A Forward-Genetic Screen And Dynamic Analysis Of Lambda Phage ...

Infection dynamics of E. coli strains showing reduced infectivity

We wondered how best to characterize the functional roles of new genes implicated in lambda infection. One weakness of the previous screens for host gene requirements in viral infection is that the information produced by these screening experiments is highly limited, generally involving only a few measurements per gene.

We hypothesized that higher time-resolution monitoring of infection dynamics would facilitate validation and further characterization of the roles these host genes play in the lambda phage lifecycle. We monitored E. coli growth and lysis over a full course of lambda infection in liquid culture (see Figure 3A). Infected K-12 WT bacteria grow exponentially for about 3 hours, after which the rate of bacterial lysis briefly outpaces growth. During this time, many phage have also induced lysogeny in their E. coli hosts, which then become resistant to further lytic infection. The lysogenic strains eventually take over the culture, growing exponentially until stationary phase.

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Figure 3. E. coli growth curves under normal conditions and incubated with lambda phage.

All four replicates are shown with the average displayed as a bolded line. These growth curves have been normalized by growth rate and maximum growth capacity to facilitate comparison between strains (see Materials and Methods). (A) A typical set of K-12 WT uninfected (gray) and infected (blue) growth curves. (B) A sample of infected knockout strain growth curves to demonstrate the variability between strains, with respect to the response to infection. (C) The ΔlamB, ΔmalI, and ΔmalT strain growth curves, which appear in the same pathway (see Figure 2A), also exhibit very similar infected growth dynamics.

https://doi.org/10.1371/journal.pgen.1001017.g003

We monitored growth of infected and uninfected cultures for all 57 strains that showed reduced infectivity in our plaque assay. We found that the infection dynamics varied significantly between strains. Figure 3B shows selected time course data, normalized by growth rate and maximum carrying capacity (i.e., optical density at stationary phase) to highlight the difference between infected and uninfected strains (The infection time courses for all 57 strains can be found in Figure S1).

We observed that genes with directly cooperative roles in lambda infection often exhibited very similar growth and clearance dynamics. As a simple example, Figure 3C shows the infection time courses for ΔlamB, ΔmalI, and ΔmalT. As mentioned above, all of these genes work together—malT and malI regulate the expression of the lamB transcript and functional protein product.