Cyanophages: Maximizing the Photo– and Redirecting the –Synthesis

by Daniel P. Haeusser
 

Pop Quiz, Microbial Hotshot

Happy Monday, readers! I hope you studied hard this weekend. Please put away your notes and close any open programs or browser tabs other than this one. It’s time for a Small Things Considered general biology quiz:

In which of the following would you find genes for proteins involved in photosynthesis? (Note: One answer only!)

A.) A naked mole rat

B.) A mushroom

C.) A shrub

D.) A virus

E.) Two of the above

Most people, at least not anticipating a trick question, may well answer C. If you picked option E, perhaps you figured that mushrooms are superficially ‘plant-like’, so maybe there are some species out there that do encode photosynthesis genes. To our knowledge there are none– but perhaps this is our ignorance. Has anyone looked?
 

Figure 1. Transmission electron micrographs of cyano­phage. (A) T4-like Cyanomyovirus S-PM2. (B) T7-like P-SSP7. (C) Cyanosiphovirus P-SS2. Source

If you are a longtime reader of Small Things Considered, and have an exceptional memory, perhaps you chose E because you recalled a post by Merry back in 2008 about a Catch-22 situation faced by marine cyanobacteria and their phage. As Merry explains in her short article:

"If the infecting cyanophage shuts down the synthesis of host proteins… photosynthesis declines. Continued photosynthesis is required [to generate energy for] maximal phage replication. So what's a cyanophage to do?"
 

The answer of course is that the virus encodes its own gene to permit host photosynthesis, while still shutting down general host functions during infection. Since the publication of Merry’s post eight years ago there has been research further advancing the proposed models for how cyanophages selfishly regulate their host’s metabolism, with important implications for estimates of global carbon cycling in an age of climate change.
 

Managing the two sides of photosynthesis

Before we get to that research, let’s first consider just what photosynthesis is. The term generally describes a single biological process of converting light energy into chemical energy, and the synthesis of carbohydrates. However, considering it more precisely, photosynthesis is actually two separate – albeit often linked – processes. Light harvesting and electron transport in specialized membrane structures called the thylakoids comprise the photo- component, converting solar energy into ATP and NADPH. These products are then used in the cytoplasm (or chloroplast stroma) for the –synthesis of cellular building blocks. As is true for all viruses, to generate new phage particles a cyanophage needs its cyanobacterial host for biosynthesis. In other words, cyanophages need to uncouple the photo- from the –synthesis. The phage needs to keep host light reactions going, but must redirect cellular energy into making new viral particles rather than into the synthesis of carbohydrates and other materials for host use. How could a phage evolve to effectively carry out such a trick?
 

The Generation of a Hypothesis

Researchers found a potential answer to this question by again considering the rich and assorted inventory of cyanophage genomes, and identifying other genes that seemed to encode products involved in photosynthesis regulation. In addition to the case explored by Merry linked above, cyanophage genomes reveal other homologues involved in things photo-related, namely components like light harvesting and pigment synthesis, reaction center formation, and electron transport carriers/acceptors. Additionally, cyanophages appear to sometimes carry genes that could potentially decouple these components from the normal host –synthesis. Some cyanophage genomes contain genes predicted to inhibit the Calvin Cycle and redirect energy to alternate pathways to viral proliferation.
 

Figure 2. Cartoon of the Calvin Cycle and a model for CP12 regulation of PRK and GAPDH. GAPDH uses NADPH to catalyze formation of glyceraldehyde-3-phosphate (GAP) and inorganic phosphate. PRK uses ATP to phosphorylate ribulose-5-phosphate (Ru5P) to produce RuBP. CP12 binding of GAPDH induces a conformational change that allows PRK to also bind, forming a complex with inhibited GAPDH and PRK activities. Source

CP12 is an example of such a cyanophage photosynthesis-uncoupling factor. Research in 2011 from the Chisholm lab characterized it as an inhibitor of two enzymes of the Calvin Cycle to fix carbon into sugar: PRK (phosphoribulokinase) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
 

The Chisholm group found that as phage-expressed CP12 presumably shuts down host carbon fixation, additional phage genes encoding auxiliary metabolic products are concurrently expressed, including a transaldolase shared between the Calvin Cycle and the Pentose Phosphate Pathway (PPP). Expression of these genes in concert would be predicted to shift metabolic priorities to the PPP, resulting in the synthesis of nucleotides for DNA, specifically the DNA of progeny phage genomes. Consistent with this hypothesis, the researchers found that the NADPH/NADP⁺ ratio increased two-fold in phage-infected cyanobacteria, indicative of flux through the Calvin Cycle (which consumes NADPH) and increases in flux through the PPP (which produces NADPH).
 

Testing the Paradigm

Taken together the data support a model where cyanophage carry photo-related electron transport genes to continue host energy acquisition, and also genes required to redirect that energy away from –synthesis of building blocks for the cyanobacterial host and into nucleotide production to generate more phage. It’s a great model that makes perfect sense. But it is built almost entirely on just metagenomics. The ‘paradigm hadn’t been rigorously tested’, to paraphrase the authors of a recent report that includes experiments that measure the ability of viral-infected Synechococcus to maintain photo-related electron transport and carbon fixation.
 

For their stab at more rigorous testing, these authors used Synechococcus sp. WH7802 infected with either cyanophage S-PM2d or S-RSM4 and compared levels of cellular CO₂ uptake or photophysiological yields to those in uninfected controls. There are key differences between the two cyanophages used. S-PM2d has a shorter latent period and only contains a few genes involved in photo-related electron transport. S-RSM4 contains many of those photo-related genes plus cp12 and the other genes involved in redirecting host central carbon metabolism.
 

The photo-related electron transport component can be measured based on the activity of Photosystem II. (The physics and chemistry of the assay are beyond the scope of this post. But for those curious, it involves using a pulse amplitude-modulated fluorometer to determine effective quantum yield photochemistry). As expected and consistent with data going back to the time of Merry’s post, infection with either phage had no effect on Synechococcus electron transport.
 

Figure 3. Effect of cyanophage infection on carbon fixation by Synechococcus sp. WH7803. Error bars represent standard deviations from three experiments. (A) Radiolabeled sodium bicarbonate uptake over time during fixed irradiance intensity. (B) Radiolabeled sodium bicarbonate uptake over a gradient of light intensity over five hours post-infection. Source

In contrast, CO₂ fixation was clearly different between uninfected and infected Synechococcus populations. This experiment involved measuring uptake of radiolabeled carbon in the form of sodium bicarbonate (NaH¹⁴CO₃) (Figure 3A) or over a gradient of irradiance (Figure 3B) using a "photosynthetron," a gadget that allowed the authors to measure changes in light intensity on carbon fixation rates. Their results (Figure 3) show that viral infection of Synechococcus significantly reduces its capacity to fix carbon. S-RSM4 infection has a stronger effect than S-PM2d. This is not surprising given that S-RSM4 encodes cp12 and other genes predicted to suppress the Calvin Cycle. Interestingly, S-PM2d infection still results in a partial reduction in carbon fixation, despite it not carrying obvious genes that directly alter carbon metabolism, as S-RSM4 does. One possibility raised by the authors is that some decreases in carbon fixation may occur indirectly, not through alterations in the Calvin Cycle, but by enhancing respiration or dissolved organic carbon. Regardless of the combination of mechanisms, phage infection leads to decreasing net CO₂ fixation.
 

Implications for Primary Production

Cyanobacteria, particularly Prochlorococcus and Synechococcus species, are the most abundant phototrophs on Earth, and are highly important in marine ecosystems and global carbon cycling. As noted by Forest Rohwer and others, phages surely play a significant role in the biogeochemical cycling activity of prokaryotes. The new study discussed here verifies for the first time the model that viruses inhibit carbon fixation by cyanobacteria, thus influencing global net primary production (the fixing of CO₂ into organic molecules). The authors say:

"...we estimate between 0.02 and 5.39 Pg C[arbon] per year is lost to viral-induced inhibition of CO₂ fixation. The upper figure of this range represents approximately 2.8-fold greater productivity than the sum of all coral reefs, salt marshes, estuaries, and marine macrophytes on Earth."
 

This is quite an effect on the global CO₂ metabolism! Given current predictions of drastic climate change, the impact of cyanophages on reducing carbon fixation would increase it even further. This study highlights the misconception of assuming that photosynthesis exists as single process of strict coupling between energy conversion and carbohydrate production. This assumption, along with neglecting to take the powerful contributions of viruses into account, can skew estimates of primary production. In a world where even proportionately small changes influence the climate, tiny viruses play a large role.