Pseudomonas phage MD8: genetic mosaicism and challenges of taxonomic classification of lambdoid bacteriophages
Fundamental questions of the evolution of viral genomes are the most important topic of virological research. As a result of the joint work of virologists from the Laboratory of Molecular Bioengineering of the IBCh RAS and the Limnological Institute of the RAS, a group of bacteriophages of the dangerous pathogen Pseudomonas was identified, the genomes of these bacterial viruses were studied, and it was shown that their formation was greatly influenced by multiple horizontal transfers, which led to pronounced genetic mosaicism. Scientists also put forward hypotheses about the origin of the new group and proposed basic principles for the taxonomic classification of lambdoid phages.
The genomics of lambdoid (λ-like) bacteriophages has been the focus of scrutiny of several generations of scientists, since the discovery of phage λ by Esther Lederberg, in 1949 and the subsequent explosive growth of interest in this phage. The identification of genetic mechanisms responsible for the lysogeny decision, the processes of lysis and adsorption have made a huge contribution to understanding the main principles of the nature of phage infection. Phage λ and related phages remain the focus of virology studies dedicated to the molecular details of the infection cycle of phages.
The studies that followed the discovery of phage λ showed the wide range of basic principles that were involved in the genome organisation of this phage among many temperate bacteriophages. Genomic research has demonstrated the high level of genetic mosaicism of λ-like (lambdoid) phages while sharing the same overall gene organisation. This mosaicism can be caused by homologous recombination that happens when a phage infects a cell carrying a prophage with appropriate homologies, or by extensive indiscriminate nonhomologous recombination followed by selection for functional phages. The temperate lifestyle of lambdoid phages and their recombination processes makes them important for bacterial evolution including the adaptation and genomic diversification of bacterial pathogens.
Pseudomonas phage MD8 (Figure 1) is a temperate phage with a λ-like genome, which has been obtained from the freshwater of Lake Baikal in 2005 and 2010. This phage has been sequenced but has yet to be studied comprehensively. The phage infects Pseudomonas aeruginosa, an important opportunistic pathogen that can cause chronic infections leading to significant morbidity and mortality. Recent studies have discovered new temperate bacteriophages infecting P. aeruginosa. Preliminary comparative bioinformatic analysis of the phage MD8 genome has shown the presence of both genes related to previously described lambdoid phages and genes specific to MD8 or other Pseudomonas phages, including possible virulence factors. Analysis has also exposed problems relating to the differentiation and taxonomic classification of Pseudomonas phages related to their genetic mosaicism.
Figure 1. Electron microscopy image of phage MD8. The scale bar is 100 nm
Analysis of phage genomes revealed a group of other Pseudomonas phages related to phage MD8 and the genomic layout of MD8-like phages indicated extensive gene exchange involving even the most conservative proteins and leading to a high degree of genomic mosaicism (Figure 2). The genomes of MD8-like phages encode relatively distant from phage λ proteins, but many of those proteins possess functionality analogous to the phage λ proteins. Multiple horizontal transfers and mosaicism of the genome of MD8, related phages and other λ-like phages raise questions about the principles of taxonomic classification of the representatives of this voluminous phage group.
Figure 2. Genetic map of the Pseudomonas MD8 phage and genomic modules identified by sequence comparison and phylogenetic analysis. Parts of the genomes generally more similar to the F10, JBD68 and φ phages, which belong to the MD8 group, are shown in parentheses on the left. The MD8 genes are coloured corresponding the colours of boxes on the left, which contain the names of not MD8-like phages related to MD8 according to analyses of proteins encoded by these genes
Since the first published report in 1971, updating the taxonomic classification of viruses has been the main task of the International Committee on Taxonomy of Viruses (ICTV). Since 1971, reports containing the results of thorough studies have been published, but several thousand Siphoviridae genomes remain unclassified. Various clustering approaches have been developed for bacteriophage classification, but preliminary studies of MD8 and related phages have shown what appears to be an inconsistency between the results of different techniques.
The current paper introduces the Pseudomonas phage MD8 and discusses the difficulties of evolutionary analysis and taxonomic classification of MD8, related phages and λ-like phages in general. First, the biological and morphological properties of MD8 are described. Next, a database search is made for related phages and related genomes are clustered using different methods. Then, essential genes and corresponding proteins are studied using sequence search, and phylogenetic and structural bioinformatic analysis. After that, related genomes are analysed, proteome and protein phylogenies are constructed, the modules acquired by horizontal transfer are found and the possible origins of the mosaic picture of the MD8 genome are reconstructed. Finally, a discussion of the implications of analyses performed and patterns observed for the purposes of taxonomy and classification of lambdoid bacteriophages is presented (Figure 3). Based on the evolutionary history of the majority of the proteins of MD8-like phages and intergenomic comparisons, it is proposed that the group comprising 26 phages be assigned to two novel genera.
The results are published in the International Journal of Molecular Sciences.
Figure 3. Possible criteria for the taxonomic classification of lambdoid bacteriophages
october 8, 2021