Evelien M. Adriaenssens

T4-Related Bacteriophage LIMEstone Isolates for the Control of Soft Rot on Potato Caused by 'Dickeya solani'

The bacterium ‘Dickeya solani’, an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with ‘Dickeya solani’, the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.

High-level diversity of tailed phages, eukaryote-associated viruses,and virophage-like elements in the metaviromes of Antarctic soils

ABSTRACT The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly Mycobacterium phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (Siphoviridae > Myoviridae > Podoviridae). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed. Phycodnaviridae and Mimiviridae viruses were the second-most-abundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.

Using signature genes as tools to assess environmental viral ecology and diversity.

ABSTRACT Viruses (including bacteriophages) are the most abundant biological entities on the planet. As such, they are thought to have a major impact on all aspects of microbial community structure and function. Despite this critical role in ecosystem processes, the study of virus/phage diversity has lagged far behind parallel studies of the bacterial and eukaryotic kingdoms, largely due to the absence of any universal phylogenetic marker. Here we review the development and use of signature genes to investigate viral diversity, as a viable strategy for data sets of specific virus groups. Genes that have been used include those encoding structural proteins, such as portal protein, major capsid protein, and tail sheath protein, auxiliary metabolism genes, such as psbA, psbB, and phoH, and several polymerase genes. These marker genes have been used in combination with PCR-based fingerprinting and/or sequencing strategies to investigate spatial, temporal, and seasonal variations and diversity in a wide range of habitats.

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:

Metagenomic analysis of the viral community in Namib Desert hypoliths

Hypolithic microbial communities are specialized desert communities inhabiting the underside of translucent rocks. Here, we present the first study of the viral fraction of these communities isolated from the hyperarid Namib Desert. The taxonomic composition of the hypolithic viral communities was investigated and a functional assessment of the sequences determined. Phylotypic analysis showed that bacteriophages belonging to the order Caudovirales, in particular the family Siphoviridae, were most prevalent. Functional analysis and comparison with other metaviromes revealed a relatively high frequency of cell wall-degrading enzymes, ribonucleotide reductases (RNRs) and phage-associated genes. Phylogenetic analyses of terL and phoH marker genes indicated that many of the sequences were novel and distinct from known isolates, and the class distribution of the RNRs suggests that this is a novel environment. The composition of the viral hypolith fraction containing many Bacillus-infecting phages was not completely consistent with Namib hypolith phylotypic surveys of the bacterial hosts, in which the cyanobacterial genus Chroococcidiopsis was found to be dominant. This could be attributed to the lack of sequence information about hypolith viruses/bacteria in public databases or the possibility that hypolithic communities incorporate viruses from the surrounding soil.

CIM(®) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles.

The use of anion-exchange chromatography was investigated as an alternative method to concentrate and purify bacterial viruses, and parameters for different bacteriophages were compared. Chromatography was performed with Convective Interactive Media(®) monoliths, with three different volumes and two matrix chemistries. Eleven morphologically distinct phages were tested, infecting five different bacterial species. For each of the phages tested, a protocol was optimized, including the choice of column chemistry, loading, buffer and elution conditions. The capacity and recovery of the phages on the columns varied considerably between phages. We conclude that anion-exchange chromatography with monoliths is a valid alternative to the more traditional CsCl purification, has upscaling advantages, but it requires more extensive optimization.

A suggested new bacteriophage genus: “Viunalikevirus”

We suggest a bacteriophage genus, “Viunalikevirus”, as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.

Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, Belonging to the "phiKMV-Like Viruses"†

ABSTRACT Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the “phiKMV-like viruses.” Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the “phiKMV-like viruses” and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the “phiKMV-like viruses.”

Diversity and Ecology of Viruses in Hyperarid Desert Soils

ABSTRACT In recent years, remarkable progress has been made in the field of virus environmental ecology. In marine ecosystems, for example, viruses are now thought to play pivotal roles in the biogeochemical cycling of nutrients and to be mediators of microbial evolution through horizontal gene transfer. The diversity and ecology of viruses in soils are poorly understood, but evidence supports the view that the diversity and ecology of viruses in soils differ substantially from those in aquatic systems. Desert biomes cover ∼33% of global land masses, and yet the diversity and roles of viruses in these dominant ecosystems remain poorly understood. There is evidence that hot hyperarid desert soils are characterized by high levels of bacterial lysogens and low extracellular virus counts. In contrast, cold desert soils contain high extracellular virus titers. We suggest that the prevalence of microbial biofilms in hyperarid soils, combined with extreme thermal regimens, exerts strong selection pressures on both temperate and virulent viruses. Many desert soil virus sequences show low values of identity to virus genomes in public databases, suggesting the existence of distinct and as-yet-uncharacterized soil phylogenetic lineages (e.g., cyanophages). We strongly advocate for amplification-free metavirome analyses while encouraging the classical isolation of phages from dominant and culturable microbial isolates in order to populate sequence databases. This review provides an overview of recent advances in the study of viruses in hyperarid soils and of the factors that contribute to viral abundance and diversity in hot and cold deserts and offers technical recommendations for future studies.

Integration of genomic and proteomic analyses in the classification of the Siphoviridae family

Using a variety of genomic (BLASTN, ClustalW) and proteomic (Phage Proteomic Tree, CoreGenes) tools we have tackled the taxonomic status of members of the largest bacteriophage family, the Siphoviridae. In all over 400 phages were examined and we were able to propose 39 new genera, comprising 216 phage species, and add 62 species to two previously defined genera (Phic3unalikevirus; L5likevirus) grouping, in total, 390 fully sequenced phage isolates. Many of the remainders are orphans which the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) chooses not to ascribe genus status at the time being.