Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli . Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself. Author Summary Temperate bacteriophages (or phages) are bacterial viruses that, unlike virulent phages, have the ability to enter a prophage dormant state upon infection, in which they stably replicate with the bacterial genome. A majority of bacterial genomes contain multiple active or defective prophages, and numerous bacterial phenotypes are modified by these prophages, such as increased virulence. These mobile genetic elements are subject to high levels of genetic exchanges, through which new genes are constantly imported into bacterial genomes. Phage-encoded homologous recombination enzymes, or recombinases, are potentially key actors in phage genome shuffling. In this work, we show that gene acquisition in temperate phages is strongly dependent on the presence of sequence homology, but is highly tolerant to divergence. We report that gene exchanges are mainly catalyzed by recombinases found on temperate phages, and show that four different Rad52-like recombinases have a relaxed fidelity in vivo , compared to RecA. This high capacity of exchange speeds up evolution of phages, and indirectly also the evolution of bacteria.
【초록키워드】 viruses, Evolution, Genome, Genetic, Infection, comparison, Horizontal gene transfer, Recombination, phenotype, bacteriophage, Bacteria, genetic diversity, genomes, in vivo, virulence, genomic, Bioinformatics analysis, mechanism, Bacterial, Evidence, Hypothesis, Homologous recombination, genetic determinants, bacteriophages, Efficiency, Escherichia coli, complementary, evidence of, genetic determinant, temperate phages, temperate phage, phage genome, virulent phages, no evidence of, dormant state, E. coli, Divergence, gene transfer, subject, sequences, sequence, molecular mechanisms, transfer, sequence homology, exchange, functions, acquisition, phage, element, phages, phage λ, Rad52, bacterial viruses, biosphere, gene exchanges, genetic exchanges, lambdoid phages, mosaicism, phage encoded recombinases, prophages, RecA, recombinases, Temperate bacteriophages, virulent, vivo assays, REcombinase, replicate, majority, contribute, dependent on, promote, addressed, catalyzed, horizontal gene, bacterial virus, defective prophages, dormant, gene exchange, homologous recombination enzymes, lambdoid phage, phage encoded recombinase, phage gene, Temperate bacteriophage, 【제목키워드】 DNA, temperate phages, prophage, REcombinase, role,