Nitrogen sourcing during viral infection of marine cyanobacteria

Abstract

The building blocks of a virus derived from de novo biosynthesis during infection and/or catabolism of preexisting host cell biomass, and the relative contribution of these 2 sources has important consequences for understanding viral biogeochemistry. We determined the uptake of extracellular nitrogen (N) and its biosynthetic incorporation into both virus and host proteins using an isotope-labeling proteomics approach in a model marine cyanobacterium Synechococcus WH8102 infected by a lytic cyanophage S-SM1. By supplying dissolved N as ¹⁵N postinfection, we found that proteins in progeny phage particles were composed of up to 41% extracellularly derived N, while proteins of the infected host cell showed almost no isotope incorporation, demonstrating that de novo amino acid synthesis continues during infection and contributes specifically and substantially to phage replication. The source of N for phage protein synthesis shifted over the course of infection from mostly host derived in the early stages to more medium derived later on. We show that the photosystem II reaction center proteins D1 and D2, which are auxiliary metabolic genes (AMGs) in the S-SM1 genome, are made de novo during infection in an apparently light-dependent manner. We also identified a small set of host proteins that continue to be produced during infection; the majority are homologs of AMGs in S-SM1 or other viruses, suggesting selective continuation of host protein production during infection. The continued acquisition of nutrients by the infected cell and their utilization for phage replication are significant for both evolution and biogeochemical impact of viruses.

Keywords: bacteriophage; biogeochemistry; proteomics.

Fig. 1.

Time courses of cell lysis, phage production, and protein ¹⁵N incorporation during infection of Synechococcus WH8102 by phage S-SM1 in HL conditions (126 µmol photons m⁻² s⁻¹). (A) Extracellular phage genome copies (by qPCR of g20) and phycoerythrin fluorescence (from lysed cell debris); points and error bars are averages and ±1 SD, respectively, across experimental triplicates. (B and C) Distributions of atom% ¹⁵N values of host (purple) and phage (green) proteins in (B) intracellular (i.e., cell pellet) and (C) extracellular (i.e., >10 kDa/<0.2 µm filtrate) samples; the numbers at the top indicate the number of proteins detected for host and phage at each timepoint, the gray bars show values of individual proteins (averaged across experimental triplicates), the colored horizontal lines show host and phage medians at each timepoint.

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 annotations 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.

Fig. 3.

Time courses of ¹⁵N incorporation into photosystem II reaction center proteins (A) D1/PsbA and (B) D2/PsbD during infection under HL, ML, and LL conditions.

Fig. 4.

¹⁵N incorporation time courses in the HL condition of the 12 Synechococcus WH8102 host proteins that became significantly labeled in our phage infection experiments. The circles after gene names indicate the presence of a homolog in viral (meta)genome sequences: ••• = present in the S-SM1 genome; •• = present in other viral isolate genomes; • = present in putative viral contigs from aquatic metagenomic data (Dataset S1).