The unicellular cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in the ocean and is responsible for nearly 50% of the total primary production in many oligotrophic regions. Cyanophages, viruses that infect cyanobacteria, are also abundant and play crucial roles in global biogeochemical cycles. Many cyanophages contain auxiliary metabolic genes of host origin. These genes are involved in photosynthesis, central carbon metabolism and phosphate uptake, leading to the hypothesis that cyanophages participate in host metabolic pathways to obtain more nutrients and energy for their own production.

Cyanophages infecting Prochlorococcus live in a phosphorus-limited environment, and they have been shown to exploit the host’s PhoR/PhoB two-component signaling sy...[ Read more ]

The unicellular cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in the ocean and is responsible for nearly 50% of the total primary production in many oligotrophic regions. Cyanophages, viruses that infect cyanobacteria, are also abundant and play crucial roles in global biogeochemical cycles. Many cyanophages contain auxiliary metabolic genes of host origin. These genes are involved in photosynthesis, central carbon metabolism and phosphate uptake, leading to the hypothesis that cyanophages participate in host metabolic pathways to obtain more nutrients and energy for their own production.

Cyanophages infecting Prochlorococcus live in a phosphorus-limited environment, and they have been shown to exploit the host’s PhoR/PhoB two-component signaling system to address phosphorus (P) limitation. To systematically examine the responses of stressed bacteria to phage infection, we applied RNA-Seq to study the transcriptomic responses of Prochlorococcus NATL2A and cyanophage P-SSM2 under P-limited conditions. We showed that the transcripts of P-acquisition genes, including the high-affinity phosphate-binding protein gene pstS, were enriched after P limitation in uninfected cells. In infected cells, these transcripts were still enriched under P-limited conditions relative to those under nutrient-replete conditions. The cyanophage P-SSM2 genome contains a pstS gene. Among the entire P-SSM2 genome, pstS and its adjacent gene g247 (of unknown function) were the only two genes that were enriched under P-limited conditions.

The method used to determine differentially expressed genes operates in a relative framework, wherein individual transcripts are potentially mispresented when the total pool of transcripts is altered. To circumvent this limitation, we developed a method for quantifying the absolute amounts of individual transcripts by adding a spike-in RNA as an internal control. Using this method, we showed that the number of host pstS transcripts per cell decreased in the infected cells. However, in the infected cells, this number remained higher in P-limited conditions than that in nutrient-replete conditions. Moreover, the number of phage pstS transcripts per cell was ~20 times higher than that of the host pstS. With pstS transcripts being contributed by the infecting phage, the infected host might continue to acquire phosphate in P-limited environments, which may fulfill the phage’s need for P.

In addition to affecting the phosphate uptake pathway of the host, phage infection also affects the host’s central carbon metabolism. As an intrinsically unstructured protein, CP12 binds to and inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) of the Calvin cycle in higher plants, algae and cyanobacteria. To our surprise, we found that the CP12 encoded by cyanophage P-HM2 was functional. After incubation with phage-encoded CP12, the host PRK activity dropped to approximately 60% of its original level. To test whether the host Calvin cycle is repressed during phage infection, we infected Prochlorococcus MED4 cells with the cyanomyovirus P-HM2 and measured the key metabolites in the central carbon metabolism pathway. We showed that the ATP/ADP ratio in infected cells was comparable to that in uninfected cells, which was consistent with the results of a previous study showing that the photosynthesis light reaction was sustained during infection. More importantly, we found that the carbon incorporation rate was dramatically repressed in the infected cells. Meanwhile, the level of ribulose 1,5-bisphosphate (RuBP) was decreased, while that of fructose 6-phosphate (F6P) was significantly increased, indicating inhibition of carbon fixation and a higher carbon flow in the oxidative pentose phosphate pathway during P-HM2 infection. In agreement with this result, neither RuBP nor F6P were significantly altered in the cells infected with the cyanopodovirus P-SSP7, which does not contain cp12 in its genome. Our results shed light on the mechanisms by which phage infection affects central carbon metabolism in the host.