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

Abstract

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.

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

“…The percent infection determined by the iPolony method was significantly and positively correlated with the MOI (Fig. 3a) (F = 143.1, R 2 = 0.87, p < 0.001) and with values obtained using a lysis-based culturedependent assay [37] (Fig. 3b) (F = 55.60, R 2 = 0.70, p < 0.001).…”

“…These samples were diluted 50-fold with medium, fixed, and frozen prior to analysis. The percent infection from the experiments conducted at different MOIs was also assessed by a lysis-based flow-cytometric sorting assay [37]. Briefly, infected samples were diluted 1000-fold in growth medium, and single cells were sorted by flow cytometry into individual wells of 96-well plates that contained exponentially growing host cell culture.…”

Two Synechococcus genes, Two Different Effects on Cyanophage Infection

“…We began our investigation of the impact of the host mutations on the phage infection process by assessing phage virulence, as determined from the ability of the phage to infect and lyse the different host strains [37]. This was determined from the percentage of cells lysed by the Syn9 phage when infecting each of the inactivation mutants compared to infection of the wild-type Synechococcus strain.…”

“…In order to assess whether these differences in phage genome replication translated into changes in phage fitness, we investigated the number of infective phages produced per cell using a single-cell burst size assay [37]. Similar to phage genome replication, the median burst size of the Syn9 phage on the PIN-PhoH mutant (79 phages·cell −1 ) was significantly higher than on the wild-type host (52 phages·cell −1 ) ( p = 0.001, n = 189 cells for the PIN-PhoH mutant and 174 cells for the wild-type host) (Figure 5).…”

“…Burst size and virulence assays were carried out as described by Kirzner et al [37]. Exponentially growing cultures were diluted to the same concentration (≈4 × 10 7 cells per mL) and infected with the Syn9 phage at MOI = 3 in the morning hours.…”