Sustained coevolution of phage Lambda and Escherichia coli involves inner- as well as outer-membrane defences and counter-defences

Abstract

Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In Escherichia coli and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair λ's ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

Fig. 1.

Genetic interaction networks during gene-for-gene (GFG) coevolution (a) and inverse gene-for-gene (IGFG) coevolution (b). In both scenarios, host alleles affect selection on pathogen phenotypes, and pathogen alleles influence selection on host phenotypes. However, the two models have different implications for understanding historical coevolution and predicting future changes. During GFG coevolution, hosts evolve resistance by gaining resistance genes, and pathogens evolve by losing genes that elicit host defences. GFG coevolution is common among plants and their bacterial pathogens; it may also occur in bacteria–phage interactions that involve restriction–modification and CRISPR defences. During IGFG coevolution, pathogen infectivity requires the exploitation of specific host features, and resistance involves eliminating the exploited features. Unlike in the GFG model, host defences in the IGFG model do not require pathogen recognition, and the pathogen’s evasion of host resistance does not require the loss of a defence elicitor.

Fig. 2.

Net population growth of phage λ on wild-type, ΔmanY and ΔmanZ bacteria. Whether the phage could grow was assessed by performing one-tailed t-tests on the log₁₀-transformed ratio of phage population densities at the start and end of a 1 day cycle, with the null hypothesis of zero growth (***, P <0.001; **, 0.001 <P <0.01; ns, not significant, P >0.05). Each test was based on five or six replicate assays. Phage isolates λ-A and λ-B evolved in a batch culture regime with 100-fold dilution each day, and so 100-fold growth was required for their persistence; this break-even level is indicated by the dashed line.

Fig. 3.

Temporal dynamics of man mutants in E. coli populations Pop-A (a) and Pop-B (b). Mutant malT alleles had already reached fixation in both populations by day 8 [17]. Bacteria with man mutations, which confer resistance to the ancestral phage λ, rose to high frequencies and then declined sharply in abundance in both populations after day 8, but before λ had evolved to use the alternative receptor OmpF (timing indicated by vertical dashed arrows). These data imply that the man mutations evolved on malT mutant backgrounds, and that λ evolved independence of the mannose permease – causing the precipitous decline in the frequency of man mutants – before it evolved the ability to use OmpF. The shaded regions indicate the maximum and minimum frequencies of the man mutants based on analysing two samples per population each day (mean n=90 colonies tested per sample, minimum 29 colonies). The horizontal grey dashed lines show the approximate limit of detection of the man mutants [0.019 for (a), 0.022 for (b)].

Fig. 4.

Evolution of man-related colony morphology on tetrazolium mannose agar. E. coli mutants with reduced ability to metabolize mannose form more deeply pigmented colonies than the wild-type bacteria. Three representative colonies are shown for each sample from days 1–20 of two coevolution experiments. Representative colonies within a column are from the same agar plate and shown at the same magnification after incubation for 18–21 h. (a) Pop-A. (b) Pop-B. (c) Comparison of wild-type and ∆manZ bacteria in the same E. coli strain B genetic background.

Fig. 5.

An inverse gene-for-gene model showing the structure of the genetic network for coevolving E. coli and λ populations. Columns indicate bacterial genotypes with four exploitable features, and rows indicate λ genotypes that exploit those features: mal, maltose transport across the outer membrane; man, mannose transport across the inner membrane; ompF, glucose and electrolyte transport across the outer membrane; imx, a hypothetical inner membrane feature that is exploited by λ that evolved independence of the mannose permease. The ‘+’ symbol indicates that either the bacteria have the feature or the phage exploit the feature. The ‘–’ symbol indicates that the bacteria lack the feature, express it to a reduced degree, or otherwise modify it to minimize phage infection. Asterisks (*) indicate infectivity for each host–phage pair, with more asterisks indicating greater infectivity. Adaptive changes through the network can proceed by two types of move: E. coli resistance (to the right along rows) and increased λ infectivity (downward along columns). The coevolving communities were founded by host genotype a and phage genotype vi (shown by the black circle). The communities analysed in this study appear to have moved through the shaded nodes in five steps, as indicated by the arrows.