Ecological and Genomic Study on Freshwater Bacteriophages

Abstract

Viruses, the smallest and simplest form of life, are the most abundant biological entities on the Earth. Bacteriophages (phages) are viruses that infect of infecting bacterial cells. As bacterial cells are known to be found in almost every environment known, their predators, bacteriophages are also found in diverse environments including ocean, soil, hot spring, polar areas, and deserts. However, despite their high abundance and ability to survive under extreme conditions, environmental bacteriophages had been understudied due to limitations in isolating and culturing them in laboratory settings. As a result, number of isolated and identified bacteriophages is very low relative to their high abundance in the environment. Recently, to overcome culturability restrictions, viral metagenome, also denoted as virome, was suggested to study bacteriophage population without culturing. Therefore, based on viral metagenome technique, many large-scale marine virome projects had been performed, especially in marine settings. However, most of the virome sequences remain as un-interpreted due to the dearth of known bacteriophage genome information in the public genome databases. Furthermore, only few number of bacteriophage studies in freshwater environments including both virome and isolation of phages have been performed despite the importance of inland freshwaters as highly conserved reservoirs of diverse microbial communities. For better understanding of freshwater microbial community structure and their ecological dynamics, this study performed both culture-independent and culture-dependent bacteriophage researches. Using viral metagenome, a culture-independent method, bacteriophage population distribution in Lake Soyang, the largest lake in South Korea, was observed. Since microbial community within a confined lake shifts as seasonal stratification takes place, bacteriophage community was also expected to change according to seasons. Therefore, 6 seasonal samples were collected from Lake Soyang and viral metagenome samples were prepared from them. When sequence similarity between 6 samples were compared, no clear seasonal variability was observed, however, gradual change of viral sequences was observed through time. When taxonomic annotation was performed using virome reads, up to 93.6% of them were not identifiable. Among those that were annotated with a taxonomic name, most of them were shown to be the phages that were isolated from marine environments. For more analysis of freshwater virome, viral contigs constructed from Lake Soyang virome data were grouped with reference viral sequences obtained from public databases, and 211 groups were found that showed no similarity with previously reported bacteriophages. In attempts to identify those unique viral groups, their putative host bacteria were predicted. Among 211 virome contig groups, 23 groups with the most viral contig sequences (976 contigs) were predicted to infect a host belonging to the phylum Proteobacteria and 1 group with 315 contigs was anticipated to infect a host within the phylum Actinobacteria, which are the two major bacterial phylum found in Lake Soyang. In spite of diverse attempts to interpret freshwater virome, inability to annotate virome reads and biasedly assigning the annotated freshwater virome reads to representative marine bacteriophage genomes indicated the under-representation and deficiency of freshwater bacteriophages. Therefore, to fill the gaps in the knowledge of freshwater bacteriophages, novel bacteriophages were physically isolated and cultured from Lake Soyang. As a result, total of 4 novel bacteriophages have been isolated from Lake Soyang. Two representative bacterial strains of the family Comamonadaceae, Rhodoferax and Curvibacter isolates were used as hosts to screen for novel phages from Lake Soyang. Hence, two independent phages infecting Curvibacter sp. and one phage infecting Rhodoferax sp. were isolated, and they were named as P26509A and P26059B, and P26218, respectively. The bacteriophage, P19250A that infects a strain belonging to the family Methylophilaceae, was also isolated and revealed to be the most abundant bacteriophage in Lake Soyang in winter seasons, in which its host, LD28 clade also thrives. In the binning analyses of freshwater viromes, P19250A was the most highly-assigned freshwater phage (up to 8.7%) in several viromes of foreign countries, including five viromes from Lake Soyang that were constructed in this study. These results showed that newly isolated bacteriophages would be an essential resource for analyses of freshwater viromes. One of the major ecological roles of bacteriophages is as mediators of horizontal gene transfer (HGT) between bacterial cells. Among many bacterial protein genes, antibiotic resistance gene (ARG) is one of the ecologically and clinically important genes that are transported by bacteriophages. To observe bacteriophage community structure of the lentic environment and their roles as ARG transporters in urban area, the Han River, which flows from Lake Soyang to the Yellow sea, passing through Seoul (the capital of South Korea) was selected as the study site. When overall sequence similarity was compared, all 6 samples collected from the Han River had low dissimilarity. Also, when taxonomic assignment of virome reads were analyzed, no significant change of taxonomic assignment was observed, indicating that Han River, which flows for approximately 180 km, has stably maintained viral population. As one of the auxiliary metabolic genes carried by bacteriophages, a number of ARGs was observed within from viral metagenome reads. Among virome contigs, 7 viral contigs were shown to be carrying well conserved active antibiotic resistance genes, suggesting that those genes may be transferred to bacterial cells upon phage infection and lead to the rise of antibiotic resistance bacterial strains. Through both culture-independent and dependent methods, distribution of bacteriophage sequences and their ecological roles in freshwater environments, both lentic and lotic, were observed. Through isolation of novel bacteriophages that are abundantly distributed in freshwater habitats, this study has provided a key information of interpreting global virome samples as well as that of Lake Soyang. Therefore, this study emphasized the need of isolation and culture of environmental bacteriophages to understand viral ecology and also viral metagenome data.