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.

Figure 1

(a) Sulfitobacter sp. 2047 cell density (OD₅₄₀) and phage concentration at each metabolite sampling time point reported in Figure 2. Line graphs are color coded as follows: phage-infected culture (), control culture () and phage numbers (). Turbidity declines are indicative of phage-induced lysis. Phage numbers were derived from qPCR assays. Averages and ranges of biological duplicates are reported. Estimates of (b) carbon and (c) nitrogen content and their (d) ratios for Sulfitobacter sp. 2047 cells and phage during an infection cycle. Bar graphs are color coded as follows: phage (), infected culture () and control culture (). Bacterial cell densities were determined by microscopy. Phage numbers were determined using qPCR. Bacterial carbon (149 fg C per cell) and nitrogen (35 fg N per cell) were derived from literature values of marine bacteria as reported in text. Phage carbon (0.2 fg C per phage) and nitrogen (0.076 fg N per phage) were derived from theoretical calculations (Jover et al., 2014). Values reflect the average of duplicate biological replicates.

Figure 2

Heat map of intracellular metabolites of phage-infected and control Sulfitobacter sp. 2047 populations. Metabolite concentrations are normalized to bacterial cell number and expressed relative to levels measured in the uninfected host cells at the corresponding time point. Ratios are log₂ transformed. Increases in intracellular metabolite concentrations are shown in red and decrease in blue. Columns correspond to min post infection, and rows represent specific metabolites. Values are averages of duplicate biological and technical replicates and are reported in Supplementary Table S3.

Figure 3

Variation in intracellular metabolite concentrations between phage-infected and control populations during the infection cycle shown in Figure 2. Columns indicate fold changes ⩾1.5 and P⩽0.05, and all data are shown in Supplementary Tables S3 and S4.

Figure 4

Absolute concentrations of (a) glutamate and (b) glutamine in control (♦) and phage-infected (▪) Sulfitobacter sp. 2047 populations. Values represent duplicate biological and duplicate technical replicates. Error bars show the s.e.m. (c) Glutamate to glutamine ratios for phage-infected and control populations throughout the experimental time course. Bar graphs are coded as indicated by the key in each figure. (d) Selected metabolites are shown to illustrate the relationship between the tricarboxylic acid (TCA) cycle and glutamate and glutamine metabolism in Sulfitobacter sp. 2047. The bar graphs represent concentrations for metabolites in the phage-infected culture and are expressed as fold change relative to the control at the corresponding time point as shown for malonyl-Coenzyme A. Fold changes are log₂ transformed and represent averages of duplicate biological and technical replicates. Asterisks designate significant fold changes (⩾1.5 and P⩽0.05).

Figure 5

Incorporation of acetate-derived ¹³C into (a, b) glutamate and (c, d) glutamine in phage-infected and control populations during two distinct phases of infection: (a, c) 0–60 min post infection (early) and (b, d) 240–300 min post infection (late). The graphs show the disappearance of unlabeled metabolites for phage-infected () and control () populations as well as the appearance of fully ¹³C-labeled metabolites in phage-infected () and control () populations. Error bars represent range of the data and are obscured by the data markers, in some cases.