Nitrogen Sourcing During Viral Infection of Marine Cyanobacteria

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 extracellular (atom %) protein N incorporation, 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 centrifugation) 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. Isotope incorporation was determined by proteomic liquid chromatography–mass spectrometry (LC–MS) analysis, using Topograph (17) to calculate 0 15 atom% N in each identified peptide and a classifier we developed 0248 (see Methods) to quality filter the Topograph results and generate hours after 15 N addition 15 atom% N values for individual proteins (SI Appendix,Fig.S2). 15 We generated protein-specific 15N values for roughly 1,000 Fig. 1. Time courses of cell lysis, phage production, and protein Nin- corporation during infection of Synechococcus WH8102 by phage S-SM1 in − − distinct phage and host proteins (out of 234 and 2,512 predicted HL conditions (126 μmol photons m 2 s 1). (A) Extracellular phage genome protein-coding ORFs in the phage and host genomes, respec- copies (by qPCR of g20) and phycoerythrin fluorescence (from lysed cell tively) at each time point in the HL experiment. As expected, the debris); points and error bars are averages and ±1 SD, respectively, across number of detected phage proteins increased as the lytic cycle experimental triplicates. (B and C) Distributions of atom% 15N values of host proceeded (Fig. 1 and SI Appendix, Table S1). We found that, on (purple) and phage (green) proteins in (B) intracellular (i.e., cell pellet) and average, proteins in the phage particles released by lysis were (C) extracellular (i.e., >10 kDa/<0.2 μm filtrate) samples; the numbers at the composed of 41% medium-derived 15N at 8 h, while host cell top indicate the number of proteins detected for host and phage at each proteins (with a small and specific set of exceptions, discussed timepoint, the gray bars show values of individual proteins (averaged across below) showed no isotope incorporation over the course of the experimental triplicates), the colored horizontal lines show host and phage medians at each timepoint. infection (Fig. 1). This is clear evidence that the dissolved 15N nitrate was taken up by infected cells, reduced to ammonium, and assimilated into amino acids, and those newly synthesized amino acids specifically utilized in the translation of phage that cyanophages can acquire a substantial proportion of their proteins without incorporation into the host proteome. This building blocks from the extracellular medium after infection be- ongoing activity is the work of host biosynthetic machinery, despite gins, consistent with prior E. coli results (13). The isotopic enrich- general suppression of host gene expression, since the phage does ment appeared first in phage proteins in the cell pellet and only not encode any genes for these processes. These data demonstrate later in the extracellular phage protein in the supernatant, following

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1901856116 Waldbauer et al. Downloaded by guest on October 1, 2021 cell lysis and the release of the progeny phage (Fig. 1). In con- progressive 15N incorporation into proteins produced over the junction with the absence of host labeling, this is further evidence course of this expression program with the proteins produced that the isotope incorporation occurred in infected cells and not, for earlier in the infection incorporating less 15N from the medium example, by a subpopulation of uninfected cells taking up the label than those synthesized later (Fig. 2A). This progression was also and producing amino acids that were subsequently acquired by detectable within individual late phage proteins, a number of which infected hosts. increased in 15N content steadily over the first 4 h of infection. The Uptake of extracellular N into phage protein was modulated maximum 15N incorporation for phage proteins expressed from by light intensity, while average 15N incorporation into host early genes was 8%, compared with 21% for middle genes and 36% protein remained negligible across all treatments. 15N in- for late genes (median values for each class) (Fig. 2B). The increase corporation levels into phage proteins were nearly equivalent in 15N incorporation levels over this expression time course dem- between the HL and the ML experiments, indicating that neither onstrates a shift in the source of N for phage protein synthesis from the decreased light availability nor somewhat lowered host primarily host derived in the early stages of infection to more ex- growth rate (μ = 0.61/d at ML, 0.66/d at HL) at the medium tracellularly derived for the later production of progeny virion compared with the HL condition had a strong impact on particles. sourcing of N for phage protein production (SI Appendix, Fig. S3). In the LL condition (host μ = 0.33/d), by contrast, phage Turnover of Phage-Encoded Photosystem II Proteins. The genes psbA proteins showed significantly less 15N incorporation, reaching and psbD, encoding the photosystem II reaction center proteins only 25% 15N by 9 h postinfection. This reduced incorporation of D1 and D2, were the first AMGs to be recognized in the ge- extracellular N suggests that the slower-growing host cells may nomes of cyanophages (19, 20). These proteins form the core of afford the infecting phages less nutrient acquisition capacity the water-splitting electron transport machinery for oxygenic (possibly due to limitation of photosynthetically generated re- photosynthesis, and the virus-encoded versions have been shown ductant for nitrate reductase), which, in turn, delays and/or re- to produce functional proteins during infection (21). These duces viral productivity (SI Appendix, Fig. S4). These results proteins are also susceptible to photodamage and must be con- illustrate how environmental controls on host physiology—in this tinually renewed—especially D1, which turns over as rapidly as case, light limitation of a photoautotroph—can influence the every 30 min to an hour (22–24). Expression of D1 and D2 from sourcing of nutrients for viral replication and show that the phage-encoded loci is thought to compensate for the shutdown SCIENCES balance between host- and extracellularly derived nutrients is not of host expression of these proteins and thereby enable contin- strictly “hardwired” into the viral infection program. ued photosynthetic electron transport and energy metabolism ENVIRONMENTAL during infection, enhancing phage production (24–26). Later-Expressed Viral Proteins Derive Less N from Host. Our molecular- We detected 15N incorporation into both D1 and D2, indicating level isotope incorporation data also show how extracellularly de- de novo synthesis of these reaction center proteins during S-SM1 rived N is funneled into the translational program of phage S-SM1. infection of Synechococcus WH8102 (Fig. 3). The peptides we de- Genes in T4-like cyanomyoviruses can be categorized into early, tected are common to both host- and phage-encoded versions of middle, and late expression clusters based on the timing of peak the proteins, so they cannot be unequivocally assigned to one or the transcription and on shared promoter motifs (18). We observed other. However, given that phage PSII genes are known to yield

A T0 T2 T4 T8 B 39

35 100 ● ●

30 75

25 ● ation (atom %) r po r 50 20 ●

15 ● 15 25

10 maximum N inco Expression Cluster ● Late 0 5 Early Middle Late Middle n=8 n=14 n=19 Early 1 02040600 20406002040600204060 15N incorporation (atom %)

Fig. 2. (A) Incorporation of medium-derived 15N into 39 proteins of phage S-SM1 during infection under HL conditions (see SI Appendix, Fig. S5 for an- notations and the position of 2 outliers excluded from the middle cluster). Panels show isotope incorporation detected at each timepoint (t = 0, 2, 4, and 8 h after 15N addition) into proteins that are classified by their transcriptional timing into early, middle, and late expression clusters. The colored points show the atom% 15N determined for a given protein at the timepoint indicated; the gray points show the atom% 15N values for that protein at all other timepoints for comparison. (B) Distributions of the maximum atom% 15N values observed across timepoints for proteins in each of the early, middle, and late expression categories. The boxes show medians and 25th/75th percentiles, the whiskers are ±1.5×IQR; the gray dots show outliers.

Waldbauer et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 1, 2021 ABto encode peptide deformylases that remove the N-formyl group from these sites, the first step in the NME pathway (32, 33). NME is necessary for stable assembly of the D1/D2 reaction center of photosystem II (34), so the continued production of methionyl aminopeptidase may be linked to phage PSII protein expression. One amino acid biosynthesis protein, cysteine synthase A, also acquired the 15N label; continued turnover of this enzyme might support production of Cys-rich pigment-linked phycobiliproteins (28) or could have an alternative regulatory function (35). We also detected 15N labeling of host ribosomal protein S21, which is necessary for mRNA binding and translation initiation (36). Homologs of these 3 proteins—methionyl aminopeptidase, cysteine synthase 15 Fig. 3. Time courses of N incorporation into photosystem II reaction A, and ribosomal protein S21—have been identified in viral ge- center proteins (A) D1/PsbA and (B) D2/PsbD during infection under HL, ML, nomes or putative viral contigs, consistent with the idea that their and LL conditions. expression during infection enhances viral fitness (32, 37). The pentose phosphate pathway (PPP), which respires stored functional protein during infection (21), and here D1 and D2 show carbon and generates reducing power and nucleotide biosynthesis 15N labeling patterns consistent with other phage proteins, we be- precursors, has been implicated as a major target of metabolic lieve the synthesis observed here is most likely from the phage remodeling during cyanophage infection (38). Three host-encoded 15 enzymes are involved in glycogen degradation via the PPP acquired genes. Both proteins became more N enriched over the course of 15 the experiment at a higher rate with increasing light intensity (Fig. N label: glycogen phosphorylase, phosphogluconate dehydroge- 3). Additionally, D1 was always more 15N enriched than D2, con- nase, and transaldolase. The latter 2 (gnd and talC) are widespread sistent with its higher rate of turnover due to photodamage. These AMGs among marine cyanophages (15), and their continued syn- results provide the first experimental validation of the assumption thesis from the host loci during infection is particularly interesting of light-responsive PSII expression/repair during cyanophage in- given that S-SM1 encodes its own copies of these enzymes. While fection made in previous modeling studies (25, 26). Notably, while the phage-encoded TalC protein was not detected in our datasets, 15 phage proteins were labeled to similar overall extents in the ML the phage-encoded Gnd was also clearly N labeled (SI Appendix, and HL experiments (SI Appendix,Fig.S4), D1 and D2 were Fig. S8), indicating that de novo synthesis of this enzyme proceeded consistently more labeled at HL, suggesting either higher rates of from both host and phage loci during infection. The host-encoded 15 photodamage that drove faster turnover or greater net synthesis of CP12 repressor of the Calvin cycle also acquired the Nlabel(SI theproteinsathigherintensity. Appendix,Fig.S8)—albeit to an extent just slightly below the P value threshold for the set of most-significantly labeled host genes Exogenous N Incorporation into Specific Host Proteins. While the (SI Appendix,Fig.S6)—anditscontinuedexpressionmayactto vast majority of host proteins showed little to no 15N in- steer carbon flux toward the PPP (38). Our results here suggest that corporation during infection (Fig. 1 and SI Appendix, Fig. S3), we enhancement of metabolic flux through the PPP during cyanophage found that 12 host-encoded proteins did incorporate significant 15N, indicating their continued production from newly synthesized amino acids despite the general suppression of host gene expression (Fig. 4 and SI Appendix, Figs. S6 and S7). While transcriptional studies in related cyanophage systems have documented continued mRNA synthesis of select host genes during infection (18, 27), our results identified a distinct set of host response proteins. Intriguingly, the majority of these host proteins (8/12) have homologs (i.e., AMGs) in the genomes of S-SM1 or other phages and/or on putative viral genome fragments from aquatic viral metagenome datasets (Dataset S1). This finding suggests that continued expression of these proteins may be advantageous for the phage. Consistent with this hypothesis, there is prior experimental evidence for several of these proteins demonstrating their activity during phage infection and their importance in maximizing progeny phage production. One of the most strongly labeled host proteins was heme oxygenase, which catalyzes the first step in phycobilin pigment biosynthesis, a pathway for which multiple AMGs exist in phage genomes (28, 29); there is also biochemical evidence for ongoing synthesis (30) and degradation (31) of phycobilisomes during infection. Heme oxygenase is found in the genomes of several Prochlorococcus phages but only a single Synechococcus phage S-SM7 (15). While the function of bilin biosynthesis during phage infection remains unclear (29), the 15N incorporation we observed suggests that some phages without these genes (such as

S-SM1) can maintain expression of host phycobilin metabolism. 15 We detected 15N labeling of several host proteins involved in Fig. 4. N incorporation time courses in the HL condition of the 12 Syn- echococcus WH8102 host proteins that became significantly labeled in our protein production or maturation (Fig. 4). Methionyl amino- phage infection experiments. The circles after gene names indicate the peptidase removes initiator methionine residues, the second step presence of a homolog in viral (meta)genome sequences: ••• = present in in the N-terminal methionine excision (NME) pathway of pro- the S-SM1 genome; •• = present in other viral isolate genomes; • = present tein maturation. Many marine phage genomes have been found in putative viral contigs from aquatic metagenomic data (Dataset S1).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1901856116 Waldbauer et al. Downloaded by guest on October 1, 2021 infection could be mediated by simultaneous protein expression particles. In contrast to the conclusions of Wikner et al. (14), from both phage and host copies of homologous genes. our results indicate that de novo biosynthesis during infection Continued de novo synthesis of other host proteins may point does contribute substantially to productivity of at least some to cellular defense mechanisms against phage infection. The Rho marine phages. termination factor, which acquired the 15N label in our experi- Extracellular N was a substantial but minority source of N for ments, has been implicated in silencing of foreign DNA elements phage replication in these experiments; under all conditions as a defense against deleterious effects of their expression (39, tested, more than half of phage protein N was host cell derived. 40). Likewise, we detected labeling of ribonuclease J, which may A bacterial cell generally contains many times the protein N act to degrade phage transcripts and inhibit infection progress, required by even large phage bursts: the roughly 1 fmol of pro- or, conversely, to drive mRNA turnover and enable phage tein N per Synechococcus cell (47, 48) could theoretically be transcription (41). Three hypothetical proteins—SYNW1071, converted (at ∼1 amol N per phage; ref. 8) into 1,000 myophage SYNW1249, and SYNW0829, the last of which has a homolog in progeny. Even setting aside some portion of the host proteome a putative cyanophage contig from the North Pacific (Dataset (e.g., in ribosomes) as required for phage replication and, S1)—were also found to become significantly labeled. It is clear therefore, “off limits,” extracellular N incorporation would not that we are just beginning to recognize the diversity of host de- seem to be necessary on a mass-balance basis. Our results sug- fense systems and components of the host metabolism that in- gest that, at some point, it becomes energetically or kinetically teract with phage replication (42, 43); the highly labeled preferable for the phage replication program to shift from host hypothetical proteins observed in this study could be useful protein catabolism toward de novo amino acid synthesis. targets for future functional characterization. The experiments of Kozloff et al. (13) on T6 infection of E. Given the striking correspondence between the small number coli found even higher levels of medium-derived N in phage 15 of host proteins found to be N labeled during S-SM1 infection particles, up to 91%. Host growth rates in those experiments and the set of metabolic processes known or inferred to be were not reported but were likely higher than those in this study. influenced by phage-encoded AMGs, it is possible that this ap- This earlier evidence as well as the lower level of isotope in- parently selective continuation of host protein production during corporation we observed in the LL experiment (SI Appendix, Fig. infection represents either an early or a late stage in the process S3) are consistent with the notion that phages infecting slower- of phage AMG acquisition (27). For example, this specific ex- growing hosts take less advantage of exogenous N than those

pression from a host gene could be an early metabolic rewiring infecting fast-growing hosts. In this, our results are consistent with SCIENCES

strategy evolved by phages before investing in carrying an AMG Wikner et al. (14), who also found lowered extracellular P in- ENVIRONMENTAL encoding the induced function in their own genome. Alterna- corporation when phages infected slow-growing hosts. This may be tively, gaining the ability to selectively express certain host pro- because slow-growing host cells contain less machinery for nutrient teins during infection may allow phages to dispense with their uptake, monomer synthesis, and/or macromolecule polymerization, own AMG copies and, thus, represent a later stage of the process. and so phages cannot acquire as much additional N or P over the Discriminating between these scenarios will require elucidation of constrained time frame of the infection. This limitation could be the mechanism of the seemingly targeted continuation of expres- one factor that contributes to correlation between host growth rate sion of particular host-encoded genes during infection. This could and phage productivity. be mediated via transcription (18) or post-transcriptionally through Current ecosystem and biogeochemical models that incorpo- specific mRNA stabilization or translational-level mechanisms, such rate the effects of viral lysis (e.g., refs. 6 and 7) often assume that as ribosome recruitment (44). the N/P content of virions released upon lysis cannot exceed the N/P content of uninfected hosts, providing a key boundary con- Discussion dition in such models. Our results indicate that, at least for N and This work has demonstrated that phages of cyanobacteria ac- potentially also for P, the nutrient content of host cells is, in fact, quire substantial amounts of N for protein synthesis from the not a reliable bound on the resources available for phage repli- extracellular environment after infection begins, through con- cation and that ongoing host cell metabolism during infection tinued operation of host nutrient acquisition and biosynthesis contributes substantially to viral productivity (49). It seems in- machinery. Our results are consistent with earlier findings of ex- creasingly clear that virally infected cells must be treated as a tracellular N deprivation suppressing phage productivity (45). separate category of agents in ecosystem models as their me- Phage nucleic acids, known to be produced mainly during the early tabolism, which remains highly active in many respects, has been stages of infection before synthesis and assembly of virion struc- so extensively rewired by viral takeover (50). When considering tural proteins, contain less extracellularly derived N than phage observed rates of nutrient uptake, for example, in microbial protein does (13), in accord with the temporal shift we observed ecosystems, biogeochemical models need to acknowledge that a toward more medium-derived N sourcing. These results parallel proportion of that uptake—for which the kinds of methods those of Stent and Maaløe (12) regarding the shift in the sourcing employed here can start to provide constraints—is flowing di- of P for phage DNA synthesis in the E. coli-T4 system and suggest rectly to viral production. In nutrient-limited conditions, both that this shift from more host-derived to more extracellularly de- the intracellular quotas and the extracellular availability of nu- rived nutrients for phage production over the course of infection trients will be lower than in the replete conditions employed could be a general feature of a range of lytic phages. here, and the balance of nutrient sourcing for viral replication is Pasulka et al. (46) recently documented enhanced incorpora- 15 likely to shift. Whether viral replication is limited by the same tion of N into Syn1 viral particles using NanoSIMS during a resources as host growth depends on the relative abilities of the multiday infection experiment using Synechococcus WH8101 as a phage to exploit the standing stock of host biochemical resources host but with the extended duration of that experiment and the and to rewire host metabolic machinery to acquire additional inability to measure isotopic compositions of specific host- and nutrients from the environment. virus-derived biomolecules that study could not differentiate between concurrent labeling of host and phage proteins and Methods direct incorporation of the exogenous nitrogen into viral parti- Cell Cultivation and Phage Infection. Axenic Synechococcus WH8102 was cles. We have shown that such labeling of viral biomolecules by 14 − grown in L1 medium (51) with 882 μM added NO3 under 3 light regimes − − an extracellular isotope tracer can occur in the absence of de- (14.4 ± 0.6, 39.5 ± 2.3, and 126.0 ± 4.6 μmol photons m 2 s 1) and infected tectable incorporation of the label into host biomass, clearly with phage S-SM1 at a multiplicity of infection (i.e., ratio of infective phage demonstrating direct transfer of dissolved nutrients to viral to host cells) of 3. Following phage adsorption, infected cells were pelleted

Waldbauer et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 1, 2021 by centrifugation, and the pellets resuspended in L1 medium with 882 μM peptide. Peptide-level data were mapped to the proteomes of WH8102 and 15 − 15 NO3 (99 atom% N) (SI Appendix, Fig. S1). All experiments were per- S-SM1 and filtered using the calibration-based classification model (SI Ap- formed in triplicate. Samples were taken immediately (T0 with respect to 15N pendix). Protein-level data were averaged across the experimental tripli- addition) and 2, 4, and 7 to 9 h later for intra- and extracellular proteomic cates, and the error taken as ±1 SD between the replicates; see SI Appendix analysis and hourly for fluorescence and quantitative PCR monitoring of for further details of proteomics analyses. Raw proteomic mass spectral data infection progress (further details in SI Appendix). are deposited in the MassIVE repository under accession MSV000083830.

15 Proteomic MS and Atom% N Determination. Cell pellets (intracellular protein ACKNOWLEDGMENTS. We are grateful to Gerry Olack, Xiufeng Ma, and fraction) were extracted, and supernatants (extracellular protein fraction) Stewart Edie for discussions and advice and to anonymous reviewers whose were concentrated before spin filter purification, trypsin digestion, and LC- comments improved the paper. This work was supported by the Gordon & MS/MS analysis. Peptides were identified via SEQUEST HT (52) and Percolator Betty Moore Foundation Marine Microbiology Initiative (Award 3305) and (53). Topograph (17) was used to calculate the atom% 15N in each identified the Simons Foundation (Award 402971).

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