Efficient dilution-to-extinction isolation of novel virus–host model systems for fastidious heterotrophic bacteria

Abstract

Microbes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus–host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages, including the first known siphophage-infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host ranges. Similar to the abundant human-associated phage ɸCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes, or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus–host systems in culture collections and genomic databases will improve both our understanding of virus–host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

Genomic Characterization and Distribution Pattern of a Novel Marine OM43 Phage

“…The relative abundances of phage genomes were normalized by total recruited nucleotides (kb) per kb of genome per gigabase of metagenome (KPKG). MEP301-type genomes for which <40% of the genomes was covered by recruited reads in a given viromic dataset were given a RPKG value of 0 (Buchholz et al, 2021).…”

“…In recent years, bacteriophages that infect ecologically important and abundant marine bacterioplankton have received increasing attention. For example, pelagiphages, RCA phages and SAR116 phages have been shown to be diverse and dominate the viral communities (Kang et al, 2013;Zhao et al, 2013Zhao et al, , 2019Zhang et al, 2019Zhang et al, , 2021Buchholz et al, 2021). Other than the abovementioned phages, phages infecting many important marine bacteria still remain poorly investigated.…”

“…To date, only one OM43 phage has been reported, limiting our understanding on their diversity, evolution, ecology, and impacts. Venkman, the first OM43 phage isolated on the OM43 strain H5P1, was isolated from the Western English Channel (Buchholz et al, 2021). Venkman exhibits significant genomic similarity with freshwater Methylophilales LD28 phage P19250A (Buchholz et al, 2021).…”

“…Venkman, the first OM43 phage isolated on the OM43 strain H5P1, was isolated from the Western English Channel (Buchholz et al, 2021). Venkman exhibits significant genomic similarity with freshwater Methylophilales LD28 phage P19250A (Buchholz et al, 2021). HTCC2181 was the first isolate from the OM43 clade, which served as a good example of bacterioplanktons with reduced genome size to adapt to the nutrition limited environments (Giovannoni et al, 2008;Halsey et al, 2012).…”

Metaviromes reveal the dynamics of Pseudomonas host-specific phages cultured and uncultured by plaque assay

“…One of the reasons for the lack of high-quality phage genomes is that the methodology for isolating single phage strains with known hosts is laborious, time-consuming, and often the limiting step [17][18][19]. One of two methods (I) direct plating or (II) enrichment approach is most often used to isolates single phages.…”