Twenty Years of Collaboration to Sort out Phage Mu Replication and Its Dependence on the Mu Central Gyrase Binding Site.

For 20 years, the intricacies in bacteriophage Mu replication and its regulation were elucidated in collaboration between Ariane Toussaint and her co-workers in the Laboratory of Genetics at the Université Libre de Bruxelles, and the groups of Martin Pato and N. Patrick Higgins in the US. Here, to honor Martin Pato's scientific passion and rigor, we tell the history of this long-term sharing of results, ideas and experiments between the three groups, and Martin's final discovery of a very unexpected step in the initiation of Mu replication, the joining of Mu DNA ends separated by 38 kB with the assistance of the host DNA gyrase.

Transposable Prophages in Leptospira: An Ancient, Now Diverse, Group Predominant in Causative Agents of Weil's Disease.

The virome associated with the corkscrew shaped bacterium Leptospira, responsible for Weil's disease, is scarcely known, and genetic tools available for these bacteria remain limited. To reduce these two issues, potential transposable prophages were searched in Leptospiraceae genomes. The 236 predicted transposable prophages were particularly abundant in the most pathogenic leptospiral clade, being potentially involved in the acquisition of virulent traits. According to genomic similarities and phylogenies, these prophages are distantly related to known transposable phages and are organized into six groups, one of them encompassing prophages with unusual TA-TA ends. Interestingly, structural and transposition proteins reconstruct different relationships between groups, suggesting ancestral recombinations. Based on the baseplate phylogeny, two large clades emerge, with specific gene-contents and high sequence divergence reflecting their ancient origin. Despite their high divergence, the size and overall genomic organization of all prophages are very conserved, a testimony to the highly constrained nature of their genomes. Finally, similarities between these prophages and the three known non-transposable phages infecting L. biflexa, suggest gene transfer between different Caudovirales inside their leptospiral host, and the possibility to use some of the transposable prophages in that model strain.

PHROG: families of prokaryotic virus proteins clustered using remote homology.

Viruses are abundant, diverse and ancestral biological entities. Their diversity is high, both in terms of the number of different protein families encountered and in the sequence heterogeneity of each protein family. The recent increase in sequenced viral genomes constitutes a great opportunity to gain new insights into this diversity and consequently urges the development of annotation resources to help functional and comparative analysis. Here, we introduce PHROG (Prokaryotic Virus Remote Homologous Groups), a library of viral protein families generated using a new clustering approach based on remote homology detection by HMM profile-profile comparisons. Considering 17 473 reference (pro)viruses of prokaryotes, 868 340 of the total 938 864 proteins were grouped into 38 880 clusters that proved to be a 2-fold deeper clustering than using a classical strategy based on BLAST-like similarity searches, and yet to remain homogeneous. Manual inspection of similarities to various reference sequence databases led to the annotation of 5108 clusters (containing 50.6 % of the total protein dataset) with 705 different annotation terms, included in 9 functional categories, specifically designed for viruses. Hopefully, PHROG will be a useful tool to better annotate future prokaryotic viral sequences thus helping the scientific community to better understand the evolution and ecology of these entities.

Extension of the transposable bacterial virus family: two genomic organisations among phages and prophages with a Tn552-related transposase.

Mu-like transposable phages and prophages have been isolated from, or predicted, in a wide range of bacterial phyla. However, related B3-like transposable phages, which differ in their genome organisation and the DDE transposase and transposition activator they code for have thus far been restricted to a very limited set of hosts. Through sequence similarity searches, we have now expanded the number of predicted B3-like prophages and uncovered a third genomic organisation. These new genomes, although only prophages, further illustrate the previously reported mosaicism existing in the proposed "Saltoviridae" family of Caudovirales, and further support the proposal to move morphology criteria (contractile vs. flexible or short tail, i.e. Myo-vs. Sipho- or Podoviridae) from the family to the subfamily level in the taxonomic classification of the Caudovirales.

Transposable Bacteriophages as Genetic Tools.

Phage Mu is the paradigm of a growing family of bacteriophages that infect a wide range of bacterial species and replicate their genome by replicative transposition. This molecular process, which is used by other mobile genetic elements to move within genomes, involves the profound rearrangement of the host genome [chromosome(s) and plasmid(s)] and can be exploited for the genetic analysis of the host bacteria and the in vivo cloning of host genes. In this chapter we review Mu-derived constructs that optimize the phage as a series of genetic tools that could inspire the development of similarly efficient tools from other transposable phages for a large spectrum of bacteria.

Bacterial Virus Ontology; Coordinating across Databases.

Bacterial viruses, also called bacteriophages, display a great genetic diversity and utilize unique processes for infecting and reproducing within a host cell. All these processes were investigated and indexed in the ViralZone knowledge base. To facilitate standardizing data, a simple ontology of viral life-cycle terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address unique viral replication cycle processes, and existing terminology was modified and adapted. Classically, the viral life-cycle is described by schematic pictures. Using this ontology, it can be represented by a combination of successive events: entry, latency, transcription/replication, host-virus interactions and virus release. Each of these parts is broken down into discrete steps. For example enterobacteria phage lambda entry is broken down in: viral attachment to host adhesion receptor, viral attachment to host entry receptor, viral genome ejection and viral genome circularization. To demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases.

Transposable phages, DNA reorganization and transfer.

Transposable bacteriophages have long been known to necessarily and randomly integrate their DNA in their host genome, where they amplify by successive rounds of replicative transposition, profoundly reorganizing that genome. As a result of such transposition, a conjugative element (plasmid or genomic island), can either become integrated in the chromosome or receive chromosome segments, which can then be transferred to new hosts by conjugation. In recent years, more and more transposable phages have been isolated or detected by sequence similarity searches in a wide range of bacteria, supporting the idea that this mode of HGT may be pervasive in natural bacterial populations.

A structured annotation frame for the transposable phages: a new proposed family "Saltoviridae" within the Caudovirales.

Enterobacteriophage Mu is the best studied and paradigm member of the transposable phages. Mu-encoded proteins have been annotated in detail in UniProtKB and linked to a controlled vocabulary describing the various steps involved in the phage lytic and lysogenic cycles. Transposable phages are ubiquitous temperate bacterial viruses with a dsDNA linear genome. Twenty-six of them, that infect α, ß and γ-proteobacteria, have been sequenced. Their conserved properties are described. Based on these characteristics, we propose a reorganization of the Caudovirales, to allow for the inclusion of a "Saltoviridae" family and two newly proposed subfamilies, the "Myosaltovirinae" and "Siphosaltovirinae". The latter could temporarily be included in the existing Myoviridae and Siphoviridae families.

Transposable Mu-like phages in Firmicutes: new instances of divergence generating retroelements.

Sequences of the most conserved proteins encoded by transposable (pro)phages were used to search recently sequenced Firmicute genomes for candidate transposable prophages. One complete Mu-like prophage, SglyMu-1, was identified, in four copies, in the Syntrophobotulus glycolicus DSM 8271 chromosome sequence. Related prophages were also found in partially assembled genomic sequences of other Firmicutes and newly sequenced Proteobacteria genomes, opening the road to the use of Mu-like derived genetic tools in Gram(+) bacteria. SglyMu-1 appears to carry a host variation system related to the DGR tropism switching retroelements, first characterized in Bordetella phages BPP-1, BIP-1 and BMP-1. Transposable phages are thus thriving among Firmicutes and can harness either of two host variation systems, the fiber genes inversion and reverse transcriptase-mediated site-directed, adenine-specific mutagenesis.

Towards a more accurate annotation of tyrosine-based site-specific recombinases in bacterial genomes.

BACKGROUND: Tyrosine-based site-specific recombinases (TBSSRs) are DNA breaking-rejoining enzymes. In bacterial genomes, they play a major role in the comings and goings of mobile genetic elements (MGEs), such as temperate phage genomes, integrated conjugative elements (ICEs) or integron cassettes. TBSSRs are also involved in the segregation of plasmids and chromosomes, the resolution of plasmid dimers and of co-integrates resulting from the replicative transposition of transposons. With the aim of improving the annotation of TBSSR genes in genomic sequences and databases, which so far is far from robust, we built a set of over 1,300 TBSSR protein sequences tagged with their genome of origin. We organized them in families to investigate: i) whether TBSSRs tend to be more conserved within than between classes of MGE types and ii) whether the (sub)families may help in understanding more about the function of TBSSRs associated in tandem or trios on plasmids and chromosomes. RESULTS: A total of 67% of the TBSSRs in our set are MGE type specific. We define a new class of actinobacterial transposons, related to Tn554, containing one abnormally long TBSSR and one of typical size, and we further characterize numerous TBSSRs trios present in plasmids and chromosomes of α- and ß-proteobacteria. CONCLUSIONS: The simple in silico procedure described here, which uses a set of reference TBSSRs from defined MGE types, could contribute to greatly improve the annotation of tyrosine-based site-specific recombinases in plasmid, (pro)phage and other integrated MGE genomes. It also reveals TBSSRs families whose distribution among bacterial taxa suggests they mediate lateral gene transfer.

Bacterial molecular networks: bridging the gap between functional genomics and dynamical modelling.

This introductory review synthesizes the contents of the volume Bacterial Molecular Networks of the series Methods in Molecular Biology. This volume gathers 9 reviews and 16 method chapters describing computational protocols for the analysis of metabolic pathways, protein interaction networks, and regulatory networks. Each protocol is documented by concrete case studies dedicated to model bacteria or interacting populations. Altogether, the chapters provide a representative overview of state-of-the-art methods for data integration and retrieval, network visualization, graph analysis, and dynamical modelling.

Prokaryote genome fluidity: toward a system approach of the mobilome.

The importance of horizontal/lateral gene transfer (LGT) in shaping the genomes of prokaryotic organisms has been recognized in recent years as a result of analysis of the increasing number of available genome sequences. LGT is largely due to the transfer and recombination activities of mobile genetic elements (MGEs). Bacterial and archaeal genomes are mosaics of vertically and horizontally transmitted DNA segments. This generates reticulate relationships between members of the prokaryotic world that are better represented by networks than by "classical" phylogenetic trees. In this review we summarize the nature and activities of MGEs, and the problems that presently limit their analysis on a large scale. We propose routes to improve their annotation in the flow of genomic and metagenomic sequences that currently exist and those that become available. We describe network analysis of evolutionary relationships among some MGE categories and sketch out possible developments of this type of approach to get more insight into the role of the mobilome in bacterial adaptation and evolution.

A modular view of the bacteriophage genomic space: identification of host and lifestyle marker modules.

Bacteriophage genomes can be regarded as an ensemble of modules which are accessible to the whole phage population via recombination. The time spent by prophages in the bacterial host provides them with the opportunity to exchange modules with other prophages or infecting phages. Here we analyze the modular structure of a set of 457 phages and 760 prophages extracted from completely sequenced bacterial genomes using the ACLAME database and its associated tools. We identified 91 modules of proteins with similar phylogenetic profiles. Of these, 25 and 6 are associated with temperate and virulent phages, respectively; 57 are restricted to a host or small group of hosts; and 55 could be annotated with a phage function. We use the transposable phages as a study case and show how the inclusion of prophages allows us to unveil new types of genome organization (i.e. novel module combinations) and obtain insight into the host range for this particular group, highlighting the utility of prophage prediction to better characterize phage diversity.

Characterization of a novel temperate phage originating from a cereulide-producing Bacillus cereus strain.

A novel temperate bacteriophage was isolated from a Bacillus cereus cereulide-producing strain and named vB_BceS-IEBH. vB_BceS-IEBH belongs to the Siphoviridae family. The complete genome sequence (53 kb) was determined and annotated. Eighty-seven ORFs were detected and for 28, a putative function was assigned using the ACLAME database. vB_BceS-IEBH replicates as a plasmid in the prophage state. Accordingly, a 9-kb plasmid-like region composed of 13 ORFs was identified. A fragment of around 2000 bp comprising an ORF encoding a putative plasmid replication protein was shown to be self-replicating in Bacillus thuringiensis. Mass spectrometry analysis of the purified vB_BceS-IEBH particle identified 8 structural proteins and enabled assignment of a supplementary ORF as being part of the morphogenesis module. Genome analysis further illustrates the diversity of mobile genetic elements and their plasticity within the B. cereus group.

Genome sequence of the plant-pathogenic bacterium Dickeya dadantii 3937.

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.

ACLAME: a CLAssification of Mobile genetic Elements, update 2010.

The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobile genetic elements (MGEs, in particular phages and plasmids). In addition to providing information on the MGEs content, classifications are available at various levels of organization. At the gene/protein level, families group similar sequences that are expected to share the same function. Families of four or more proteins are manually assigned with a functional annotation using the GeneOntology and the locally developed ontology MeGO dedicated to MGEs. At the genome level, evolutionary cohesive modules group sets of protein families shared among MGEs. At the population level, networks display the reticulate evolutionary relationships among MGEs. To increase the coverage of the phage sequence space, ACLAME version 0.4 incorporates 760 high-quality predicted prophages selected from the Prophinder database. Most of the data can be downloaded from the freely accessible ACLAME web site (http://aclame.ulb.ac.be). The BLAST interface for querying the database has been extended and numerous tools for in-depth analysis of the results have been added.

Prophinder: a computational tool for prophage prediction in prokaryotic genomes.

UNLABELLED: Prophinder is a prophage prediction tool coupled with a prediction database, a web server and web service. Predicted prophages will help to fill the gaps in the current sparse phage sequence space, which should cover an estimated 100 million species. Systematic and reliable predictions will enable further studies of prophages contribution to the bacteriophage gene pool and to better understand gene shuffling between prophages and phages infecting the same host. AVAILABILITY: Softare is available at http://aclame.ulb.ac.be/prophinder

Reticulate representation of evolutionary and functional relationships between phage genomes.

Bacteriophage genomes show pervasive mosaicism, indicating the importance of horizontal gene exchange in their evolution. Phage genomes represent unique combinations of modules, each of them with a different phylogenetic history. The traditional classification, based on a variety of criteria such as nucleic acid type (single/double-stranded DNA/RNA), morphology, and host range, appeared inconsistent with sequence analyses. With the genomic era, an ever increasing number of sequenced phages cannot be classified, in part due to a lack of morphological information and in part to the intrinsic incapability of tree-based methods to efficiently deal with mosaicism. This problem led some virologists to call for a moratorium on the creation of additional taxa in the order Caudovirales, in order to let virologists discuss classification schemes that might better suit phage evolution. In this context, we propose a framework for a reticulate classification of phages based on gene content. Starting from gene families, we built a weighted graph, where nodes represent phages and edges represent phage-phage similarities in terms of shared genes. We then apply various measures of graph topology to analyze the resulting graph. Most double-stranded DNA phages are found in a single component. The values of the clustering coefficient and closeness distinguish temperate from virulent phages, whereas chimeric phages are characterized by a high betweenness coefficient. We apply a 2-step clustering method to this graph to generate a reticulate classification of phages: Each phage is associated with a membership vector, which quantitatively characterizes its membership to the set of clusters. Furthermore, we cluster genes based on their "phylogenetic profiles" to define "evolutionary cohesive modules." In virulent phages, evolutionary modules span several functional categories, whereas in temperate phages they correspond better to functional modules. Moreover, despite the fact that modules only cover a fraction of all phage genes, phage groups can be distinguished by their different combination of modules, serving the bases for a higher level reticulate classification. These 2 classification schemes provide an automatic and dynamic way of representing the relationships within the phage population and can be extended to include newly sequenced phage genomes, as well as other types of genetic elements.