A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

Ohno, Sanae; Okano, Hironori; Tanji, Yasunori; Ohashi, Akiyoshi; Watanabe, Kazuya; Takai, Ken; Imachi, Hiroyuki

doi:10.1007/s00253-012-4174-1

Cited by 12 publications

(13 citation statements)

References 46 publications

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“…SYBR gold at 80°C for 10 min (38) demonstrated the highest fluorescence intensity and least background noise (data not shown); thus, these conditions were chosen as the standard for the rest of the study. As in a previous study (32), we found that the SYBR gold-labeled cyanophages produced a number of plaques on host lawn agarose plates equivalent to that obtained with unlabeled phage (see Fig. S2 in the supplemental material).…”

Section: Resultssupporting

confidence: 85%

“…Third, fluorescently labeled viruses (FLVs) have been used as probes to tag their host cells for examination under the microscope (29–31), a method that is limited only by the fact that it is low throughput. Additionally, while FLVs and flow cytometry have been combined to increase throughput (32), labeling of nonhost cells has plagued these experiments even when a thymidine analog (EdU [5-ethynyl-2′-deoxyuridine]) was used as an alternative virus-labeling agent (32). …”

Section: Introductionmentioning

confidence: 99%

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Contrasting Life Strategies of Viruses that Infect Photo- and Heterotrophic Bacteria, as Revealed by Viral Tagging

Deng

Gregory

Yilmaz

et al. 2012

mBio

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Ocean viruses are ubiquitous and abundant and play important roles in global biogeochemical cycles by means of their mortality, horizontal gene transfer, and manipulation of host metabolism. However, the obstacles involved in linking viruses to their hosts in a high-throughput manner bottlenecks our ability to understand virus-host interactions in complex communities. We have developed a method called viral tagging (VT), which combines mixtures of host cells and fluorescent viruses with flow cytometry. We investigated multiple viruses which infect each of two model marine bacteria that represent the slow-growing, photoautotrophic genus Synechococcus (Cyanobacteria) and the fast-growing, heterotrophic genus Pseudoalteromonas (Gammaproteobacteria). Overall, viral tagging results for viral infection were consistent with plaque and liquid infection assays for cyanobacterial myo-, podo- and siphoviruses and some (myo- and podoviruses) but not all (four siphoviruses) heterotrophic bacterial viruses. Virus-tagged Pseudoalteromonas organisms were proportional to the added viruses under varied infection conditions (virus-bacterium ratios), while no more than 50% of the Synechococcus organisms were virus tagged even at viral abundances that exceeded (5 to 10×) that of their hosts. Further, we found that host growth phase minimally impacts the fraction of virus-tagged Synechococcus organisms while greatly affecting phage adsorption to Pseudoalteromonas. Together these findings suggest that at least two contrasting viral life strategies exist in the oceans and that they likely reflect adaptation to their host microbes. Looking forward to the point at which the virus-tagging signature is well understood (e.g., for Synechococcus), application to natural communities should begin to provide population genomic data at the proper scale for predictively modeling two of the most abundant biological entities on Earth.

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Section: Resultssupporting

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Contrasting Life Strategies of Viruses that Infect Photo- and Heterotrophic Bacteria, as Revealed by Viral Tagging

Deng

Gregory

Yilmaz

et al. 2012

mBio

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“…Estimating viral production using Viral BONCAT Accurate estimates of viral production in the ocean are critical for determining the influence of viral lysis on phytoplankton and bacterial mortality. A variety of methods have been employed to estimate viral production; these include, but are not limited to the incorporation of radiolabelled substrates into viral DNA (e.g., 3 H-thymidine or 32 P; Steward et al, 1992a,b;Fuhrman and Noble, 1995), TEMbased observations of visibly infected cells (e.g., Proctor et al, 1993;Steward et al, 1996), quantifying viral decay rates (e.g., Heldal and Bratbak, 1991;Bratbak et al, 1992), using fluorescently labelled viruses as tracers (e.g., Noble and Fuhrman, 2000;Ohno et al, 2012), and virus reduction approaches (e.g., Wilhelm et al, 2002;Helton et al, 2005;Winget et al, 2005;Weinbauer et al, 2010).…”

Section: Tracking Elemental Exchanges Between Viruses and Their Hostsmentioning

confidence: 99%

“…Fluorescence based methods such as the general proteintargeted Viral-BONCAT are comparatively inexpensive and easy to employ with minimal sample manipulation and experimental set up. Nucleic-acid based fluorescence staining of viruses is widely used in microbial ecology studies to visualize and count populations (e.g., Sherr et al, 1987;Noble and Fuhrman, 1998;Hennes et al, 1995;Zhang et al, 2010;Ohno et al, 2012;Allers et al, 2013). By targeting proteins rather than nucleic acids, Viral-BONCAT potentially targets a broader range of viruses, such as single stranded RNA/DNA viruses, which are currently missed with SYBR staining methods, but may represent a large proportion of the viral assemblage in the surface ocean (Steward et al, 2013;Roux et al, 2016b).…”

Section: Tracking Elemental Exchanges Between Viruses and Their Hostsmentioning

confidence: 99%

Interrogating marine virus‐host interactions and elemental transfer with BONCAT and nanoSIMS‐based methods

Pasulka

Thamatrakoln

Kopf

et al. 2017

Environmental Microbiology

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Summary While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus‐host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single‐virus level. We describe two new complementary approaches – stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence‐based biorthogonal non‐canonical amino acid tagging (BONCAT) – for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1 and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.

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“…This method is also difficult to apply to environmental samples where lysogenic infections are prevalent, because the method relies on observations of visible plaque formations, which are often absent from lysogenic infections [3, 58, 59]. Recently, Deng et al [52] demonstrated a new technique, known as "viral tagging," for identifying the interactions between cultivated host bacteria and their phages, which used the nucleic acid stain SYBR Gold to generate fluorescently labeled phages, so that the host cells fluoresced with viral tagging, thereby allowing the sorting of virus-tagged cells by flow cytometry [52, 60]. This emerging technique is undoubtedly helpful for not only exploring virus-host interactions in their natural habitats when the method is combined with other experimental tools, such as single viral genomics [61] and phageFISH [62], but also identifying viral receptors in macro-organisms (e.g., the mammalian gut) if the method is combined with a fluorescently labeled receptor protein during histological examinations.…”

Section: Virus-host Interactions and Emerging Technologiesmentioning

confidence: 99%

Comparative Viral Metagenomics of Environmental Samples from Korea

2013

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The introduction of metagenomics into the field of virology has facilitated the exploration of viral communities in various natural habitats. Understanding the viral ecology of a variety of sample types throughout the biosphere is important per se, but it also has potential applications in clinical and diagnostic virology. However, the procedures used by viral metagenomics may produce technical errors, such as amplification bias, while public viral databases are very limited, which may hamper the determination of the viral diversity in samples. This review considers the current state of viral metagenomics, based on examples from Korean viral metagenomic studies-i.e., rice paddy soil, fermented foods, human gut, seawater, and the near-surface atmosphere. Viral metagenomics has become widespread due to various methodological developments, and much attention has been focused on studies that consider the intrinsic role of viruses that interact with their hosts.

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A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

Cited by 12 publications

References 46 publications

Contrasting Life Strategies of Viruses that Infect Photo- and Heterotrophic Bacteria, as Revealed by Viral Tagging

Contrasting Life Strategies of Viruses that Infect Photo- and Heterotrophic Bacteria, as Revealed by Viral Tagging

Interrogating marine virus‐host interactions and elemental transfer with BONCAT and nanoSIMS‐based methods

Comparative Viral Metagenomics of Environmental Samples from Korea

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