of Yong-L2-223 were studied. [Results] The host range tests against 31 strains of cyanobacteria showed that Yong-L2-223 could infect the indicator host PCC 7002 (Synechococcales) and two freshwater strains Microcystis viridis FACHB-1342 (Chroococcales) and Aphanizomenon flos-aquae FACHB-1209 (Nostocales) from the Dianchi Lake. The infection of the cyanobacterial strains from both the seawater and freshwater samples indicated that Yong-L2-223 was a euryhaline cyanophage. Yong-L2-223 was myovirus-like, consisting of an icosahedral head (about 60 nm in diameter) and a contractile tail (about 136 nm in length). The genome (double-stranded DNA) of Yong-L2-223 had a length of 65 725 bp, with the G+C content of 58.6% and 100 ORFs. It was predicted to carry the Cas4 gene, gene transfer factor (GTA) gene, auxiliary metabolic genes (AMGs), and a gene cluster for the synthesis of pre-Q0. These genes may contribute to the adaptation and infection of the cyanophage in cyanobacteria of three orders. The pairwise sequence comparison (PASC) illustrated that the highest similarity sharing by cyanophage Yong-L2-223 and all the viruses in the current genome databases was only 3.78%, far below the genus boundary cut-off of 70% defined by the International Committee on Taxonomy of Viruses. In the phylogenetic tree based on the whole proteomes, Yong-L2-223 formed an independent branch, with long evolutionary distances from other phages. [Conclusion] Yong-L2-223 is a new genus of the Caudoviricetes class. For the first time, we used a marine cyanobacterial strain as the indicator host to isolate and obtain a novel cyanophage from freshwater, which broadened the understanding of cyanophages, enriched cyanophage genome database, and laid a foundation for the development of cyanophage resources.