Compensatory evolution for a gene deletion is not limited to its immediate functional network

Abstract

Background: Genetic disruption of an important phenotype should favor compensatory mutations that restore the phenotype. If the genetic basis of the phenotype is modular, with a network of interacting genes whose functions are specific to that phenotype, compensatory mutations are expected among the genes of the affected network. This perspective was tested in the bacteriophage T3 using a genome deleted of its DNA ligase gene, disrupting DNA metabolism.

Results: In two replicate, long-term adaptations, phage compensatory evolution accommodated the low ligase level provided by the host without reinventing its own ligase. In both lines, fitness increased substantially but remained well below that of the intact genome. Each line accumulated over a dozen compensating mutations during long-term adaptation, and as expected, many of the compensatory changes were within the DNA metabolism network. However, several compensatory changes were outside the network and defy any role in DNA metabolism or biochemical connection to the disruption. In one line, these extra-network changes were essential to the recovery. The genes experiencing compensatory changes were moderately conserved between T3 and its relative T7 (25% diverged), but the involvement of extra-network changes was greater in T3.

Conclusion: Compensatory evolution was only partly limited to the known functionally interacting partners of the deleted gene. Thus gene interactions contributing to fitness were more extensive than suggested by the functional properties currently ascribed to the genes. Compensatory evolution offers an easy method of discovering genome interactions among specific elements that does not rest on an a priori knowledge of those elements or their interactions.

Figure 1

Fitness evolution of T3 deleted for the ligase gene and adapted to the ligase-defective host (points A, B) or to the ligase-normal host (C). Final fitness equals initial fitness for the oblique line, thus the vertical distance from the oblique line to the labeled data point represents the total improvement during adaptation. Adaptation of A was initiated from T3Δ1.3A₀, adaptations of B and C from T3Δ1.3B₀. Initial fitness of T3Δ1.3B₀ was negative on the ligase-defective host, so the phage could not initially maintain its numbers and had to be propagated initially on plates for the B adaptation. In contrast, initial fitness of T3Δ1.3A₀ on the ligase normal host was over 20 doublings/hr. Initial fitness of T3Δ1.3A₀ was higher, due to the acquisition of 5 changes by the first step at which the isolate could be identified as carrying the deletion. Final fitnesses of all three were substantially improved over initial. However, final fitnesses of A and B fell short of the fitness limit (28.9) of the ligase+ T3 adapted to the same host [designated limit (A, B)]. Adaptation of T3Δ1.3B₀ to the ligase-normal host (point C) resulted in a final fitness approaching the presumed fitness limit. Standard errors in both the vertical and horizontal dimensions are indicated, often obscured by the symbols.

Figure 2

Genome organization and location of mutations in T3 and T7. The identities and order of all essential genes and most non-essential genes are the same in both phages. Genes shown in blue function in DNA metabolism (dark blue indicates major roles, light blue lesser roles). Genes in red encode virion proteins (the light red gene is for scaffolding, absent in the mature virion). The three internal core genes have unique functions and identities. Genes shown in gray have other functions or their functions are not known. Asterisks are shown above the genes in which compensatory substitutions were observed in either T3 adaptation and shown below the genes of T7 that experienced compensatory evolution. Overlapping genes are offset (the overlap of helicase and primase and the overlap of the two forms of the capsid gene are each shown as a single gene).

Figure 3

Genome network of phages T3 and T7. Genes (ovals) are identified with partial names or numbers; the only gene shown that is not found in both phages is 1.05, found only in T3. Light blue genes are those that evolved compensatory changes for ligase deletion in either T7 or T3; dark blue genes evolved compensatory changes in both T7 and at least one line of T3. Solid lines indicate direct contacts known from biochemistry, contacts inferred from yeast-2-hybrid data, or contacts inferred from known associations. For example, the three core proteins are found inside the phage head, and it is not known which of them contact each other and which contact other head proteins. Dashed lines indicate known functional interactions for two discrete phenotypes (DNA metabolism, lysis). The phage RNAP obviously interacts functionally with most of these genes through its expression of them, and those interactions are not shown. Many non-essential genes are omitted; the few listed with no connections are non-essential under lab growth conditions but evolved compensatory changes. This network represents the state of knowledge for T7 (and thus T3), but the T7 network has not been extensively explored, so this network should be acknowledged as incomplete. Furthermore, there is yet no structure of the T3 or T7 virion that reveals the locations of gp6.7, gp7.3, or gp13 nor of the relative locations of tail A to tail B, so many of the connections shown here for those proteins have been assigned by relatively weak inference. Sources include [5,8-14]. Gene numbers and functions are given in Additional file 1. Abbreviations (gene name, number): Lys (lysozyme, 3.5), Holin (17.5), RNAP (RNA polymerase, 1), DNAP (DNA polymerase, 5), Tnase (terminase, 19), Scaffold (9), Capsid (major/minor capsid, 10A, 10B), T Fiber (tail fiber, 17), Tail A (11), Tail B (12), Endo (endonuclease, 3), Exo (exonuclease, 6), H/P (helicase/primase, 4A, 4B), ssB (ss DNA binding protein, 2.5).