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 a new SAR11 and three new members of the methylotrophic bacterial clade OM43, and used these as bait to isolate 117 new phages including the first known siphophage infecting SAR11, and the first known phages for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that SAR11 viruses have ecotype-specific host-ranges. Similar to the abundant human-associated phage ϕCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny, irrespective of detectable associated genes, and/or host phenotypic bistability, with lysis putatively maintained within a susceptible subpopulation. Broader representation of 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

“…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, 2013 , 2019 ; Zhang et al, 2019 , 2021 ; Buchholz et al, 2021 ). Other than the above-mentioned 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 ).…”

Draft Genome Sequences of Pelagimyophage Mosig EXVC030M and Pelagipodophage Lederberg EXVC029P, Isolated from Devil’s Hole, Bermuda

“…Viruses infecting the heterotrophic bacterial clade of Pelagibacterales are an important component of marine microbial communities throughout global oceans ( 1 ). Since the discovery and first isolation of four pelagiphages in 2013 ( 2 ), 38 more have been isolated and sequenced ( 3 – 5 ). Out of the 38 isolated pelagiphages, 36 belong to the Podoviridae family, with only one species each of Myoviridae and Siphoviridae .…”

“…Viruses were concentrated by tangential flow filtration (50R VivaFlow 100-kDa Hydrosart filter; Sartorius Lab Instruments, Göttingen, Germany). We used previously described dilution-to-extinction-based methods ( 4 ) with HTCC1062 as a bait host (grown in artificial seawater medium ASM1 [ 7 ]) in 96-well Teflon plates, which does not rely on plaque formation, because the host does not grow on solid medium. The purification process was repeated five times; nonetheless, final sequence data contained two genomes, suggesting an impure culture.…”

“…For DNA isolation, a 50-ml HTCC1062 culture (10 6 cells/ml), amended with 5 ml of 0.1-μm-filtered lysate, was grown in ASM1 (18°C) until cell death (detected via flow cytometry). Debris was removed using 0.1-μm-pore polyvinylidene difluoride (PVDF) filters, and lysate was subjected to PEG8000/NaCl DNA isolation (modified from https://doi.org/10.17504/protocols.io.c36yrd , as described previously ( 4 ).…”