Effects of Escherichia coli physiology on growth of phage T7 in vivo and in silico

Abstract

Phage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions. We established Escherichia coli at different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection. We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased. To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host. Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume. The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay. When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes. Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions.

FIG. 1.

Intracellular one-step growth of phage T7 on E. coli BL21 growing at different rates. The host cells were grown at 0.7, 1.0, 1.2, 1.5, and 1.7 doublings per h. Experimental data of the intracellular PFU for each host growth rate are from three separate infections indicated by black circles, white triangles, black diamonds, black traingles, white squares, in the order of the growth rates above; Bars indicate standard deviations. Output from the simulation is shown by solid lines.

FIG. 2.

Phage T7 growth dependence on host growth rate. (A) An intracellular one-step growth curve can be characterized by three variables: eclipse time is the period between infection initiation and the time point when phage progeny first appear, the rise rate is the slope of the straight line starting from the end of the eclipse period, and the burst size is the final number of phage progeny produced from a single infection. (B) The intracellular rise rate; (C) the eclipse time. Both are extracted using a three-parameter model from the data in Fig. 1, as a function of the E. coli growth rate. The experimental results are shown in black circles, and 95% confidence intervals are indicated. Results of processing the computer simulation growth curves are shown by solid lines. A one-parameter adjustment to the eclipse time is shown by a dashed line. This adjustment incorporated a constant delay in the initiation of phage adsorption to the host cell.

FIG. 3.

Sensitivity of the intracellular rise rate to host physiological parameters. The parameters were normalized to their base case values, which were calculated based on an E. coli growth rate of 1.5 doublings per h using equations from Table 1. The rise rate was normalized to the value calculated from the base case parameters. Because the NTP number did not affect the rise rate over the parameter range examined, it is omitted from the figure.

FIG. 4.

Effect of the EcRNAP number on the procapsid assembly process (A) and the allocation of ribosomes to different mRNAs (B) (a snapshot taken at 21 min post-infection initiation). The total procapsid number accounts for both mature capsids and procapsids.

FIG. 5.

A “bottleneck landscape” for phage T7 growth with respect to two host parameters, the levels of the host RNA polymerase (EcRNAP) and ribosome. At any point on this figure, phage growth is limited by the rate of translation by the host ribosomes or of transcription by the EcRNAP or of phage DNA synthesis. The base case setting is labeled by the filled circle.