Nitrogen sourcing during viral infection of marine cyanobacteria Jacob R. Waldbauera,1,2, Maureen L. Colemana,1,2, Adriana I. Rizzoa, Kathryn L. Campbella, John Lotusa, and Lichun Zhanga aDepartment of the Geophysical Sciences, University of Chicago, Chicago, IL 60637 Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved June 21, 2019 (received for review January 31, 2019) The building blocks of a virus derived from de novo biosynthesis biology. The Hershey–Chase experiment (9), which showed that during infection and/or catabolism of preexisting host cell bio- only the atomic constituents of the parental virion nucleic acid, mass, and the relative contribution of these 2 sources has impor- and not of its protein, enter the host cell at the beginning of tant consequences for understanding viral biogeochemistry. We infection, is widely regarded as the first definitive evidence that determined the uptake of extracellular nitrogen (N) and its bio- DNA is the essential genetic material (10). Contemporaneously, synthetic incorporation into both virus and host proteins using an a number of groups were using isotopic labels to understand the isotope-labeling proteomics approach in a model marine cyano- relative amounts of phage material that derived from the host bacterium Synechococcus WH8102 infected by a lytic cyanophage cell as opposed to the medium with similar goals of un- S-SM1. By supplying dissolved N as 15N postinfection, we found that proteins in progeny phage particles were composed of up to derstanding the fundamental roles of biochemicals. Cohen (11) 41% extracellularly derived N, while proteins of the infected host showed that roughly 70% of the P in the DNA of the T2 and T4 cell showed almost no isotope incorporation, demonstrating that phages of E. coli derives from the medium after infection, and de novo amino acid synthesis continues during infection and con- Stent and Maaløe (12) demonstrated that phage DNA produced tributes specifically and substantially to phage replication. The early in the infection derives its P more from preexisting host source of N for phage protein synthesis shifted over the course of DNA than does later-synthesized phage DNA, whose P comes infection from mostly host derived in the early stages to more primarily from the medium. The only published measurements SCIENCES medium derived later on. We show that the photosystem II reac- that specifically address the question of host-versus-medium- ENVIRONMENTAL tion center proteins D1 and D2, which are auxiliary metabolic derived N for viral protein synthesis (13), which examined T6 genes (AMGs) in the S-SM1 genome, are made de novo during phage infection of E. coli, found that as much as 91% of phage infection in an apparently light-dependent manner. We also iden- protein N could derive from the medium postinfection. Follow- tified a small set of host proteins that continue to be produced ing this formative period of molecular biology, few studies ex- during infection; the majority are homologs of AMGs in S-SM1 or plored viral nutrient sourcing in other phage–host systems. One other viruses, suggesting selective continuation of host protein production during infection. The continued acquisition of nutri- study suggested that marine bacteriophages, in contrast to those ents by the infected cell and their utilization for phage replica- of enteric bacteria, could derive nearly all their nucleotides from tion are significant for both evolution and biogeochemical impact of viruses. Significance biogeochemistry | proteomics | bacteriophage Viral infection drives microbial mortality and nutrient recycling in many ecosystems. Despite the importance of this process, little is iruses are increasingly recognized as ubiquitous, abundant, known about how viruses obtain the resources they need to Vand integral players in microbial communities. In addition to produce progeny. Here, we assess the balance between 2 basic their influence on population dynamics and host evolution, they sources of nutrients: the biomass of the infected host cell and the also play important roles in the biogeochemistry of microbial extracellular environment. Using an ecologically relevant marine – ’ ecosystems, particularly with regard to nutrient cycling (1–3). phage host system, we show that the phage uses the host cell s Viruses essentially compete with microbial cells for the nutrients nutrient uptake and biosynthetic machinery to acquire N from the that limit biological production, and viral productivity has been extracellular environment and incorporate it specifically into viral found to correlate with environmental nutrient availability in a proteins. We also show that certain host proteins continue to be variety of settings (4, 5). There are 2 basic sources for the nutrients, produced during infection, suggesting specific roles in viral pro- such as N and phosphorus (P), that viruses need for replication: (1) duction or host defense. Our findings illustrate virus-driven nu- breakdown and recycling of host cell biomass, and (2) de novo trient flow in marine ecosystems. biosynthesis using host metabolic machinery and nutrients derived Author contributions: J.R.W. and M.L.C. designed research; J.R.W., M.L.C., A.I.R., K.L.C., from the extracellular environment. The balance of nutrients de- and L.Z. performed research; J.R.W., M.L.C., and A.I.R. contributed new reagents/analytic riving from each of these sources underpins the relationship among tools; J.R.W., M.L.C., A.I.R., K.L.C., J.L., and L.Z. analyzed data; and J.R.W. and M.L.C. wrote host physiology, environmental nutrient availability, and viral pro- the paper. ductivity. Furthermore, the degree of host biomass degradation and The authors declare no conflict of interest. acquisition of extracellular nutrients during infection influences the This article is a PNAS Direct Submission. composition and stoichiometry of dissolved organic matter released Published under the PNAS license. upon lysis. Assumptions about the proportions of host- versus ex- Data deposition: Proteomic mass spectral data are available in the Mass Spectrometry tracellularly sourced nutrients for viral replication have a strong Interactive Virtual Environment (MassIVE) repository, ftp://massive.ucsd.edu/MSV000083830. influence on the predictions of viral ecology and biogeochemistry 1J.R.W. and M.L.C. contributed equally to this work. models (6, 7), yet empirical constraints on this balance for most 2To whom correspondence may be addressed. Email: [email protected] or mlcoleman@ virus-host systems are lacking (8). uchicago.edu. There is, however, a long history of tracking the source and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fate of viral biomolecule constituents in Escherichia coli and its 1073/pnas.1901856116/-/DCSupplemental. phages, and this work lies at the foundation of modern molecular www.pnas.org/cgi/doi/10.1073/pnas.1901856116 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 the host cell, perhaps as a result of adaptation to much lower A extracellular PE fluorescence (AU) ambient nutrient availability outside the cell (14). 104 Because phage particles are composed primarily of protein 1010 and nucleic acids, they are enriched in N and P, the nutrients that limit phytoplankton growth throughout much of the oceans, and have higher elemental N:C and P:C ratios compared with cellular 109 biomass (8). Phages of marine bacteria have undergone selection 103 for efficient replication under nutrient-limited conditions, which is 8 likely to influence how the viruses acquire nutrients during in- 10 fection. It is clear from marine viral genomes, which bear a variety of AMGs hypothesized to enhance the efficiency of nutrient ac- 107 quisition or utilization during infection, that adaptation to chronic extracellular phage 102 genome copies (per ml) nutrient scarcity has shaped viral evolution just as much as that of 02468 their hosts (15, 16) and expression of AMGs during infection could 15 also shape the balance between intra- and extracellularly derived hours after N addition nutrients. Here, we used a model marine cyanobacterium, Syn- B echococcus WH8102, and T4-like myovirus, S-SM1, to explore the sourcing of N for phage protein production during infection. Using 100 939 12 925 25 840 25 876 26 15N isotopic labeling and novel proteomics techniques, we tracked host phage N flow from the extracellular medium into individual phage and 80 host proteins over the course of infection. Results 60 Direct Incorporation of Acquired N into Viral Proteins. We differ- entiated host biomass-derived versus extracellularly derived N in 40 proteins using an isotope labeling approach (SI Appendix, Fig. S1). Briefly, Synechococcus WH8102 host cells and the S-SM1 phage, each prepared in natural abundance (i.e., 0.4 atom% 15N) 20 7 media, were mixed at high concentrations [∼7to9× 10 cells/mL, 15 multiplicity of infection (i.e., ratio of infective phages to host 0 cells) of 3]. After allowing the phage to adsorb for 30 min, the protein N incorporation, intracellular (atom %) 0248 cells were pelleted and then resuspended in medium containing 15 − 98 atom% NO3 as the sole N source. Thus, any N acquired C protein N incorporation, extracellular (atom %) from the medium during the infection would be 15N, while the 14 39 5 43 3 80 10 261 34 100 preexisting host biomass contained N. Immediately after resus- host phage 15 pensioninthelabeledmediumandat2,4,and7to9hlater, samples were collected for protein 15N-incorporation measure- 80 ments in both intracellular (by pelleting infected cells by centrifu- gation) and extracellular (by filtering the supernatant) fractions. 60 The infection experiment was performed at 3 different light in- − − tensities (126, 40, and 14 μmol photons m 2 s 1 using host cells that had been grown at the same respective light levels; herein abbre- 40 viated high light (HL), medium light (ML), and low light (LL), respectively) to explore the effects of host growth rate and light 20 availability on phage nutrient incorporation.