Freshwater Viral Metagenome Analyses Targeting dsDNA Viruses.

Viral metagenomics is one of the most widely used approaches to study viral population genomics. With the recent development of bioinformatic tools, the number of molecular biological methods, programs, and software to analyze viral metagenome data have greatly increased. Here, we describe the basic analysis workflow along with bioinformatic tools that can be used to analyze viral metagenome data. Although this chapter assumes that the viral metagenome data are prepared from the freshwater samples and are subjected to dsDNA sequencing, the protocol can be applied and modified for other types of metagenome data collected from a variety of sources.

Biodiesel production and simultaneous treatment of domestic and livestock wastewater using indigenous microalgae, Chlorella sorokiniana JD1-1.

In this study, the potential of Chlorella sorokiniana JD1-1 for biodiesel production was evaluated using domestic wastewater (DWW) as a diluent for locally-generated livestock wastewater (LWW). This strategy aimed to provide sustainable wastewater treatment, reduce environmental impacts, enhance cost-effectiveness, and promote biodiesel production. LWW was diluted with tap water and DWW at ratios of 75%, 50%, and 25% (v/v), and the effects on microalgal growth, nutrient removal efficiency, and lipid yield were evaluated. Although the maximum biomass concentration was observed in the artificial growth medium (BG-11) (1170 mg L-1), 75% dilution using tap water (610 mg L-1) and DWW (780 mg L-1) yielded results comparable to the exclusive use of DWW (820 mg L-1), suggesting a potential for substitution. Total nitrogen (TN) removal rates were consistently high under all conditions, particularly in samples with higher concentrations of LWW. Conversely, total phosphorus (TP) concentrations decreased under most conditions, although some displayed large increases. Further studies are necessary to optimize the nutrient balance while maintaining economic feasibility and maximizing biodiesel production.

Isolation and screening of indigenous microalgae species for domestic and livestock wastewater treatment, biodiesel production, and carbon sequestration.

The use of indigenous microalgae strains for locally generated domestic (DWW) and livestock wastewater (LWW) treatment is essential for effective and economical applications. Phototrophic microalgae-based biofuel production also contributes to carbon sequestration via CO2 fixation. However, simultaneous consideration of both isolation and screening procedures for locally collected indigenous microalgae strains is not common in the literature. We aimed to isolate indigenous microalgae strains from locally collected samples on coastlines and islands in South Korea. Among five isolated strains, Chlorella sorokiniana JD1-1 was selected for DWW and LWW treatment due to its ability to grow in waste resources. This strain showed a higher specific growth rate in DWW than artificial growth medium (BG-11) with a range of 0.137-0.154 d-1. During cultivation, 96.5%-97.1% of total nitrogen in DWW and 89.2% in LWW was removed. Over 99% of total phosphorus in DWW and 96.4% in LWW was also removed. Finally, isolated C. sorokiniana JD1-1 was able to fix CO2 within a range of 0.0646-0.1043 g CO2 L-1 d-1. These results support the domestic applications of carbon sequestration-efficient microalgae in the waste-to-energy nexus.

Metaviromics coupled with phage-host identification to open the viral 'black box'.

Viruses are found in almost all biomes on Earth, with bacteriophages (phages) accounting for the majority of viral particles in most ecosystems. Phages have been isolated from natural environments using the plaque assay and liquid medium-based dilution culturing. However, phage cultivation is restricted by the current limitations in the number of culturable bacterial strains. Unlike prokaryotes, which possess universally conserved 16S rRNA genes, phages lack universal marker genes for viral taxonomy, thus restricting cultureindependent analyses of viral diversity. To circumvent these limitations, shotgun viral metagenome sequencing (i.e., metaviromics) has been developed to enable the extensive sequencing of a variety of viral particles present in the environment and is now widely used. Using metaviromics, numerous studies on viral communities have been conducted in oceans, lakes, rivers, and soils, resulting in many novel phage sequences. Furthermore, auxiliary metabolic genes such as ammonic monooxygenase C and ß-lactamase have been discovered in viral contigs assembled from viral metagenomes. Current attempts to identify putative bacterial hosts of viral metagenome sequences based on sequence homology have been limited due to viral sequence variations. Therefore, culture-independent approaches have been developed to predict bacterial hosts using single-cell genomics and fluorescentlabeling. This review focuses on recent viral metagenome studies conducted in natural environments, especially in aquatic ecosystems, and their contributions to phage ecology. Here, we concluded that although metaviromics is a key tool for the study of viral ecology, this approach must be supplemented with phage-host identification, which in turn requires the cultivation of phage-bacteria systems.

Viral metagenomes of Lake Soyang, the largest freshwater lake in South Korea.

A high number of viral metagenomes have revealed countless genomes of putative bacteriophages that have not yet been identified due to limitations in bacteriophage cultures. However, most virome studies have been focused on marine or gut environments, thereby leaving the viral community structure of freshwater lakes unclear. Because the lakes located around the globe have independent ecosystems with unique characteristics, viral community structures are also distinctive but comparable. Here, we present data on viral metagenomes that were seasonally collected at a depth of 1 m from Lake Soyang, the largest freshwater reservoir in South Korea. Through shotgun metagenome sequencing using the Illumina MiSeq platform, 3.08 to 5.54-Gbps of reads per virome were obtained. To predict the viral genome sequences within Lake Soyang, contigs were constructed and 648 to 1,004 putative viral contigs were obtained per sample. We expect that both viral metagenome reads and viral contigs would contribute in comparing and understanding of viral communities among different freshwater lakes depending on seasonal changes.

Freshwater viral metagenome reveals novel and functional phage-borne antibiotic resistance genes.

BACKGROUND: Antibiotic resistance developed by bacteria is a significant threat to global health. Antibiotic resistance genes (ARGs) spread across different bacterial populations through multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages. ARGs carried by bacteriophages are considered especially threatening due to their prolonged persistence in the environment, fast replication rates, and ability to infect diverse bacterial hosts. Several studies employing qPCR and viral metagenomics have shown that viral fraction and viral sequence reads in clinical and environmental samples carry many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes. Owing to the wide application of viral metagenomics, nevertheless, different classes of ARGs are being continuously found in viral metagenomes acquired from diverse environments. As such, the presence and functionality of ARGs encoded by bacteriophages remain up for debate. RESULTS: We evaluated ARGs excavated from viral contigs recovered from urban surface water viral metagenome data. In virome reads and contigs, diverse ARGs, including polymyxin resistance genes, multidrug efflux proteins, and ß-lactamases, were identified. In particular, when a lenient threshold of e value of ≤ 1 × e-5 and query coverage of ≥ 60% were employed in the Resfams database, the novel ß-lactamases blaHRV-1 and blaHRVM-1 were found. These genes had unique sequences, forming distinct clades of class A and subclass B3 ß-lactamases, respectively. Minimum inhibitory concentration analyses for E. coli strains harboring blaHRV-1 and blaHRVM-1 and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow- and extended-spectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages. CONCLUSION: Our results showed that viruses in the environment carry as-yet-unreported functional ARGs, albeit in small quantities. We thereby suggest that environmental bacteriophages could be reservoirs of widely variable, unknown ARGs that could be disseminated via virus-host interactions. Video abstract.

Genomic and ecological study of two distinctive freshwater bacteriophages infecting a Comamonadaceae bacterium.

Bacteriophages of freshwater environments have not been well studied despite their numerical dominance and ecological importance. Currently, very few phages have been isolated for many abundant freshwater bacterial groups, especially for the family Comamonadaceae that is found ubiquitously in freshwater habitats. In this study, we report two novel phages, P26059A and P26059B, that were isolated from Lake Soyang in South Korea, and lytically infected bacterial strain IMCC26059, a member of the family Comamonadaceae. Morphological observations revealed that phages P26059A and P26059B belonged to the family Siphoviridae and Podoviridae, respectively. Of 12 bacterial strains tested, the two phages infected strain IMCC26059 only, showing a very narrow host range. The genomes of the two phages were different in length and highly distinct from each other with little sequence similarity. A comparison of the phage genome sequences and freshwater viral metagenomes showed that the phage populations represented by P26059A and P26059B exist in the environment with different distribution patterns. Presence of the phages in Lake Soyang and Lake Michigan also indicated a consistent lytic infection of the Comamonadaceae bacterium, which might control the population size of this bacterial group. Taken together, although the two phages shared a host strain, they showed completely distinctive characteristics from each other in morphological, genomic, and ecological analyses. Considering the abundance of the family Comamonadaceae in freshwater habitats and the rarity of phage isolates infecting this family, the two phages and their genomes in this study would be valuable resources for freshwater virus research.

Genome characteristics and environmental distribution of the first phage that infects the LD28 clade, a freshwater methylotrophic bacterial group.

Bacteriophages infecting major groups of freshwater heterotrophic bacteria have been rarely isolated, hampering analyses of freshwater viromes. Here, we report the isolation and genomic characterization of P19250A, the first phage that infects the LD28 clade, an abundant freshwater methylotrophic bacterial group. P19250A was isolated from Lake Soyang, an oligotrophic reservoir, using an LD28 strain as a host. Morphological and genomic analyses revealed that P19250A is a lytic siphovirus with a ∼38.6-kb genome. To analyze the distribution of P19250A genome within its habitat, six seasonal viral metagenome (virome) samples were prepared from Lake Soyang. Through binning analysis of freshwater viromes, P19250A was shown to be the most highly assigned freshwater phage that infects heterotrophic bacteria (up to 8.21%) in five viromes. Furthermore, when freshwater virome data collected worldwide were analyzed, P19250A genome also showed high abundance, especially in Lough Neagh, UK, where P19250A genome was recorded as the most abundant bacteriophage. From metagenome analysis, the proportion of P19250A-assigned reads showed seasonal fluctuation following the abundance of the LD28 clade in Lake Soyang. These results showed that P19250A would be an essential resource for analyses of freshwater viromes, and also suggest that phages of other abundant freshwater bacteria need to be isolated for better understanding of freshwater viruses.

Complete genome sequence of bacteriophage P26218 infecting Rhodoferax sp. strain IMCC26218.

Bacteriophage P26218 is a virus that thrives in freshwater and infects Rhodoferax sp. strain IMCC26218, both of which were isolated from Soyang Lake, Korea. The bacterial host, IMCC26218, belongs to the genus Rhodoferax and is closely related to R. saidenbachensis, with 98.7 % 16S rRNA gene sequence similarity. Bacteriophage P26218 has an icosahedral head structure with a diameter of ~52 nm and short tail of ~9 nm, which is a typical morphology of the Podoviridae family. Its complete dsDNA genome was 36,315 bp with 56.7 % G + C content. This is the first genome sequence reported for a lytic phage of the genus Rhodoferax.