微囊藻的原噬藻体基因组特征分析

Genomic characterization of the procyanophages in Microcystis spp.

摘要:

蓝细菌(cyanobacteria)，俗称蓝藻，是水生生态系统中重要的初级生产者，也是常见的水华优势藻。噬藻体(cyanophage)，尤其是原噬藻体(prophage)是重要的浮游生态因子，影响蓝藻的进化和水生微生物群落结构。然而，有关原噬藻体的信息极为有限，迄今尚未有关于微囊藻(Microcystis)原噬藻体的研究报道。目的 探讨微囊藻的溶原性及其原噬藻体的基因组特征。方法 从NCBI数据库中下载了所有微囊藻基因组(共计354个)序列，利用PHASTER软件预测其携带的原噬藻体，并分析原噬藻体的携带率、基因组大小和G+C含量；通过毒力因子数据库(virulence factors of bacterial pathogens, VFDB)和综合抗生素研究数据库(comprehensive antibiotic resistance database, CARD)平台，分析完整型原噬藻体与疑似型原噬藻体中携带的耐药因子与毒力因子；综合运用生物信息学工具对完整型原噬藻体和疑似型原噬藻体进行基因功能注释和系统进化分析。选取含有完整型原噬藻体的水华微囊藻(Microcystis aeruginosa) FACHB-1326，用丝裂霉素C进行原噬藻体活化诱导，并采用点斑法进一步验证活化噬藻体的感染活性。结果 在全部354个微囊藻基因组中，98.3%的藻株被预测出携带原噬藻体或原噬藻体样片段；共发现完整型原噬藻体13个，疑似型原噬藻体5个，不完整型原噬藻体725个。将完整型原噬藻体和疑似型原噬藻体分别命名为WZ1-WZ13和YS1-YS5。在其基因组中均未发现耐药因子与毒力因子基因。蛋白谱发育树(phylogenomic tree)显示，这18株微囊藻原噬藻体与其他已知病毒的进化距离较远。生物信息学分析提示，YS5揭示了一个以往未知的新属；WZ2、WZ3、WZ4、WZ5、WZ6、WZ7、WZ9、WZ10、WZ11、WZ12与YS3共同揭示了一个以往未知的新科；WZ1与YS1共同揭示了一个新科；YS2、YS4、WZ8和WZ13则各自揭示了一个新科。FACHB-1344和FACHB-1326各被预测出一个完整型原噬藻体，试验表明丝裂霉素C可成功将其诱导活化。结论 溶原现象在微囊藻中普遍存在，微囊藻基因组中整合的原噬藻体代表了以往未知的病毒进化谱系。本研究拓宽了对浮游病毒的认知。

Abstract:

Cyanobacteria, commonly known as blue-green algae, are important primary producers in aquatic ecosystems and common dominant algae causing algal blooms in freshwater. Cyanophages, especially procyanophages, are important planktonic ecological factors that affect the evolution of blue-green algae and aquatic microbial communities. Yet studies on procyanophages in cyanobacteria remain scarce. So far, few studies have reported the procyanophages in Microcystis.Objective To investigate the prevalence of lysogeny in Microcystis and characterize the genomic features of procyanophages in Microcystis.Methods All the 354 genome sequences of Microcystis spp. in GenBank were downloaded. PHASTER was used to predict procyanophage regions in the Microcystis genomes. Resistance and virulence factors in intact procyanophages and questionable procyanophages were annotated via the virulence factors of bacterial pathogens (VFDB) and comprehensive antibiotic resistance database (CARD). Bioinformatics tools were used for gene annotation and phylogenetic analysis of the procyanophages in Microcystis. Microcystis flos-aquae FACHB-1344 and M. aeruginosa FACHB-1326 each was predicted to harbor an intact procyanophage by PHASTER. To evaluate the infection activities of these two procyanophages, mitomycin C induction assays and dot-ELISA were conducted.Results Among all the 354 Microcystis genomes, 98.3% were predicted to harbor intact procyanophages, questionable or imcomplete procyanophages. A total of 13 intact procyanophages, 5 questionable procyanophages and 725 incomplete procyanophages were predicted by PHASTER. The 13 intact procyanophages and 5 questionable procyanophages were named as WZ1-WZ13 and YS1-YS5, respectively. No antibiotic resistance or virulence gene was detected in them. The phylogenomic tree displayed distant evolutionary relationships between the 18 procyanophages and other known viruses. Bioinformatics analysis suggested that YS5 revealed a previously unknown novel genus. WZ2, WZ3, WZ4, WZ5, WZ6, WZ7, WZ9, WZ10, WZ11, WZ12 and YS3 together revealed a novel family. WZ1 and YS1 together revealed a novel family. YS2, YS4, WZ8 and WZ13 each revealed a novel family. The procyanophages in M. flos-aquae FACHB-1344 and FACHB-1326 were verified to be activated by mitomycin C.Conclusion Lysogeny widely exists in Microcystis spp. The novel procyanophages in Microcystis spp. unlock novel viral evolutionary lineages previously unknown. This study enriches the understanding about cyanobacterium-virus interactions and the diversity of aquatic viruses.