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Darin R. Rokyta - One of the best experts on this subject based on the ideXlab platform.
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Distributed under Creative Commons CC-BY 4.0 Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant
2020Co-Authors: Victoria M. Pearson, Brian S Caudle, Darin R. RokytaAbstract:ABSTRACT Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such as Circoviridae, Geminiviridae, and Microviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group
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Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant
PeerJ, 2016Co-Authors: Victoria M. Pearson, S. Brian Caudle, Darin R. RokytaAbstract:Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such as Circoviridae, Geminiviridae, and Microviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.
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Intergenic incompatibilities reduce fitness in hybrids of extremely closely related bacteriophages
PeerJ Inc., 2015Co-Authors: Andrew M. Sackman, Danielle Reed, Darin R. RokytaAbstract:Horizontal gene transfer and recombination occur across many groups of viruses and play key roles in important viral processes such as host-range expansion and immune-system avoidance. To have any predictive power regarding the ability of viruses to readily recombine, we must determine the extent to which epistasis restricts the success of recombinants, particularly as it relates to the genetic divergence between parental strains. In any hybridization event, the evolutionary success or failure of hybrids is largely determined by the pervasiveness of epistasis in the parental genomes. Recombination has previously been shown to incur steep fitness costs in highly divergent viruses as a result of disrupted epistatic interactions. We used a pair of bacteriophages of the family Microviridae to demonstrate that epistasis may evidence itself in the form of fitness costs even in the case of the exchange of alleles at a locus with amino acid divergence as low as 1%. We explored a possible biophysical source of epistasis in the interaction of viral coat and scaffolding proteins and examined a recovery mutation that likely repairs interactions disrupted by recombination
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horizontal gene transfer and the evolution of microvirid coliphage genomes
Journal of Bacteriology, 2006Co-Authors: Darin R. Rokyta, Christina L Burch, S B Caudle, Holly A WichmanAbstract:ABSTRACT Bacteriophage genomic evolution has been largely characterized by rampant, promiscuous horizontal gene transfer involving both homologous and nonhomologous source DNA. This pattern has emerged through study of the tailed double-stranded DNA (dsDNA) phages and is based upon a sparse sampling of the enormous diversity of these phages. The single-stranded DNA phages of the family Microviridae, including φX174, appear to evolve through qualitatively different mechanisms, possibly as result of their strictly lytic lifestyle and small genome size. However, this apparent difference could reflect merely a dearth of relevant data. We sought to characterize the forces that contributed to the molecular evolution of the Microviridae and to examine the genetic structure of this single family of bacteriophage by sequencing the genomes of microvirid phage isolated on a single bacterial host. Microvirids comprised 3.5% of the detectable phage in our environmental samples, and sequencing yielded 42 new microvirid genomes. Phylogenetic analysis of the genes contained in these and five previously described microvirid phages identified three distinct clades and revealed at least two horizontal transfer events between clades. All members of one clade have a block of five putative genes that are not present in any member of the other two clades. Our data indicate that horizontal transfer does contribute to the evolution of the microvirids but is both quantitatively and qualitatively different from what has been observed for the dsDNA phages.
Bentley A. Fane - One of the best experts on this subject based on the ideXlab platform.
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Cross-functional Analysis of the Microviridae Internal Scaffolding Protein
2020Co-Authors: April D Burch, Bentley A. FaneAbstract:The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (ùX174, G4 and a3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70 % in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently¯exible. There was one condition in which a B protein could not cross-function. The ùX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 C. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the ®rst B protein mediated reaction. Two G4 mutants, which can productively utilize the ùX174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more ef®cient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region
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the Microviridae diversity assembly and experimental evolution
Virology, 2016Co-Authors: Sarah M Doore, Bentley A. FaneAbstract:The Microviridae, comprised of ssDNA, icosahedral bacteriophages, are a model system for studying morphogenesis and the evolution of assembly. Historically limited to the φX174-like viruses, recent results demonstrate that this richly diverse family is broadly divided into two groups. The defining feature appears to be whether one or two scaffolding proteins are required for assembly. The single-scaffolding systems contain an internal scaffolding protein, similar to many dsDNA viruses, and have a more complex coat protein fold. The two-scaffolding protein systems (φX174-like) encode an internal and external species, as well as an additional structural protein: a spike on the icosahedral vertices. Here, we discuss recent in silico and in vivo evolutionary analyses conducted with chimeric viruses and/or chimeric proteins. The results suggest 1) how double scaffolding systems can evolve into single and triple scaffolding systems; and 2) how assembly is the critical factor governing adaptation and the maintenance of species boundaries.
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The fX174 Protein J Mediates DNA Packaging and Viral Attachment to Host Cells
2014Co-Authors: Ricardo A. Bernal, Bentley A. Fane, Susan Hafenstein, Raquel Esmeralda, Michael G. RossmannAbstract:Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages fX174, G4, and a3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The fX174 DNA-binding protein, J, is 13 amino acid residues longer than the a3 and G4 J proteins by virtue of an additional nucleic acidbinding domain at the amino terminus. Chimeric fX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric a3 and G4 phages, containing the fX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein
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Structural Studies of Bacteriophage a3 Assembly
2014Co-Authors: Ricardo A. Bernal, Bentley A. Fane, Susan Hafenstein, Paul R Chipman, Timothy S Baker, Norman H. Olson, Valorie D. Bowman, Michael G. RossmannAbstract:Bacteriophage a3 is a member of the Microviridae, a family of small, singlestranded, icosahedral phages that include fX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108 S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The a3 “open ” procapsid structural intermediate was determined to 15 A ˚ resolution by cryo-electron microscopy (cryo-EM). Unlike the fX174 “closed ” procapsid and the infectious virion, the a3 open procapsid has 30 A ˚ wide pores at the 3-fold vertices and 20 A ˚ wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobi
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Microviridae microviruses and gokushoviruses
eLS, 2011Co-Authors: James E Cherwa, Bentley A. FaneAbstract:Members of the Microviridae comprise two subfamilies. The microviruses (Greek for small), which infect free-living bacteria, are among the fastest known replicating viruses. Gokushoviruses (Japanese for very small) occupy a unique niche, infecting obligate intracellular bacteria, such as Chlamydia and Bdellovibrio, or mollicutes, bacteria without a cell wall. All members of the family contain small (4000–6000 bases), circular, single-stranded deoxyribonucleic acid (ssDNA) genomes of positive polarity, which are packaged inside small (∼25 nm diameter) T=1 icosahedral capsids. The other icosahedral, ssDNA virus families: Parvoviridae, Circoviridae, Nanoviridae and Geminiviridae; share most of these properties, suggesting a large super family spanning several domains of life. The most well known member of the Microviridae, ϕX174, has been extensively used to study the fundamental mechanisms of DNA replication and capsid assembly. The latter is uniquely dependent on two scaffolding proteins, and has become a model system for experimental evolution. Key Concepts: Whilst overlapping reading frames increase the amount of genetic information encoded in small genomes, they do not appear to significantly impact the ability of the virus to genetically adapt to selective pressures. Due to the genome's positive polarity, DNA replication must commence before viral genes can be transcribed. Microvirus DNA replication occurs in three distinct stages: (1) ssDNA is first converted to a double-stranded molecule, (2) amplification of the double-stranded molecule, (3) single-stranded genomic DNA synthesis and packaging. Genomic DNA synthesis and packaging are concurrent processes; thus, a genome is not synthesised unless there exists a capsid in which to package it. Gene expression is controlled by the finely tuned interplay of cis-acting genetic elements: promoters, ribosome binding sites, mRNA stability sequences and transcription terminators. Microviruses are distinguished by their two scaffolding protein system, whereas Gokushoviruses utilise a single scaffolding protein. Capsid assembly is mediated by scaffolding proteins, which induce conformational switches in the viral coat protein to control the timing and fidelity of morphogenesis. Cell lysis is achieved by inhibiting host cell wall biosynthesis, a mechanism reminiscent of some antibiotics. Keywords: bacteriophage; icosahedral; ssDNA; atomic structure; scaffolding protein; procapsid
Mart Krupovic - One of the best experts on this subject based on the ideXlab platform.
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Evolution and Diversity of the Microviridae Viral Family through a Collection of 81 New Complete Genomes Assembled from Virome Reads
2014Co-Authors: Simon Roux, Mart Krupovic, Axel Poulet, Didier Debroas, François EnaultAbstract:Recent studies suggest that members of the Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three subfamilies described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an importan
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Networks of evolutionary interactions underlying the polyphyletic origin of ssDNA viruses
Current Opinion in Virology, 2013Co-Authors: Mart KrupovicAbstract:Viruses with single-stranded (ss) DNA genomes infect hosts from all three domains of life and are present in all imaginable environments. Many new ssDNA viruses have been recently isolated, including those infecting algae, fungi, insects and even archaea. In parallel, culture-independent metagenomic approaches have illuminated the tremendous genetic diversity of these viruses, yielding valuable insights into their evolution. Here, I integrate this knowledge to propose a scenario in which certain groups of ssDNA viruses (including Geminiviridae, Circoviridae, Parvoviridae and Microviridae) have originated from plasmids via acquisition of jelly-roll capsid protein genes from ssRNA viruses. This scenario places structurally related viruses with DNA and RNA genomes into an evolutionary continuum and highlights general evolutionary trends in the virosphere.
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Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads.
PLoS ONE, 2012Co-Authors: Simon Roux, Mart Krupovic, Axel Poulet, Didier Debroas, François EnaultAbstract:Recent studies suggest that members of the Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three sub-families described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an important role in these ecosystems and an ancient origin.
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Major capsid protein (MCP) variation within the Microviridae.
2012Co-Authors: Simon Roux, Mart Krupovic, Axel Poulet, Didier Debroas, François EnaultAbstract:(A) Three-dimensional model of the SpV4 virion (PDB ID:1KVP). Three capsomers donating long insertion loops to form the ‘mushroom-like’ protrusions at the three-fold axes of symmetry of the icosahedral capsid are highlighted in blue, green, and red. (B) A boxplot illustrating the variation of MCP sizes between the four subgroups of the Microviridae. (C) Three-dimensional models of the MCPs from viruses representing the four subgroups of the Microviridae: Microvirus (ΦX174 protein F; PDB ID:1CD3), Gokushovirinae (SpV4 VP1, PDB ID:1KVP), Alpavirinae (Prevotella bucalis prophage BMV5 protein VP1; GI:282877220), Pichovirinae (Pavin_279 protein VP1). The insertions within the VP1 proteins of gokusho-, alpa- and pichoviruses relative to the F protein of ΦX174 are highlighted in green.
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Abundance and distribution of VP1-like sequences in the environment.
2012Co-Authors: Simon Roux, Mart Krupovic, Axel Poulet, Didier Debroas, François EnaultAbstract:The number of viromes and the origin of the samples used for virome preparation are indicated. VP1 sequences were affiliated by best BLAST hit against a database including VP1 sequences from both the previously published Microviridae genomes and the complete genomes assembled in this study.
Nianzhi Jiao - One of the best experts on this subject based on the ideXlab platform.
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A Virus Infecting Marine Photoheterotrophic Alphaproteobacteria (Citromicrobium spp.) Defines a New Lineage of ssDNA Viruses.
Frontiers in microbiology, 2018Co-Authors: Qiang Zheng, Qi Chen, Curtis A. Suttle, Nianzhi JiaoAbstract:In recent metagenomic studies, single-stranded DNA (ssDNA) viruses that infect bacteria have been shown to be diverse and prevalent in the ocean; however, there are few isolates of marine ssDNA phages. Here, we report on a cultivated ssDNA phage (vB_Cib_ssDNA_P1) that infects Citromicrobium bathyomarinum RCC1878 (family Sphingomonadaceae), and other members of the genus. This is the first ssDNA phage reported to infect marine alphaproteobacteria, and represents a newly recognized lineage of the Microviridae infecting members of Sphingomonadaceae, the Amoyvirinae. The ~26 nm diameter polyhedral capsid contains a 4,360 bp genome with 6 open reading frames (ORFs) and a 59.3% G+C content. ORF1 encodes the capsid protein and ORF3 encodes the replication initiator protein. The replication cycle is ~5 hours, followed by a burst releasing about 180 infectious particles. The closest relative of vB_Cib_ssDNA_P1 is a prophage within the genome of Novosphingobium tardaugens strain NBRC16725. Phylogenetic analysis indicates that the vB_Cib_ssDNA_P1 phage and two related prophages, as well as an environmental sequence, form a novel group within the Microviridae. Our results indicate that this is a previously unknown lineage of ssDNA viruses which also supplies a new model system for studying interactions between ssDNA phages and marine bacteria.
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Image_2_A Virus Infecting Marine Photoheterotrophic Alphaproteobacteria (Citromicrobium spp.) Defines a New Lineage of ssDNA Viruses.TIF
2018Co-Authors: Qiang Zheng, Qi Chen, Curtis A. Suttle, Nianzhi JiaoAbstract:In recent metagenomic studies, single-stranded DNA (ssDNA) viruses that infect bacteria have been shown to be diverse and prevalent in the ocean; however, there are few isolates of marine ssDNA phages. Here, we report on a cultivated ssDNA phage (vB_Cib_ssDNA_P1) that infects Citromicrobium bathyomarinum RCC1878 (family Sphingomonadaceae), and other members of the genus. This is the first ssDNA phage reported to infect marine alphaproteobacteria, and represents a newly recognized lineage of the Microviridae infecting members of Sphingomonadaceae, the Amoyvirinae. The ∼26 nm diameter polyhedral capsid contains a 4,360 bp genome with 6 open reading frames (ORFs) and a 59.3% G+C content. ORF1 encodes the capsid protein and ORF3 encodes the replication initiator protein. The replication cycle is ∼5 h, followed by a burst releasing about 180 infectious particles. The closest relative of vB_Cib_ssDNA_P1 is a prophage within the genome of Novosphingobium tardaugens strain NBRC16725. Phylogenetic analysis indicates that the vB_Cib_ssDNA_P1 phage and two related prophages, as well as an environmental sequence, form a novel group within the Microviridae. Our results indicate that this is a previously unknown lineage of ssDNA viruses which also supplies a new model system for studying interactions between ssDNA phages and marine bacteria.
Qiang Zheng - One of the best experts on this subject based on the ideXlab platform.
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A Virus Infecting Marine Photoheterotrophic Alphaproteobacteria (Citromicrobium spp.) Defines a New Lineage of ssDNA Viruses.
Frontiers in microbiology, 2018Co-Authors: Qiang Zheng, Qi Chen, Curtis A. Suttle, Nianzhi JiaoAbstract:In recent metagenomic studies, single-stranded DNA (ssDNA) viruses that infect bacteria have been shown to be diverse and prevalent in the ocean; however, there are few isolates of marine ssDNA phages. Here, we report on a cultivated ssDNA phage (vB_Cib_ssDNA_P1) that infects Citromicrobium bathyomarinum RCC1878 (family Sphingomonadaceae), and other members of the genus. This is the first ssDNA phage reported to infect marine alphaproteobacteria, and represents a newly recognized lineage of the Microviridae infecting members of Sphingomonadaceae, the Amoyvirinae. The ~26 nm diameter polyhedral capsid contains a 4,360 bp genome with 6 open reading frames (ORFs) and a 59.3% G+C content. ORF1 encodes the capsid protein and ORF3 encodes the replication initiator protein. The replication cycle is ~5 hours, followed by a burst releasing about 180 infectious particles. The closest relative of vB_Cib_ssDNA_P1 is a prophage within the genome of Novosphingobium tardaugens strain NBRC16725. Phylogenetic analysis indicates that the vB_Cib_ssDNA_P1 phage and two related prophages, as well as an environmental sequence, form a novel group within the Microviridae. Our results indicate that this is a previously unknown lineage of ssDNA viruses which also supplies a new model system for studying interactions between ssDNA phages and marine bacteria.
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Image_2_A Virus Infecting Marine Photoheterotrophic Alphaproteobacteria (Citromicrobium spp.) Defines a New Lineage of ssDNA Viruses.TIF
2018Co-Authors: Qiang Zheng, Qi Chen, Curtis A. Suttle, Nianzhi JiaoAbstract:In recent metagenomic studies, single-stranded DNA (ssDNA) viruses that infect bacteria have been shown to be diverse and prevalent in the ocean; however, there are few isolates of marine ssDNA phages. Here, we report on a cultivated ssDNA phage (vB_Cib_ssDNA_P1) that infects Citromicrobium bathyomarinum RCC1878 (family Sphingomonadaceae), and other members of the genus. This is the first ssDNA phage reported to infect marine alphaproteobacteria, and represents a newly recognized lineage of the Microviridae infecting members of Sphingomonadaceae, the Amoyvirinae. The ∼26 nm diameter polyhedral capsid contains a 4,360 bp genome with 6 open reading frames (ORFs) and a 59.3% G+C content. ORF1 encodes the capsid protein and ORF3 encodes the replication initiator protein. The replication cycle is ∼5 h, followed by a burst releasing about 180 infectious particles. The closest relative of vB_Cib_ssDNA_P1 is a prophage within the genome of Novosphingobium tardaugens strain NBRC16725. Phylogenetic analysis indicates that the vB_Cib_ssDNA_P1 phage and two related prophages, as well as an environmental sequence, form a novel group within the Microviridae. Our results indicate that this is a previously unknown lineage of ssDNA viruses which also supplies a new model system for studying interactions between ssDNA phages and marine bacteria.