Cyanophages from a less virulent clade dominate over their sister clade in global oceans.

Environmental virus communities are highly diverse. However, the infection physiology underlying the evolution of diverse phage lineages and their ecological consequences are largely unknown. T7-like cyanophages are abundant in nature and infect the marine unicellular cyanobacteria, Synechococcus and Prochlorococcus, important primary producers in the oceans. Viruses belonging to this genus are divided into two distinct phylogenetic clades: clade A and clade B. These viruses have narrow host-ranges with clade A phages primarily infecting Synechococcus genotypes, while clade B phages are more diverse and can infect either Synechococcus or Prochlorococcus genotypes. Here we investigated infection properties (life history traits) and environmental abundances of these two clades of T7-like cyanophages. We show that clade A cyanophages have more rapid infection dynamics, larger burst sizes and greater virulence than clade B cyanophages. However, clade B cyanophages were at least 10-fold more abundant in all seasons, and infected more cyanobacteria, than clade A cyanophages in the Red Sea. Models predicted that steady-state cyanophage abundances, infection frequency, and virus-induced mortality, peak at intermediate virulence values. Our findings indicate that differences in infection properties are reflected in virus phylogeny at the clade level. They further indicate that infection properties, together with differences in subclade diversity and host repertoire, have important ecological consequences with the less aggressive, more diverse virus clade having greater ecological impacts.

A single-cell polony method reveals low levels of infected Prochlorococcus in oligotrophic waters despite high cyanophage abundances.

Long-term stability of picocyanobacteria in the open oceans is maintained by a balance between synchronous division and death on daily timescales. Viruses are considered a major source of microbial mortality, however, current methods to measure infection have significant methodological limitations. Here we describe a method that pairs flow-cytometric sorting with a PCR-based polony technique to simultaneously screen thousands of taxonomically resolved individual cells for intracellular virus DNA, enabling sensitive, high-throughput, and direct quantification of infection by different virus lineages. Under controlled conditions with picocyanobacteria-cyanophage models, the method detected infection throughout the lytic cycle and discriminated between varying infection levels. In North Pacific subtropical surface waters, the method revealed that only a small percentage of Prochlorococcus (0.35-1.6%) were infected, predominantly by T4-like cyanophages, and that infection oscillated 2-fold in phase with the diel cycle. This corresponds to 0.35-4.8% of Prochlorococcus mortality daily. Cyanophages were 2-4-fold more abundant than Prochlorococcus, indicating that most encounters did not result in infection and suggesting infection is mitigated via host resistance, reduced phage infectivity and inefficient adsorption. This method will enable quantification of infection for key microbial taxa across oceanic regimes and will help determine the extent that viruses shape microbial communities and ecosystem level processes.

Adaptation to sub-optimal hosts is a driver of viral diversification in the ocean.

Cyanophages of the Myoviridae family include generalist viruses capable of infecting a wide range of hosts including those from different cyanobacterial genera. While the influence of phages on host evolution has been studied previously, it is not known how the infection of distinct hosts influences the evolution of cyanophage populations. Here, using an experimental evolution approach, we investigated the adaptation of multiple cyanophage populations to distinct cyanobacterial hosts. We show that when infecting an "optimal" host, whose infection is the most efficient, phage populations accumulated only a few mutations. However, when infecting "sub-optimal" hosts, different mutations spread in the phage populations, leading to rapid diversification into distinct subpopulations. Based on our results, we propose a model demonstrating how shifts in microbial abundance, which lead to infection of "sub-optimal" hosts, act as a driver for rapid diversification of viral populations.

Variability in progeny production and virulence of cyanophages determined at the single-cell level.

Little information regarding viral progeny production (burst size) and host mortality (viral virulence) is currently available for environmentally relevant phages. This is partially due to the difficulty in accurately measuring these infection properties with existing methods. Here, we set up a simple system for determining viral virulence and burst size at the single-cell level following flow cytometric separation of infected cells. We applied this assay to two distinct cyanomyoviruses, Syn9 and S-TIM5, during infection of two marine Synechococcus strains each. We found that virulence ranged from 44%-82%, differing for the same phage on different hosts. Average burst sizes ranged from 21-43 infective viruses/cell, and differed with host for Syn9, whereas the burst size of S-TIM5 was similar for both hosts. In addition, virulence and burst sizes were different for the two phages when infecting their common host. Furthermore, wide-ranging cell-to-cell variability was found for single-cell burst sizes in each of the four interactions, ranging from 2 to over 100 infective viruses/cell. This variability, discerned at both the population and single-cell levels under controlled laboratory conditions, is likely to be much more complex in natural environments.

Diversity and evolutionary relationships of T7-like podoviruses infecting marine cyanobacteria.

Phages are extremely abundant in the oceans, influencing the population dynamics, diversity and evolution of their hosts. Here we assessed the diversity and phylogenetic relationships among T7-like cyanophages using DNA polymerase (replication), major capsid (structural) and photosynthesis psbA (host-derived) genes from isolated phages. DNA polymerase and major capsid phylogeny divided them into two discrete clades with no evidence for gene exchange between clades. Clade A phages primarily infect Synechococcus while clade B phages infect either Synechococcus or Prochlorococcus. The major capsid gene of one of the phages from clade B carries a putative intron. Nearly all clade B phages encode psbA whereas clade A phages do not. This suggests an ancient separation between cyanophages from these two clades, with the acquisition or loss of psbA occurring around the time of their divergence. A mix and match of clustering patterns was found for the replication and structural genes within each major clade, even among phages infecting different host genera. This is suggestive of numerous gene exchanges within each major clade and indicates that core phage functions have not coevolved with specific hosts. In contrast, clustering of phage psbA broadly tracks that of the host genus. These findings suggest that T7-like cyanophages evolve through clade-limited gene exchanges and that different genes are subjected to vastly different selection pressures.