Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism

Abstract

Cyanophages infecting the marine cyanobacteria Prochlorococcus and Synechococcus encode and express genes for the photosynthetic light reactions. Sequenced cyanophage genomes lack Calvin cycle genes, however, suggesting that photosynthetic energy harvested via phage proteins is not used for carbon fixation. We report here that cyanophages carry and express a Calvin cycle inhibitor, CP12, whose host homologue directs carbon flux from the Calvin cycle to the pentose phosphate pathway (PPP). Phage CP12 was coexpressed with phage genes involved in the light reactions, deoxynucleotide biosynthesis, and the PPP, including a transaldolase gene that is the most prevalent PPP gene in cyanophages. Phage transaldolase was purified to homogeneity from several strains and shown to be functional in vitro, suggesting that it might facilitate increased flux through this key reaction in the host PPP, augmenting production of NADPH and ribose 5-phosphate. Kinetic measurements of phage and host transaldolases revealed that the phage enzymes have k(cat)/K(m) values only approximately one third of the corresponding host enzymes. The lower efficiency of phage transaldolase may be a tradeoff for other selective advantages such as reduced gene size: we show that more than half of host-like cyanophage genes are significantly shorter than their host homologues. Consistent with decreased Calvin cycle activity and increased PPP and light reaction activity under infection, the host NADPH/NADP ratio increased two-fold in infected cells. We propose that phage-augmented NADPH production fuels deoxynucleotide biosynthesis for phage replication, and that the selection pressures molding phage genomes involve fitness advantages conferred through mobilization of host energy stores.

Fig. 1.

(A) The PPP and Calvin cycle in cyanobacteria showing genes carried by cyanophages. Genes found in phages are boxed and denoted with the number of genomes among 24 in which they are found. CP12 (cp12) is grouped with the PPP, because it shuts off the competing Calvin cycle, inhibiting PRK (prkB) and GAPDH (gap2). Both pathways run clockwise, with the shared reactions (blue) running downward (Calvin cycle) or upward (PPP), and therefore the two pathways are not expected to run concurrently. Transaldolase (talC in phage, talB in host) is the only reaction in the intersecting part of the pathways that is needed only in one direction. Arrows show proposed directionality of the reactions, and one-way arrows do not necessarily indicate irreversibility. P, phosphate; (genes) cbbA, fructose-1,6-bis-P/sedoheptulose-1,7-bis-P aldolase; glpX, fructose-1,6/sedoheptulose-1,7-bisphosphatase; pgi, P-glucose isomerase; pgk, P-glycerate kinase; pgl, 6-P-gluconolactonase; prkB, P-ribulokinase; rbcLS, ribulose-1,5-bis-P carboxylase/oxygenase; rpe, ribulose-5-P epimerase; rpiA, ribulose-5-P isomerase; tktA, transketolase; tpi, triose-P isomerase; (substrates) BPG, 2,3-bis-P-glycerate; DHAP, DHA P; E4P, erythrose 4-P; FBP, fructose 1,6-bis-P; F6P, fructose 6-P; GAP, glyceraldehyde 3-P; G6P, glucose 6-P; PGA, 3-P-glyceric acid; R5P, ribose 5-P; RuBP, ribulose 1,5-bis-P; Ru5P, ribulose 5-P; SBP, sedoheptulose 1,7-bis-P; 6PG, 6-P-gluconate; 6PGL, 6-P-gluconolactone; S7P, sedoheptulose 7-P; and X5P, xylulose 5-P. (B) Relative abundance of T4-like cyanophage genes in the GOS database. For each gene found in a T4-like cyanophage (red circles represent genes found in all sequenced genomes, i.e., core genes, and gray circles represent noncore genes), the number of times it was observed as a sequence read is plotted as a function of gene size. Core genes show a linear increase with gene size in the number of observed reads, as expected, as the likelihood of cloning and sequencing fragments of larger genes is greater than for smaller genes. The linear regression (with 95% confidence intervals) of this pattern is shown for core genes.

Fig. 2.

Cyanophage Syn9 infection of Synechococcus WH8109, showing expression of a phage gene for the Calvin cycle inhibitor CP12 alongside other phage metabolic genes. Error bars represent SDs of three technical and three biological replicates. (A) Degradation of host gDNA and replication of phage gDNA (free and intracellular) as determined by qPCR of chromosomal genes in host (rnpB) and phage (g20). (B) mRNA of T4-like early genes g61 (DNA primase) and g43 (DNA polymerase) and late genes g20 (portal protein) and g23 (major coat protein). (C) mRNA of PSII gene psbA (D1 subunit) and RNR gene nrdA (α subunit). (D) mRNA of PPP genes zwf (glucose-6-P dehydrogenase), gnd (6-P-gluconate dehydrogenase), talC (transaldolase), and cp12 (Calvin cycle inhibitor CP12).

Fig. 3.

Comparison of phage and host gene sizes for shared orthologues. For each gene orthologue found in at least three cyanophage genomes and at least three Prochlorococcus or Synechococcus genomes, the median gene size across all phages and across all hosts is plotted. Bars represent the range of sizes observed. The line shows a 1:1 ratio, whereby phage and host gene sizes are identical. Median gene sizes for each host–phage pair were compared by using the Mann–Whitney–Wilcoxon test (P < 0.05) with correction for false discovery rate (SI Methods). Blue circles indicate genes that were shorter in phages than in hosts, red circles those that were shorter in hosts than in phages, and unfilled circles those that were statistically the same size in phages and hosts.

Fig. 4.

Dynamics of the intracellular NADPH/NADP ratio (Top) and NADP(H)/NAD(H) ratio (Bottom) during infection of Prochlorococcus MED4 by cyanophage P-HM2 in the light or in the dark. Error bars represent SDs of two technical and two biological replicates.

Fig. P1.

Model of cyanobacterial metabolism during cyanophage infection. (A) In uninfected cells, four interrelated pathways combine to make DNA building blocks (nucleotides): the light reactions of photosynthesis, the Calvin cycle (the so-called “dark reactions” of photosynthesis), the pentose phosphate pathway, and nucleotide biosynthesis. (B) In infected cells, host-like genes (blue ovals) acquired by phage over evolutionary time are proposed to augment or inhibit key steps in host metabolism, leading to increased nucleotide biosynthesis for phage reproduction.