A novel phage with unusual pyrimidine nucleotide substitution: Q&A with Dr. Branko Rihtman and Dr. Richard Puxty

Issue 135 | July 16, 2021
19 min read
Capsid and Tail

Graphical abstract for Rihtman et al. (2021). Read the full paper at https://doi.org/10.1016/j.cub.2021.05.014

This week, Madhav Madurantakam Royam interviewed Dr. Branko Rihtman and Dr. Richard Puxty about their new paper describing an unusual nucleotide substitution from deoxythymidine to deoxyuridine in a novel family of phages belonging to Naomiviridae.

What’s New

Rebekah Dedrick (University of Pittsburgh) and colleagues published a new paper in Nature Medicine on how antibody-mediated neutralization limited phage treatment of a pulmonary M. abscessus infection. They used the same phages that worked well in their well-known 2019 UK patient case, but they didn’t work well in this new patient, which led to important insights into the immune system’s impact on phage therapy. Paper | Behind-the-Paper by Graham Hatfull and Keira Cohen | Public digest article, Ars Technica

Phage TherapyPhage-immune interactionsResearch paper

Matthias Hoetzinger (Linnaeus University, Sweden) and colleagues published a paper in Environmental Microbiology on how the dynamics of Baltic Sea phages are driven by environmental changes. They isolated 121 phage strains infecting three bacterial hosts during a Baltic Sea mesocosm experiment, and found tons of insights, including that phytoplankton blooms may influence succession of heterotrophic bacteria through their phages.

Marine phagesResearch paper

Hayley Nordstrom (University of Pittsburgh School of Medicine) and colleagues published a preprint showing genomic and functional characterization of P. aeruginosa-targeting phages isolated from hospital wastewater. They used a panel of clinical isolates (most from people with CF) to isolate over a dozen phages from hospital wastewater, characterized the isolates, the phages, and phage-resistant mutants, and got tons of new functional genomics insights! Tweet thread.

Phage characterizationPhage genomicsPreprint

Karen Fong (Summerland Research and Development Centre, Canada) and colleagues published a review article in PHAGE on the host range of phages and its impact on the agri-food sector. They discuss the research progress, applications, and implications of broad host range phages, asking ‘how broad is broad enough?’, with a focus on tailed phages infecting human pathogens of concern in the agri-food sector.

Agri-foodHost rangeReview

Diana Pires (University of Minho, Portugal) and colleagues published a review in Annual Review of Virology on understanding the complex phage-host interactions in biofilm communities. They summarize the main features affecting phage-biofilm interactions, highlight currently available methods of studying these interactions, and address the applications of phages for biofilm control in different contexts.

BiofilmsPhage-biofilm interactionsReview

Latest Jobs

Sponsored Ad Phage Biotech

Multiple positions

Adaptive Phage Therapeutics

Gaithersburg, MD, USA

Adaptive Phage Therapeutics, a clinical-stage, phage-based biotech company in Gaithersburg, Maryland, USA, is hiring for several positions. To apply, email your cover letter and resume to [email protected].

Clinical Data Manager

Design and manage an electronic database, including data verification, data cleaning, and quality assurance.

Project Manager, Project & Alliance Management

Lead and manage teams developing drug and vaccine candidates to ensure integration of project and company goals.

Quality Control Manager

Manage QC operations, direct staff, assist in designing QC systems to ensure patient safety and data integrity.

Quality Assurance Documentation Specialist

Manage QAD operations to ensure compliant and efficient management of controlled cGMP documents.

Quality Assurance (QA) Specialist/Sr. Specialist

Participate in QA operations and act as QA point-person.

Manufacturing Associate I/II

Amplify and purify clinical phages, including propagating, maintaining, and cataloguing bacterial cell and phage lines.

Manufacturing Technician I/II

Operate standard processing equipment (e.g. peristaltic pumps, filtration systems, liquid handlers), and perform microbiological assays.

Manufacturing Fill Finish Technician I/II

Perform Fill/Finish operations, routinely monitor manufacturing processes, recognize potential issues.

BioinformaticsPhage genomicsPost Doc
The Mostowy lab at the Jagiellonian University in Kraków, Poland is hiring a postdoc to work on bioinformatics and genomics of phages that infect opportunistic bacterial pathogens like Klebsiella pneumoniae and interact with their polysaccharide capsules.
Gut phageResearch Assistant
The Hill lab at APC Microbiome (Cork, Ireland) is hiring a research assistant to collaborate with a major pharma company to explore the role of phages in shaping the human gut microbiome.

Community Board

Anyone can post a message to the phage community — and it could be anything from collaboration requests, post-doc searches, sequencing help — just ask!

Evergreen Logo

There’s still time to register for Evergreen!

Sign up here to be part of the longest running phage meeting.

Phagebiotics Research Foundation and Phage Directory present the 24th biennial Evergreen International Phage Meeting August 2-5th 2021. This year you can participate in person or virtually. The meeting will include primary session talks, live and virtual poster sessions, daily flash talks and Q&A for all participants.

As always, we look forward to hearing about all the exciting phage work going on worldwide!

Thanks to our sponsors, Phagebiotics and TAILOR! Want to sponsor Evergreen? There’s still time!


Note: date/time change!

For PHAVES #20 next week, Dr. Gina Suh, MD (Mayo Clinic, Rochester, MN) and John Haverty will present a doctor-patient experience with phage therapy on July 22 at 2:30 PM Eastern! Gina treated John’s leg with phages, which led him to avoid amputation. Hear about their experience and ask them all your questions!

Register here!

PHAVESVirtual Event

Thanks so much to those who signed up for Instill Science since we launched last week! For those who missed it, read about our new peer feedback matching initiative here!

We’re thrilled to have the community’s support as we get going on this; if you signed up, you’ll hear from us soon with next steps! And if you’d like to join the movement, whether you want to improve the impact of your phage research, or have specific expertise you’d like to share with others in the community, we’d love to bring you on board!

Join the Instill movement.

Have a specific question about your in-progress manuscript that you think someone in the phage community could answer? Want to get advice on whether you should shoot for a higher journal?

Get advice from phage experts about your manuscript!

Phage DirectoryNew initiativeCollaboration

Tired of manually counting plaques on petri dishes? Harness the power of AI to count plaques in near real-time with OnePetri, an automated plaque counting iOS app. Here’s a short clip of the app in action.

I am looking for beta testers to help test OnePetri before publishing the app for free, alongside its source code, next month. If you have a device running iOS 13 or newer, please fill out this form to register for beta access and start saving time with OnePetri! Questions? Don’t hesitate to reach out by email: [email protected] .

Looking forward to sharing some more news in the weeks ahead! — Michael Shamash (McGill University)

Phage AppsSeeking beta testersTool

The FDA’s Center for Biologics Evaluation and Research and NIH’s NIAID have announced a public workshop entitled “Science and Regulation of Bacteriophage Therapy.”

The purpose of the public workshop is to exchange information with the medical and scientific community about the regulatory and scientific issues associated with phage therapy. It will run Aug 30-Sep 1, and is free to attend. Sessions will include choosing and characterizing phages for therapy, manufacturing for stability and clinical use, using phages clinically, and research gaps that remain.

Register here.

Phage TherapyVirtual EventWorkshop

I observed lysis plaques all over ATCC P. aeruginosa 27853 soft agar overlay prepared from overnight bacterial cultures. The bacterial strain is reported to contain prophage sequences in its genome. ATCC website also mentions that phage may be present in the culture. Could it be prophage induction (maybe due to stress conditions from high cell densities or spontaneous?) or phage contamination? Please email [email protected] if you can help!

Experimental helpSeeking advice

A novel phage with unusual pyrimidine nucleotide substitution: Q&A with Dr. Branko Rihtman and Dr. Richard Puxty

Profile Image
Graduate Student
Puxty Lab,
University of Warwick, Coventry, UK

Biotechnology, Phage isolation, Phage Therapy, Phage-host interactions, Data Analytics, Molecular Biology

I am Madhav Madurantakam Royam, a first year Ph.D. student at Puxty’s Lab at the School of Life Sciences, University of Warwick, UK. My Ph.D. work focuses on elucidating the role of tRNA’s in mycobacteriophages with respect to its virulence and host range. My work involves evolution of mycobacteriophages against their hosts and generating tRNA mutants in them. I have finished my Master of Science (MSc) and Master of Technology [By Research] degrees from Vellore Institute of Technology, Vellore, India. I have had experiences on working with Gram-negative pathogens such as Citrobacter sp. Pseudomonas aeruginosa, Klebsiella sp, and Escherichiacoli. During my Master of Technology [By Research] degree, I have isolated lytic bacteriophages against Citrobacter sp. and characterized their in vitro & in vivo efficacy in a zebrafish model.

My research interests are to understand the evolution of phages against their hosts and characterise their defence mechanism them using various molecular biology and bioinformatic tools.

Profile Image
Postdoctoral Researcher
Scanlan Lab,
University of Warwick, Coventry, UK

Interested in AMGs and functional metagenomics involving all phages. Currently working on marine phages of Cyanobacteria and Roseobacter

Profile Image
Assistant Professor
Puxty Lab (PI),
University of Warwick, Coventry, UK

Anti-phage systems in non-model organisms, viral auxiliary metabolic genes, viral photosynthesis, phage ecology and evolution

Branko Rihtman and Richard Puxty (University of Warwick) recently published a new paper in Current Biology documenting an unusual nucleotide substitution from deoxythymidine to deoxyuridine in a novel family of phages belonging to Naomiviridae. These substitutions render the phage DNA resistant to digestion by common restriction enzymes such as EcoRV, XbaI and HindIII.

Madhav: Could you give us a little background about yourself and your research interests, to start?

Branko Rihtman: I studied for my undergraduate and MSc degrees at Hebrew University of Jerusalem, Israel, focusing on transcriptional regulation of nitrogen stress in marine cyanobacteria in the lab of Prof. Anton Post. I completed my PhD under supervision of Prof. David Scanlan at the University of Warwick and Prof. Martha Clokie at the University of Leicester, studying the transcriptional landscape of cyanophage infection under phosphate stress conditions, as well as characterising some of the phage auxiliary metabolic genes. Since then, I have worked as a postdoctoral researcher in the lab of Prof. Yin Chen at the University of Warwick, isolating and studying roseophages and their effect on lipid content of roseobacter cells, work which has resulted, among others, in this paper. My main research interest is the presence and function of auxiliary metabolic genes in phage genomes and different ways in which phages affect the metabolism of their host over the course of infection.

Richard Puxty: I am a molecular microbiologist. My early interest was in photosynthesis in prokaryotes, but since a PhD at Warwick (2010-2014), I have become interested in phage biology, predominantly through the discovery that phage genomes encode core genes of photosynthesis. Since then, I have fallen in love with phage research and in particular, the molecular genetic arms race between phage and their hosts.

M: Can you give us a quick summary of your new article?

B & R: In this article, we describe a novel family of roseophages — Naomiviridae — which were isolated from sea water from two very distant locations: water from the canal under the Rialto bridge in Venice and coastal water from Puerto Morelos beach in Mexico. The fact that two very similar phages, which are at the same time so distinct from any other currently known phages, were isolated from such distant locations hints towards global distribution of this viral family.

This observation then raises the question: why were they not previously seen in numerous metagenomic studies? The reason probably lies in the same problem that we encountered when trying to sequence these phages: the library preparation step, which is based on a transposon insertion step, has failed over several attempts.

We have finally succeeded to sequence the genomes by performing an initial whole genome amplification step, but this has alerted us to the fact that these genomes may have some unique modifications that are preventing transposon insertion in the initial steps of the sequencing process.

To our surprise, we found that these phages have completely replaced deoxythymidine in their genomes with deoxyuridine. Genome sequencing of the phages has shown that they possess in their genomes genes encoding some of the enzymes which would potentially be required for this conversion of dTTP to dUTP, such as cytosine deaminase and dTTPase, located in proximity to the viral DNA polymerase. Querying of environmental metagenomes has discovered other viral contigs which contain putative cytosine deaminase and dTTPase orthologues in a similar genetic context to the one found in DSS3_VP1 and DSS3_PM1, pointing to the presence of the same or similar genomic modification in other phages in marine environments.

Finally, uniqueness of these phages’ features, as well as large phylogenetic distance from all other currently known phages, has helped us classify them as members of a novel viral family Naomiviridae, forming a new genus Noahvirus within that family. Hopefully, these results will help inform future metagenomic studies of marine environments and will facilitate identification of additional phages belonging to this viral family.

M: Can you share more about roseophages and their importance?

B & R: Compared to other viral groups, very few roseophages have been isolated and characterised in detail. Since the members of the Roseobacter clade are important participants in the biogeochemistry of the ocean, participating, among other important biogeochemical processes, in the production of methylated amines, which are important precursors in nucleating particles involved in cloud formation above marine environments, thus having a global effect on the climate.

Due to the importance of their bacterial hosts, roseophages also play an important role in these biogeochemical processes by shaping the Roseobacter community dynamics and affecting the relative presence of these bacteria in the ocean. They also influence the flux of dissolved organic and inorganic compounds via the viral shunt of the microbial loop, where lysis of the bacterial host cells releases nutrients into the water and makes them available to other trophic levels of the ecosystem.

In addition to their role in the bacterial life cycle, these phages possess orthologs to bacterial metabolic genes which can provide insight into specific niche adaptation mechanisms and teach us about evolution of bacterial enzymes under nutrient stress conditions, processes which are much more pronounced in phages than in their bacterial hosts due to the preponderance of evolutionary pressures exerted on viruses compared to the bacteria they infect.

Finally, constant arms race between the bacteria and the viruses that infect them have produced a large number of viral defense systems in bacteria, which have been finding their way into different biotechnological applications over the years. Therefore, it is very important to study the novel phage genera, especially those that show novel features and unique adaptations to compete against their host defense mechanisms.

M: How was the nucleotide substitution discovered, and what previous research has been done on such novel nucleotides?

B&R: The nucleotide substitution was discovered due to the fact that the library preparation step of Illumina sequencing has failed a number of times. The only way we could sequence the genomes was if we performed a whole genome amplification step prior to the library prep.

After sequencing the genomes, we were intrigued about the nature of the modifications, and so we sent the purified viral DNA to Dr. Peter Weigele at New England Biolabs in Cambridge, MA, USA. He and his team performed a mass spectrometry analysis of separate nucleotides forming the DNA of Naomivirus phages, and have shown that dTTP is completely absent from the genome, while being replaced by dUTP.

dTTP-to-dUTP genome modification has been shown to be present in Bacillus (Takahasi et al. 1963) and Yersinia phages (Kiljunen et al., 2005), but this is the first time it has been described in marine phages.

M: Could you expand on the inhibition of restriction digestion by common endonucleases, on the UDG endonuclease VIII assay, and on why this assay was chosen?

B&R: We hypothesised that the purpose of the phage genome modification was to protect the phage DNA from host endonucleases, and therefore tried to digest it with some common dUTP-sensitive endonucleases.

Together with endonuclease assay, we performed the UDG-endonuclease VIII assay, in which dUTP is removed from the genome by the action of UDG, creating an abasic site in the genome which is then open for digestion by endonuclease VIII.

We performed these experiments as an additional, independent way to experimentally demonstrate the presence of dUTP in the phage genome, using the whole-genome amplified DNA as a control that shows that sensitivity of native phageDNA to UDG-endonuclease VIII assay is not sequence-dependent.

M: Could you share a few insights into this phage’s genome sequence and lifecycle?

B&R: DSS3_VP1 and DSS3_PM1 belong to the siphoviral morphotype and have a latent period of 110 and 210 minutes respectively. Their capsids possess an icosahedral structure measuring 47–55 nm in diameter, with the tail measuring approximately 230–240 nm in length. One-step infection experiments in liquid culture show that DSS3_VP1 and DSS3_PM1 have infection kinetics that differ from previously reported double-stranded DNA (dsDNA) roseophages, which were shown to not fully lyse the infected bacterial culture, while Naomivirus phages achieved complete lysis of the liquid culture.

From the genome sequence perspective, Naomivirus phages are relatively conventional, apart from the genes required for conversion of dTTP to dUTP. Similarly to quite a few other dsDNA phages, they possess a putative type I self-splicing intron, containing a type VII homing endonuclease within the sequence of the large terminase gene TerL. They also possess both a DNA and an RNA polymerase, which is expected due to the unconventional nature of their genome. Similarly to almost all known sequenced phages, about 80% of the predicted ORFs encode hypothetical proteins with no known orthologues in other genomes.

M: What is the importance of studying these nucleotide substitutions, and how can this impact our understanding of life in nature?

B & R: There are several important aspects of studying unconventional nucleotides in general. Firstly, they are a part of life’s alphabet, and learning about different ‘letters’ used by different organisms shows us the boundaries of adaptations of living beings.

Since these modifications are possibly a part of the phage adaptation to constantly evolving bacterial viral defense mechanisms, learning about them contributes to our knowledge of the way viruses have adapted to avoid those defenses, and can instruct us in developing novel antivirals against eukaryotic viruses of concern. Additionally, previous research into bacterial anti-viral defense mechanisms have yielded important biotechnological molecular tools, such as CRISPR-Cas.

Since Naomivirus phages possess unconventional nucleotides, it is reasonable to assume that their DNA and RNA polymerases will be adapted to copy and transcribe from unconventional templates, so overexpressing and purifying these two enzymes can add to an arsenal of polymerases capable of producing unconventional DNA molecules, which would be resistant to existing host endonucleases and therefore be easier to transform into different bacterial hosts for cloning purposes, for example. Therefore, it is important to tap into this potential treasure trove of possible future biotechnological tools.

Finally, genomes of organisms that contain both conventional deoxynucleotides and unconventional ones, such as dUTP, can potentially teach us about the evolutionary origins of self-replicating molecules, especially if they contain nucleotides that bridge the gap between RNA and DNA.


Rihtman, B., Puxty, R. J., Hapeshi, A., Lee, Y. J., Zhan, Y., Michniewski, S., … & Chen, Y. (2021). A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution. Current Biology.

  • Main paper described in this feature

Takahashi, I. and Marmur, J., 1963. Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature197(4869), pp.794-795.

Kiljunen, S., Hakala, K., Pinta, E., Huttunen, S., Pluta, P., Gador, A., Lönnberg, H. and Skurnik, M., 2005. Yersiniophage ϕR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine. Microbiology151(12), pp.4093-4102.

Many thanks to Atif Khan for finding and summarizing this week’s phage news, jobs and community posts!

Capsid & Tail

Follow Capsid & Tail, the periodical that reports the latest news from the phage therapy and research community.

We send Phage Alerts to the community when doctors require phages to treat their patient’s infections. If you need phages, please email us.

In collaboration with

Mary Ann Liebert PHAGE

Supported by

Leona M. and Harry B. Helmsley Charitable Trust

Crossref Member Badge