Annika Gillis

Taxonomy of prokaryotic viruses: update from the ICTV bacterial and archaeal viruses subcommittee

The prokaryotic virus community is represented on the International Committee on Taxonomy of Viruses (ICTV) by the Bacterial and Archaeal Viruses Subcommittee. In 2008, the three caudoviral families Myoviridae, Podoviridae, and Siphoviridae included only 18 genera and 36 species. Under the able chairmanship of Rob Lavigne (KU Leuven, Belgium), major advances were made in the classification of prokaryotic viruses and the order Caudovirales was expanded dramatically, to reflect the genome-based relationships between phages. Today, the order includes six subfamilies, 80 genera, and 441 species. This year, additional changes in prokaryotic virus taxonomy have been brought forward under the new subcommittee chair, Andrew M. Kropinski (University of Guelph, Canada). These changes are:

Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

Prevalence, genetic diversity and host range of tectiviruses among members of the Bacillus cereus group

ABSTRACT GIL01, Bam35, GIL16, AP50, and Wip1 are tectiviruses preying on the Bacillus cereus group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive bacterial hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group by assessing their occurrence and genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 isolates were evaluated using primers designed to be specific to two variable regions detected in previously described elements. PCR and propagation tests revealed that tectivirus-like elements occurred in less than 3% of the isolates. Regardless of this limited distribution, several novel tectiviruses were found, and partial DNA sequencing indicated that a greater diversity exists within the family Tectiviridae. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship between the infection patterns of the tectiviruses and their diversity was found.

Identification of five novel tectiviruses in Bacillus strains: analysis of a highly variable region generating genetic diversity

Our biosphere is abundant with unique and small genes for which no homologs are known. These genes, often referred to as orphans or ORFans, are commonly found in bacteriophage genomes but their origins remain unclear. We discovered five novel tectivirus-like genetic elements by screening more than five-hundred Bacillus strains. A highly variable region (HVR) of these viruses was shown to harbor ORFans in most of these otherwise well-conserved bacteriophages. Previous studies demonstrated that mutations close to this region dramatically alter bacteriophage gene regulation, suggesting that the acquisition of those ORFans may provide a source of genetic diversity that is then subject to genetic selection during bacteriophage evolution.

Influence of lysogeny of tectiviruses GIL01 and GIL16 on Bacillus thuringiensis growth, biofilm formation, and swarming motility

ABSTRACT Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages.

Atomic force microscopy: A powerful tool for studying bacterial swarming motility

Swarming motility is a fascinating phenomenon by which some bacteria use flagella to move over solid surfaces. Understanding the molecular mechanisms underlying swarming motility requires studying the factors that induce and control flagella expression in swarming cells. Traditionally, flagella are observed by optical or electron microscopy, but none of these techniques combine versatility and easiness, with quantitative and high-resolution information. We report an atomic force microscopy (AFM)-based approach for the fast imaging of bacterial phenotypes (cell shape, flagella expression) in swarming motility studies. Cells from the gram-positive bacterium Bacillus thuringiensis sv. israelensis were inoculated on energy-rich media containing increasing agar concentrations. Following swarming assays (2 days), the cell morphology and the amount of flagella were directly observed by AFM imaging in air. Consistent with the macroscopic swarming behavior, cells harvested from the rim of colonies spreading on soft agar were hyperflagellated, elongated and arranged in chains. Increasing the agar concentration led to much lower amounts of flagella and to shorter rod-shaped cells, a finding consistent with the slower swarming motility of the cells. Cells taken from colony centers on soft and hard agar surfaces were generally non-flagellated, rod-shaped, rarely arranged in chains, and exhibited lysis and sporulation. This study shows that AFM imaging can readily discriminate between swarming and non-swarming cells, and quantify their morphological details, thus offering an important tool to study the dynamics of bacterial populations.

pXO16 from Bacillus thuringiensis serovar israelensis: almost 350 kb of terra incognita

Bacillus thuringiensis strains usually harbor large sets of plasmids, some of which carrying the entomopathogenic δ-endotoxins. B. thuringiensis serovar israelensis, active on Dipteran larvae, carries the very large conjugative plasmid pXO16 (350 kb). pXO16 displays a macroscopic aggregation phenotype when liquid cultures of conjugative partners are mixed. Its conjugative apparatus is able of transferring itself and other non-conjugative and non-mobilizable plasmids in a fast and very efficient manner. Even though its conjugative kinetics and capabilities have been extensively studied, the genetic bases for this unique transfer system remain largely unknown. In this work, the sequence of pXO16 has been identified in the existing sequenced genome of B. thuringiensis sv. israelensis HD-789 as corresponding to the p01 plasmid. Despite pXO16 sequence being highly coding, few CDS possess homologs in the databases. However, potential regions responsible for the aggregation phenotype and the plasmid replication have been highlighted. The common orientation of all CDS and the presence of a high number of potential paralogs suggested a phage-like nature. Concerning conjugative functions, no significant type IV secretion system homologs have been found, indicating that pXO16 encodes an unforeseen conjugative system.

Detection of the cryptic prophage-like molecule pBtic235 in Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis has long been recognized to carry numerous extrachromosomal molecules. Of particular interest are the strains belonging to the B. thuringiensis subsp. israelensis lineage, as they can harbor at least seven extrachromosomal molecules. One of these elements seems to be a cryptic molecule that may have been disregarded in strains considered plasmid-less. Therefore, this work focused on this cryptic molecule, named pBtic235. Using different approaches that included transposition-tagging, large plasmid gel electrophoresis and Southern blotting, conjugation and phage-induction experiments, in combination with bioinformatics analyses, it was found that pBtic235 is a hybrid molecule of 235,425 bp whose genome displays potential plasmid- and phage-like modules. The sequence of pBtic235 has been identified in all sequenced genomes of B. thuringiensis subsp. israelensis strains. Here, the pBtic235 sequence was considered identical to that of plasmid pBTHD789-2 from strain HD-789. Despite the fact that the pBtic235 genome possesses 240 putative CDSs, many of them have no homologs in the databases. However, CDSs coding for potential proteins involved in replication, genome packaging and virion structure, cell lysis, regulation of lytic-lysogenic cycles, metabolite transporters, stress and metal resistance, were identified. The candidate plasmidial prophage pBtic235 exemplifies the notable diversity of the extrachromosomal realm found in B. thuringiensis.

Genomic, proteomic, and phylogenetic analysis of spounaviruses indicates paraphyly of the order Caudovirales

ABSTRACT It is almost a cliche that tailed bacteriophages of the order Caudovirales are the most abundant and diverse viruses in the world. Yet, their taxonomy still consists of a single order with just three families: Myoviridae, Siphoviridae, and Podoviridae. Thousands of newly discovered phage genomes have recently challenged this morphology-based classification, revealing that tailed bacteriophages are genomically even more diverse than once thought. Here, we evaluate a range of methods for bacteriophage taxonomy by using a particularly challenging group as an example, the Bacillus phage SPO1-related viruses of the myovirid subfamily Spounavirinae. Exhaustive phylogenetic and phylogenomic analyses indicate that the spounavirins are consistent with the taxonomic rank of family and should be divided into at least five subfamilies. This work is a case study for virus genomic taxonomy and the first step in an impending massive reorganization of the tailed bacteriophage taxonomy.Since the mid-20th century, prokaryotic double-stranded DNA viruses producing tailed particles (“tailed phages”) were grouped according to virion tail morphology. In the early 1980s, these viruses were classified into the families Myoviridae, Siphoviridae, and Podoviridae, later included in the order Caudovirales. However, recent massive sequencing of prokaryotic virus genomes revealed that caudovirads are extremely diverse. The official taxonomic framework does not adequately reflect caudovirad evolutionary relationships. Here, we reevaluate the classification of caudovirads using a particularly challenging group of viruses with large dsDNA genomes: SPO1-like viruses associated with the myovirid subfamily Spounavirinae. Our extensive genomic, proteomic, and phylogenetic analyses reveal that some of the currently established caudovirad taxa, especially at the family and subfamily rank, can no longer be supported. Spounavirins alone need to be elevated to family rank and divided into at least five major clades, a first step in an impending massive reorganization of caudovirad taxonomy.

Complete Genome Sequence of Bacillus velezensis CN026 Exhibiting Antagonistic Activity against Gram-Negative Foodborne Pathogens

ABSTRACT We report here the complete genome sequence of Bacillus velezensis strain CN026, a member of the B. subtilis group, which is known for its many industrial applications. The genome contains 3,995,812 bp and displays six gene clusters potentially involved in strain CN026’s activity against Gram-negative foodborne pathogens.