Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition

ABSTRACT Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host

cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material

released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected _Sulfitobacter_ sp. 2047 was

studied using a variety of techniques, including liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and

nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven

time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux

measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile

of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these

small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has

implications for microbial community ecology and biogeochemistry. SIMILAR CONTENT BEING VIEWED BY OTHERS METABOLITES FROM INTACT PHAGE-INFECTED _SYNECHOCOCCUS_ CHEMOTACTICALLY ATTRACT

HETEROTROPHIC MARINE BACTERIA Article 15 November 2024 ECOLOGY OF INORGANIC SULFUR AUXILIARY METABOLISM IN WIDESPREAD BACTERIOPHAGES Article Open access 09 June 2021 LARGE FRESHWATER PHAGES

WITH THE POTENTIAL TO AUGMENT AEROBIC METHANE OXIDATION Article Open access 24 August 2020 INTRODUCTION It is estimated that 20–30% of the marine microbial community is infected by viruses

at any given time. This contributes to as much as 3 Gt of carbon that are released in the world’s oceans each year as a result of virus-mediated lysis (Suttle, 2007). The released dissolved

organic matter (DOM) is a major contributor to marine microbial activity (Weinbauer et al., 2011), stimulating both primary and secondary productivity (see, for example, Middelboe and Lyck,

2002; Riemann and Middelboe, 2002; Middelboe and Jorgensen, 2006). Through their lytic activities, viruses influence the flow of carbon, nitrogen and other nutrients in the marine

environment (Brussaard et al., 2008). To better define the role of viruses as global drivers of nutrient flow, we need an improved understanding of the impact of infection on host metabolic

processes. Presently, little is known of the influences of bacteriophage infection on global host metabolism beyond isolated model systems. A few marine phage–host systems appear to

sequester sufficient resources for nucleic acid synthesis entirely from degradation of host DNA (Wikner et al., 1993), although the sources of other macromolecular building blocks are

unknown. Much of the knowledge concerning phage–host interactions comes from studies in _Escherichia coli_. Both lysogenic and lytic coliphages initially promote similar alterations in host

metabolism (Calendar, 2006) that halt host cell DNA synthesis (Kazmierczak and Rothman-Denes, 2006), degrade host DNA (Miller et al., 2003) and assemble the machinery for viral production

(Taylor, 1995). These activities drastically alter aspects of host metabolism in a specific manner that favors virus replication (see, for example, Poranen et al., 2006). In fact, a number

of viruses contain auxiliary metabolic genes to overcome rate-limiting steps in host biosynthesis (Breitbart, 2012). A dramatic example of host manipulation has been demonstrated in marine

cyanophage that encode and express photosynthesis proteins homologous to those found in their hosts (Thompson et al., 2011). However, the extent to which pathway-specific alterations of host

metabolism is a universal strategy among phages remains unknown. Furthermore, the hijacking of host metabolic processes may not be fully sufficient to supply the demand for rapid virus

production, necessitating the acquisition of resources from the environment. For example, the breakdown of host DNA does not fully supply precursors needed for coliphage DNA synthesis. Thus,

an initial lag in _E. coli_ metabolism is followed by rapid uptake of nutrients to be used for _de novo_ synthesis of macromolecular building blocks (Friedman and Gots, 1953). Similarly, it

has been found that thymidine uptake by infected _Pseudoalteromonas_ sp. SKA18 occurred at rates similar to uninfected cells during the early stages of infection, yet became markedly

enhanced just before onset of cell lysis (Middelboe, 2000). These observations suggest an interplay between the viral life cycle and environmental nutrient availability. From the standpoint

of characterizing the influence of virus activity on ocean biogeochemistry, most studies have focused on characterizing the bulk material properties of lysate DOM (for example, total C, N,

Fe, Se; see Gobler et al., 1997; Bratbak et al., 1998; Poorvin et al., 2004; Lønborg et al., 2013) or monitoring a few select molecules (for example, dimethyl sulfide, acrylate) or compound

classes, such as amino acids and carbohydrates (see, for example, Weinbauer and Peduzzi, 1995; Middelboe and Jorgensen, 2006; Shelford et al., 2012). In general, these studies reported

increases in these molecules in lysates (see, for example, Poorvin et al., 2004; Lønborg et al., 2013) and have hypothesized that viral lysates are rich in free and combined amino acids

(Middelboe and Jorgensen, 2006) and may be an important source of labile organic nitrogen (Shelford et al., 2012). Only recently has the technology necessary to monitor metabolism at the

systems level become available via nuclear magnetic resonance (Fiehn, 2002) or liquid chromatography–mass spectrometry (LC-MS/MS)-based metabolomics (Siuzdak, 1994; Coon et al., 2005; Bajad

et al., 2006), and these same tools can be used to help characterize the chemical composition of DOM. Bacteria belonging to the _Roseobacter_ lineage, which includes _Sulfitobacter_ species,

are abundant in marine systems and carry out critical biogeochemical transformations (Buchan et al., 2005). Herein we report the study of a _Sulfitobacter_ sp. 2047 roseophage infection

system in which LC-MS/MS-based metabolomics techniques were employed to both compare the metabolism of an infected population with an uninfected control and to gain insight into the

interaction between active infection and small metabolites within the DOM pool. As nonlipid-containing phages comprise primarily nucleic acid and protein, typically in an equal mass ratio

(see, for example, Bancroft and Freifelder, 1970), we hypothesized that any influences of viral infection on host metabolism would be manifested in pathways that contribute to the synthesis

of the biochemical building blocks for these macromolecules and that this would be reflected in the suite of metabolites released from lysed cells. MATERIALS AND METHODS BACTERIUM AND PHAGE

ISOLATION _Sulfitobacter_ sp. 2047 was isolated from a mesocosm study in Raunefjorden, Norway, in 2008 by enrichment with dimethylsufoniopropionate (Pagarete et al., 2011; Budinoff, 2012).

Agar plates (0.8% w/v Noble Agar; Difco, Sparks, MD, USA) were made using 0.22 μm filtered fjord water and 10 mM dimethylsufoniopropionate (kindly provided by J Henrikson and W Whitman, UGA,

Athens, GA, USA). The strain was subsequently maintained in artificial sea water: 1.0% agar plates (230 mM NaCl, 5.3 mM KCl, 3.9 mM CaCl2, 0.1 mM H3BO3, 11.8 mM MgSO4, 11.2 mM MgCl2, 0.8 mM

NaHCO3, 5 mM NH4Cl, 75 μM K2HPO4 and 10 mM Tris-HCl (pH 7.5)) supplemented with 0.25% yeast extract and 0.4% tryptone at 20 °C. Filter-sterilized (0.22 μm) stock solutions were added to the

autoclaved basal salt solution along with vitamins, iron and trace metals prepared as previously described (Budinoff and Hollibaugh, 2007). A lytic phage (Φ2047B) (Budinoff, 2012; Ankrah et

al., 2014) was isolated from viral concentrates of Raunefjorden sea water using standard bacteriophage enrichment (Van Twest and Kropinski, 2009; Wommack et al., 2009). Plaque purification

and preparation of phage stocks were based on previously described methods (Kropinski et al., 2009). Concentrated lysates were made by gently washing soft agar from plaque assay plates with

MSB buffer. The final purified phage concentrate was 0.22 μm filtered and stored at 4 °C in the dark. All growth experiments with the bacterium, including infections, were performed in

artificial sea water at 20 °C. All chemicals were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Genome analysis of _Sulfitobacter_ sp. 2047 revealed it is lysogenized by a prophage

(designate as Φ2047A) that is genetically distinct from Φ2047B (HQ317387). BACTERIAL CULTURE CONDITIONS AND METABOLITE MEASUREMENTS To determine whether phage infection shifts the metabolite

repertoire of _Sulfitobacter_ sp. 2047, the bacterium was grown in artificial sea water supplemented with 10 mM sodium acetate at 25 °C in the dark with 200 r.p.m. agitation. Once cultures

reached an optical density (OD540) of ca. 0.17, phages were added at a multiplicity of infection of 4 (4 phage cell−1). Under these conditions, cell lysis is evident at ∼240 min post

infection (p.i.). To monitor host metabolites during all phases of infection, samples were collected before phage addition (_t_=0) and then 15, 30, 60, 120, 240, 360 and 480 min post phage

addition. Extracellular metabolites were measured in cell-free filtrates of the populations at 480 min. Control cultures (without added phages) were maintained and sampled in parallel.

Samples taken for microscopic analysis were fixed with gluteraldehyde (0.5%), stored at −80 °C and processed using standard techniques. Samples for quantitative polymerase chain reaction

(qPCR) were flash frozen and stored at −80 °C until processing, whereas samples for metabolite analysis were immediately processed. Following the initial experiment, a separate analysis

using stable isotope-labeled nutrients was performed to examine turnover rates and cellular fluxes for a subset of metabolites (Supplementary Table 2). For these studies, 20 mM 13C-labeled

sodium acetate (1,2-13C2, 99%, Cambridge Isotope Laboratories, Andover, MA, USA) was added to a set of control and viral infected cultures at either 0 min or 240 min p.i. These two time

points were selected to represent each of the two phases of infection that were evident from the metabolite profiles identified in the initial experiment described above. Samples for the

flux analysis were taken from each culture at 0, 5, 15, 30, 60 and 120 min post addition of 13C-acetate. However, isotope incorporation was complete at 60 min, and the data presented herein

reflect this. The targeted metabolomic methods employed to measure relative intracellular metabolite concentrations and turnover rates used slight modifications of a known metabolite

extraction procedure (Rabinowitz and Kimball, 2007; Bennett et al., 2008; Yuan et al., 2008). Briefly, 10 ml of cells were rapidly collected on Magna nylon filters (Millipore, Billerica, MA,

USA) via vacuum filtration and then extracted by placing the filter directly in a petri dish containing extraction solvent at 4 °C as previously described. Extracellular metabolites from

both infected and control cultures were collected at 480 min by filtering 10 ml of culture as previously described and collecting the filtrate. The resulting liquids were lyophilized and

then resuspended in 300 μl 40:40:20 acetonitrile/methanol/water with 0.1 M formic acid for MS analysis. Two LC-MS/MS analyses, one in each of positive and negative ion modes, were performed

for each sample, and relative metabolite levels and fluxes were analyzed as previously described (Rabinowitz and Kimball, 2007; Bennett et al., 2008; Yuan et al., 2008). METABOLITE DATA

ANALYSIS Signal intensity for all metabolites was analyzed as peak area, and each measurement was manually curated using the Xcalibur 2.0.7 Quan Browser analysis package (Thermo Fisher

Scientific, Waltham, MA, USA). Metabolite area counts were normalized to cell density to obtain per cell metabolite abundances, and a ratio of these values from each condition was used to

compare relative concentrations of metabolites. All heat maps were generated using Gene Cluster 3.0 (de Hoon et al., 2004) and viewed using Java TreeView 1.1.5 (Saldanha, 2004). Interactions

among the metabolites were visualized using resources available through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/kegg/). DATA ANALYSIS FOR STABLE ISOTOPE

INCORPORATION INTO METABOLITES Peak areas for all compounds detected in the 13C incorporation experiments were calculated as described for the pool size data above. Once the peak areas were

determined, the ratios of unlabeled, partially labeled and fully labeled metabolites were determined for each time point. These values were then used to calculate the rate of disappearance

(turnover) for the unlabeled material. The data were fit using the following equation: _y_=_Ae_(-_k_x)+_c_, where _k_ is the rate constant. This equation is most correct for populations that

have reached steady state, which may not be the case in the experiments reported herein because of the addition of a pulse of labeled nutrients. Therefore, the ratios of rate constants from

the phage-infected and control cultures were calculated and multiplied by the fold difference in metabolite concentration to determine relative fluxes. PHAGE ENUMERATION Phages were

enumerated using qPCR assays specific for each of the two phages (Φ2047A and Φ2047B). Genome sequences were obtained for the host and Φ2047B, facilitating the design and optimization of

phage-specific primer sets (see Supplementary Materials). qPCR was carried out by using a DNA Engine Opticon 2 real-time PCR detector with the Opticon Monitor 3.1.32 software package

(Bio-Rad Laboratories, Inc., Hercules, CA, USA). qPCR reactions were performed in a 25 μl volume with 12.5 μl SYBR Premix Ex _Taq_ cocktail RR041 (Perfect Real Time; Takara Bio, Inc., Shiga,

Japan), 500 nM forward and reverse primers and 10.0 μl of template dilutions. The amplification programs for each primer set are as follows: 95 °C for 3 min, 40 cycles of: 95 °C for 20 s,

56 °C or 55 °C (Φ2047A or Φ2047B, respectively) for 20 s, 72 °C for 20 s, followed by 5 min at 72 °C. Fluorescence measurements were conducted at the end of each cycle at 72 °C. Melt curves

were generated after each assay to verify the specificity of the amplification by heating from 50 °C to 100 °C, read every 1 °C. Standards were developed from plasmids containing cloned

sequences and 10-fold serial dilutions of these samples in 10 mM Tris-HCl were used in the reactions. Standard curves were determined as the correlation between the log of gene copy numbers

and the _C__t_. In all cases, correlation coefficients for standard curves assays were ⩾0.99. Full instrumentation and additional experimental details are available in the supporting

documents. RESULTS THE _SULFITOBACTER_-PHAGE INFECTION CYCLE The growth of the infected _Sulfitobacter_ sp. 2047 cultures was nearly identical to that of the uninfected controls until the

onset of cell lysis at 240 min p.i. (Figure 1a). Rapid production of phages occurred between 60 and 240 min p.i., at which point it plateaued. By the end of the 480 min experiment, 3.53 ×

1011 new virus particles had been produced, the density of bacteria in the flasks receiving phages was ∼30% of that present in the no-virus control cultures, and phages outnumbered bacteria

by ∼2500 to 1 (Figure 1). Under the growth conditions employed here, infection of the host with the lytic phage results in induction of a prophage for at least a portion of the host

population, leading to the propagation of both phages in batch culture (Supplementary Table S1). STOICHIOMETRIC SHIFTS IN CARBON AND NITROGEN ASSOCIATED WITH PHAGE INFECTION The

distributions of carbon (C) and nitrogen (N) in biological particles for the control culture and the _Sulfitobacter_-roseophage infection system were calculated based on reported estimates

of the C and N content in both marine viruses and bacteria. The total masses of bacteria (cellular) and phage C and N were calculated using previously reported elemental estimates of 0.2 fg

C and 0.076 fg N per phage and 149 fg C and 35 fg N per cell (Heldal et al., 1985; Vrede et al., 2002; Chan et al., 2012; Jover et al., 2014). At the final time point, cells in the control

culture contained 76.74 (±10.43) and 18.0 (±2.45) mg l−1 of C and N, respectively (Figures 1b and c). During the experiment, this culture was estimated to incorporate 31.33 and 7.33 mg l−1

of C and N, respectively, into new cells. In comparison, phage-infected cultures were found to have 95 mg l−1 of C in both intact bacterial cells (22.35±2.98 mg l−1) and viral particles

(72.66±2.05 mg l−1) by the final time point. As for N, 29.59 mg l−1 had been incorporated in total with 5.25 (±0.7) and 24.34 (±0.7) mg l−1 being found in bacteria and phages, respectively

(Figures 1b and c). These data were used to calculate the C/N ratio for infected and control populations throughout the course of the experiment. This ratio remained constant for the control

populations, but decreased by 32% for the infected cultures by 480 min (Figure 1d). INTRACELLULAR METABOLITE CONCENTRATIONS THROUGHOUT THE INFECTION CYCLE The relative concentrations of 82

central pathway metabolites (the ‘core metabolome’) were measured via LC-MS/MS for both infected and control populations at seven discrete time points throughout the infection cycle (Figure

2). During the early infection period (ending at 120 min p.i.), <25% of the measured metabolites were significantly different between the infected and control populations (18 at _P⩽_0.05

and ⩾1.5-fold change). At the end of the infection cycle (480 min p.i.), ∼75% of metabolite concentrations were significantly elevated (59 at _P_⩽0.05 and ⩾1.5-fold change) in phage-infected

populations (Figure 3). The intracellular metabolite concentration progressively increased in the phage-infected populations for nearly every metabolite over the course of the infection

cycle. In fact, almost no metabolites showed a significant decrease in concentration (Supplementary Table S3). For example, dramatic differences were evident in uridine diphosphate

(UDP)-activated sugars. The relative concentration of UDP-glucose in the infected population was elevated 1.7-fold 15 min after phage addition and remained fairly constant throughout the

experiment. UDP-glucuronate/galacturonate became elevated over time with a 13-fold spike in concentration at 120 min p.i., the time point preceding widespread cell lysis. Similarly, several

amino acids were elevated in concentration in the late-phase infected populations relative to controls by 1.6- to 6.0-fold, with an average of 2.9-fold, at 480 min p.i. (that is, alanine,

aspargine, aspartate, cysteine, glutamate, glutamine, lysine, proline, serine, threonine and valine). To verify that _Sulfitobacter_ sp. 2047 contained high concentrations of glutamate and

glutamine, their absolute concentrations were determined for this experiment and found to be 7.1 × 10−14 g per cell and 2 × 10−15 g per cell, respectively, in the infected culture at the

final time point (Figures 4a and b). METABOLIC TURNOVER AND FLUX DURING PHAGE INFECTION Complementary experiments to analyze metabolite turnover and metabolic flux were also performed using

a stable isotope-labeled carbon source. 13C-acetate was added to separate sets of cultures at times that matched both the early (immediately following virus addition) and late (240 min p.i.)

phases that were identified from the time course used to determine growth dynamics and overall metabolite concentration (see Figures 1a and 2). Incorporation of the label into 14 selected

metabolites representing tricarboxylic acid cycle components, N assimilation intermediates and amino acids was monitored via LC-MS/MS during the first 120 min following 13C acetate addition

in both cases. These measurements provide two useful pieces of data: (1) the rate at which the metabolite pool is converted to entirely new metabolites because of biosynthesis and (2) the

total amount of nutrients that pass through a specific compound, that is, the flux through them, which is proportional to the turnover rate and metabolite pool size. With the exception of

glutamate and glutamine (Table 1 and Figure 5), the turnover rates for all measured metabolites were indistinguishable between the control and viral-treated cultures throughout the infection

cycle (Supplementary Tables S5–12). Consequently, the fluxes for these metabolites were increased in infected populations as the larger pools were being replenished at equivalent, or only

slightly reduced, rates. The measured turnover rates for glutamate and glutamine are indistinguishable between the control and viral-treated cultures during the early infection period

(Figures 5a and c), but they are decreased in the infected cultures during the later stages of infection (Figures 5b and d). The calculated flux through glutamate in the infected cultures is

equivalent to controls throughout both phases of the infection cycle. However, flux through glutamine is 70–100% greater and 200–400% greater during the early and late infection periods,

respectively (Table 1). COMPOSITION OF EXTRACELLULAR SMALL-MOLECULE COMPONENTS FOLLOWING PHAGE-INDUCED CELL LYSIS Cell-free filtrates of phage-infected and control cultures were collected at

480 min p.i., and 56 metabolites were detected in filtrates from either infected or control cultures (Table 2). The metabolite concentrations in the infected cultures were not uniformly

higher for every molecule. Instead, there was an increase in 29%, no change in 45% and a decrease in 27% of the metabolite concentrations detected in the virus-infected cultures (Table 2).

For example, several components of C and N metabolism (that is, α-ketoglutarate, phosphoenolpyruvate, lactate and erythrose-4-phosphate) as well as small cofactors (flavin adenine

dinucleotide and nicotinamide adenine dinucleotide) are either decreased or not detected in the filtrates of virus-infected cultures relative to controls. In contrast, cell wall

constituents, such as phosphatidylcholine and phosphatidylethanolamine, were present only in the filtrates of virus-infected cultures. In addition, several amino acids (asparagine,

methionine, cysteine and homoserine) and urea were elevated by an average of 2- and 2.8-fold, respectively. Finally, all acyl-Coenzyme A metabolites were either elevated or only present in

infected culture filtrates (Table 2). DISCUSSION A way to view the potential metabolic burden of virion production on a host cell is to consider the elemental resources required by the

virus. The C/N ratio of phages (3.00) is predicted to be lower than that of bacteria (4.25) (Vrede et al., 2002; Chan et al., 2012; Jover et al., 2014). Quantitative estimates indicated that

phage particles represented a significant portion of redistributed C and N in our experiment, and as a result of phage production these elements were incorporated into biological particles

(cells and phages) at greater, but disproportionate, amounts compared with control populations. In fact, the infected cultures had incorporated 49% more C and 148% more N than the controls

by the conclusion of the experiment. Thus, partitioning of host N into phage particles represented a potential for stoichiometric disruption in the C/N of infected cells. To assess the

effect of phage production on the metabolism of the host population, we measured intracellular metabolites from both the control and infected cultures. These data highlight the distinct

metabolic trajectories of these populations, as it was observed that viral infection led to a progressive and general increase in concentration for nearly all measured compounds. Dynamics in

phage production and host population survival can be used to divide the time course of the experiment into two phases: an early infection period (0–120 min) and a later infection period

(240–480 min). The per-cell metabolite concentrations also support this division. During the second phase of the time course, phage production had leveled off, the host population had

dropped to half of its peak cell density and the variation in the core metabolome became more dramatic relative to the uninfected controls. The 66 metabolites with significantly elevated

concentrations in infected cultures clearly point to the population of infected cells being physiologically distinct from those that are uninfected. Differences in UDP-activated sugars

(UDP-glucose, UDP-glucuronate/galacturonate) may reflect alterations in cell wall integrity in phage-infected bacteria. Similarly, several amino acids were elevated in concentration in

infected populations relative to controls. Of particular interest were glutamate and glutamine, which together serve as the entry points for 99% of inorganic N into biomolecules (Yan, 2007),

and are among the most abundant metabolites in _E. coli_ (Bennett et al., 2009). We confirmed that the absolute concentrations for glutamate and glutamine in _Sulfitobacter_ sp. 2047 are

similar to those reported for _E. coli_ (1.5 × 10−14 g cell−1 and 1.1 × 10−15 g cell−1, respectively) (Bennett et al., 2009) and thus are expected to be major components of the metabolome.

The ratio of intracellular glutamate to glutamine is an indicator of N availability in many bacterial species, with an increased ratio indicating growth under N limitation (Flynn et al.,

1989). An analysis of the trends in this ratio for both cultures indicates that the control population, which shows a progressive increase in this ratio through the growth cycle, is likely

experiencing decreased N availability as the cultures reach the end of exponential phase. As sufficient ammonium was provided to supply the N required in both cultures, these data suggest

that the infected cultures had access to preferred N-containing nutrient sources not available in the control cultures. Following the measurement of metabolite concentrations during the

infection cycle, stable isotope labeling studies using 13C-acetate were performed to determine whether the general increases in metabolite concentrations were because of increased

biosynthesis or decreased utilization of each molecule. The rates of label incorporation for 12 of the 14 metabolites monitored indicated that both populations were metabolically active. As

metabolite concentrations and turnover were similar between infected and control populations during the early time points, these data further support the observation that host metabolism was

not dramatically altered within the first hour following phage addition. However, the flux through all metabolites at the later time points was altered as the higher cellular concentrations

of these metabolites in the infected cultures results in an increased flux through these molecules as the larger pools are being replaced in the same unit time. Although the glutamate and

glutamine pools turned over more slowly during the later phase of the infection, the elevated concentrations of these compounds resulted in measured fluxes for these metabolites that were

equal for glutamate and 200–400% greater for glutamine than those of the control population. We suggest that the apparent decrease in turnover rates for these two molecules is the result of

uptake and incorporation of unlabeled nutrients from lysed kin. The metabolites with decreased concentrations in the filtrates of the infected cultures were primarily those that are expected

to be readily recycled, such as components of central C and N metabolism and small cofactors. This suggests that intact cells in the virus-infected cultures remained active and were able to

rapidly consume material from lysed cells to support their own metabolism, consequently depleting selected metabolites in the extracellular milieu. Metabolites with increased relative

concentrations in the filtrates were those typically related to cellular stress or those that are potentially too large to be transported into the cell (that is, >600 Da; Benz, 1985;

Williams, 2000). The presence of bacterial cell wall constituents (that is, phosphatidylcholine and phosphatidylethanolamine) in the phage-infected culture filtrates is also consistent with

the degradation of cell wall material from lysed cells and with prior studies demonstrating the prevalence and recalcitrance of bacterial cell wall components (that is, peptidoglycan) in

marine (McCarthy et al., 1998; Pedersen et al., 2001; Jørgensen et al., 2003) and virus-derived DOM (Middelboe and Jorgensen, 2006). Dissolved and free amino acids have also been previously

identified in viral lysates (Middelboe and Jorgensen, 2006), but the contribution of individual amino acids was not reported. Of the extracellular amino acids detected here, several of those

with the largest increases in concentration for the phage-infected cultures are also part of the normal cellular stress response (for example, asparagine, cysteine, homoserine and

methionine; Jozefczuk et al., 2010). Acyl-CoAs may not be efficiently transported and assimilated because of their large size and/or low concentrations in nature, potentially explaining

their relative increase in infected culture filtrates. The repertoire of metabolites remaining in the lysate may also be a reflection of the host’s substrate preferences. For example, urea,

a nitrogen-rich byproduct of _de novo_ nucleotide biosynthesis (Berg et al., 2012), was also elevated in infected cultures. The small molecule composition of the filtrates as well as the

intracellular metabolite concentrations and turnover rates suggest that the phage-infected cultures were incorporating nutrients from recycled sources, that is, from small molecules that

were liberated during viral lysis or from breakdown of unlabeled macromolecules. The strongest evidence for this phenomenon comes from the data for glutamate and glutamine that, despite

showing high intracellular concentrations in infected populations, were undetectable in the culture filtrates. Given that these compounds are typically preferred sources of N (over ammonium)

in bacteria (Kirchman et al., 1989; Middelboe et al., 1996; Riemann and Middelboe, 2002), it is likely that these compounds were released from lysed cells and then rapidly assimilated by

metabolically active cells. ECOLOGICAL IMPLICATIONS AND CONCLUSIONS This work provides a biochemical basis from which a mechanistic understanding of the influence of phage infection on host

metabolism can be developed for extension to a biogeochemical framework. In terms of how virus activity shapes host metabolism, our results show that phage infection in _Sulfitobacter_ sp.

2047 does not lead to any pathway-specific alterations in host metabolism. Instead, a general increase in net host metabolic activity is used to meet the requirements of virus propagation

that is facilitated by recycling of metabolites from lysed siblings. Additional studies are necessary to determine whether this observed response is generalizable to other host–phage

systems. It is relevant to highlight that in the model system used here simultaneous production of two distinct phages (one lytic and one lysogenic) occurs. Furthermore, by the final

sampling time point, some unknown and undistinguishable fraction of the population is resistant to infection, or at least lysis. As lysogeny is hypothesized to be prevalent in marine

bacteria in general (Stopar et al., 2004; Leitet et al., 2006; Paul, 2008) and in roseobacters in particular (Chen et al., 2006; Zhao et al., 2010), the complexity of this infection process

may, in fact, be reflective of events that are common in nature. An increased demand for N results in a stoichiometric imbalance of C/N in infected cells that has implications for trophic

transfer and stimulation of secondary production. In fact, our observations suggest that infected cells are a functionally different entity than their noninfected counterparts. In pelagic

environments, the progression of the lytic cycle may thus be constrained by the availability of host-limiting nutrients because of increased cellular demands. Conversely, removal of

constraints on nutrient availability may stimulate host growth, thereby facilitating viral production (Higgins et al., 2009; Weinbauer et al., 2009). Indeed, the interplay between nutrient

demand and availability may influence the lytic–lysogeny decision (Hong et al., 1971; Herskowitz and Hagen, 1980; Jiang and Paul, 1998) that phages make when infecting a cell. This could

also result in the establishment of a state that may be perceived as pseudolysogeny (Łoś and Węgrzyn, 2012) where the cell is infected but there are insufficient cellular resources to

propagate new virions. Alternatively, infected cells with access to sufficient nutrients may themselves be N enriched and more susceptible to grazing. To this end, the idea of the infected

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CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to Dr Charles Budinoff for isolation of _Sulfitobacter_ sp. 2047 and its infecting phage and Dr Willie

Wilson for encouraging and facilitating our participation in the mesocosm experiment that yielded this bacterium and phage. We thank Thomas Lane for the analysis of the _Sulfitobacter_ sp.

2047 genome. We are indebted to Dr Andrew Lang for insightful comments on an earlier version of this manuscript. This work was supported by NSF Grants OCE-1061352 awarded to AB, SRC and SWW.

JLM was supported by OCE-1208784 awarded to SRC. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Microbiology, University of Tennessee, Knoxville, TN, USA Nana Yaw D Ankrah, 

Daniel R Jones, Mary K Hadden, Gary R LeCleir, Steven W Wilhelm & Alison Buchan * Department of Chemistry, University of TN, Knoxville, TN, USA Amanda L May, Jesse L Middleton, Jessica R

Gooding & Shawn R Campagna Authors * Nana Yaw D Ankrah View author publications You can also search for this author inPubMed Google Scholar * Amanda L May View author publications You

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author inPubMed Google Scholar * Alison Buchan View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Alison Buchan.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Supplementary Information accompanies this paper on The ISME Journal website

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al._ Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition. _ISME J_ 8, 1089–1100 (2014). https://doi.org/10.1038/ismej.2013.216

Download citation * Received: 05 April 2013 * Revised: 27 October 2013 * Accepted: 31 October 2013 * Published: 05 December 2013 * Issue Date: May 2014 * DOI:

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physiology * metabolomics