Microviridae Goes Temperate: Microvirus-Related Proviruses Reside in the Genomes of Bacteroidetes

Citation: Krupovic M, Forterre P ( Microviridae Goes Temperate: Microvirus-Related Proviruses Reside in the Genomes of Bacteroidetes Mart Krupovic 0 Patrick Forterre 0 Jianming Qiu, University of Kansas Medical Center, United States of America 0 1 Unite Biologie Mole culaire du Ge`ne chez les Extremophiles, Department of Microbiology, Institut Pasteur, Paris, France, 2 Institut de Ge ne tique et Microbiologie, CNRS- UMR 8621, Universite Paris-Sud 11 , Orsay , France The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated. - Funding: This work was supported by the European Molecular Biology Organization (Long-Term Fellowship ALTF 347-2010 to MK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. A number of ecological studies have revealed that microbial viruses predominate in the biosphere and outnumber their hosts by at least one order of magnitude [1,2]. Due to their abundance and consequent influence on the composition and diversity of microbial communities, viruses can be rightfully considered to be the major players in the global ecosystem [3,4]. Until recently, the majority of viruses in the environment were believed to possess double-stranded DNA genomes [2]. However, technological advances in single-stranded (ss) DNA amplification and sequencing from environmental samples revealed that viruses with ssDNA genomes are more prevalent in both soil and marine environments than previously recognized [58]. This realization precipitated an interest amongst environmental virologists in the diversity and distribution of ssDNA bacterial viruses in nature [7,9]. Among ssDNA viruses that are most often identified in the environment using metagenomic approach are those belonging to the family Microviridae. However, the host organisms have yet to be determined. The Microviridae comprises small isometric icosahedral viruses with circular single-stranded DNA genomes [10]. The members of this family are further divided into two subgroups based on structural and genomic differences. Viruses infecting enterobacteria belong to a genus Microvirus and are typified by microvirus phiX174. The other subgroup consists of viruses infecting obligate parasitic bacteria, such as Chlamydia, Bdellovibrio and Spiroplasma [11]. These viruses are grouped into subfamily Gokushovirinae (genera Chlamydiamicrovirus, Bdellomicrovirus and Spiromicrovirus) (http://www.ictvonline.org). Virions of phiX174-like microviruses are composed of four structural proteins (major capsid protein F, major spike protein G, DNA-binding protein J and DNA pilot protein H) [12]. In contrast, only two structural proteins, homologues of phiX174 proteins F and H, were identified in mature virions of gokushoviruses [13]. Furthermore, virion assembly in phiX174-like microviruses proceeds with the aid of two scaffolding proteins, internal scaffolding protein B and external scaffolding protein D [14]. The latter one does not have an equivalent in gokushoviruses. Consequently, the genomes of gokushoviruses are slightly smaller than those of microviruses (4.5 kb versus 5.36.2 kb). Viruses from both groups replicate their genomes via a rolling-circle (RCR) mechanism and encode dedicated RCR initiation proteins. All characterized members of the Microviridae are strictly lytic, unable to lysogenize their hosts [10]. However, the attempt to induce viruses from marine Synechococcus strains isolated from the Gulf of Mexico resulted in the production of icosahedral non-tailed virus-like particles that contained ssDNA [15], although detailed characterization of the virus-like particles was not performed. Furthermore, genomes of Chlamydophila caviae (formerly Chlamydia psittaci) and Chlamydia pneumoniae contain gene fragments showing sequence similarity to genes of Chlamydia-infecting gokushoviruses [16]. These observations suggest that the Microviridae might include not only lytic but also temperate members, as is the case for all other families of bacterial DNA viruses that possess circular genomes or replicate their genomes via a circular intermediate. Unexplored diversity and abundance of the Microviridae viruses in the environment fuelled our interest in this virus group. In order to obtain more information about these viruses we analyzed the genomic sequences available in public databases for the presence of proviruses related to Microviridae. The rationale behind this approach is that a provirus, defective or not, represents a molecular record that a cell has been in contact with a particular virus [17]. In this study we identified seven proviruses that are related to members of the Microviridae. The proviruses are integrated in the genomes of different species of the order Bacteroidales (phylum Bacteroidetes). The identified proviruses are only distantly related to the previously characterized microviruses and gokushoviruses and may represent a new group or subfamily within the Microviridae. Searches against metagenomic databases suggest that these new viruses might be associated with the human gut microbiota. Our results presented here extend the knowledge on the evolution, diversity and host range of microviruses. Results and Discussion Identification of Microviridae-related proviruses Bacterial and archaeal DNA viruses are often capable of integrating their genomes into the host chromosome thereby becoming proviruses. Even though proviruse (...truncated)