Frequency and fitness consequences of bacteriophage φ6 host range mutations

Abstract

Viruses readily mutate and gain the ability to infect novel hosts, but few data are available regarding the number of possible host range-expanding mutations allowing infection of any given novel host, and the fitness consequences of these mutations on original and novel hosts. To gain insight into the process of host range expansion, we isolated and sequenced 69 independent mutants of the dsRNA bacteriophage Φ6 able to infect the novel host, Pseudomonas pseudoalcaligenes. In total, we found at least 17 unique suites of mutations among these 69 mutants. We assayed fitness for 13 of 17 mutant genotypes on P. pseudoalcaligenes and the standard laboratory host, P. phaseolicola. Mutants exhibited significantly lower fitnesses on P. pseudoalcaligenes compared to P. phaseolicola. Furthermore, 12 of the 13 assayed mutants showed reduced fitness on P. phaseolicola compared to wildtype Φ6, confirming the prevalence of antagonistic pleiotropy during host range expansion. Further experiments revealed that the mechanistic basis of these fitness differences was likely variation in host attachment ability. In addition, using computational protein modeling, we show that host-range expanding mutations occurred in hotspots on the surface of the phage's host attachment protein opposite a putative hydrophobic anchoring domain.

Figure 1. Spatial models of Φ6 P3 protein mutants.

Panel A: Three host range mutation hotspots (accounting for 86% of all mutations) are highlighted in this linear representation of the 648 amino acid sequence of the Φ6 P3 gene. The remaining 14% of mutations are not shown. Panel B: Space-filling representations of the Φ6 P3 protein are shown as predicted by I-TASSER. Colored regions correspond to the mutation hotspots depicted in Panel A. A putative hydrophobic anchoring domain (HAD) is shown in orange. In our model, the hydrophobic anchoring domain penetrates Φ6's outer lipid membrane to bind inner membrane protein P6. Panel C: Surface electrical charges of E8G mutant contrasted with ancestor. Space-filling representations showing predicted surface electrical charges for the Φ6 E8G host range mutant and its ancestor were estimated using I-TASSER. Positively- and negatively-charged regions are depicted in blue and red respectively. Arrows indicate the predicted location of the mutated 8^th residue. The most prominent difference between the mutant and the ancestor is the greater surface positive charge at the presumed host binding domain.

Figure 2. Mutant absolute fitness on canonical and novel hosts.

Panel A: Absolute fitness of 13 Φ6 host range mutants on the novel host, ERA. Each point is the mean of 5 replicate measurements of fitness. Bars are ±1SE. Panel B: Absolute fitness of 13 Φ6 host range mutants on the canonical host, PP. Each point is the mean of 5 replicate measurements of fitness. Fitness of wildtype Φ6 is shown by the dotted line for comparison. Bars are ±1SE.

Figure 3. Mean ERA attachment rate (k) is plotted against phage Φ6 fitness on ERA.

Attachment to ERA was correlated with fitness on ERA for Φ6 host range mutants. Each point is the mean of 3 replicate measurements. Dotted lines show 95% confidence intervals.