0000000000053860
AUTHOR
Jaana K. H. Bamford
Did the ancient crenarchaeal viruses from the dawn of life survive exceptionally well the eons of meteorite bombardment?
The viruses of Crenarchaeota are unexpectedly diverse in their morphologies, and most have no, or few, genes related to bacterial, eukaryal, euryarchaeal, or other crenarchaeal viruses. Though several different virus morphotypes have been discovered in enrichment cultures of microbial communities collected from geothermally heated environments around the world, the origins of such differences are unknown. We present a model that combines consideration of Earth's geological history, the early emergence of hyperthermophiles, and the early formation of viruses from primordial genes with the intent to explain this vast diversity of crenarchaeal viruses. Several meteorite- or flood basalt-induce…
Association of colony morphotypes with virulence, growth and resistance against protozoan predation in the fish pathogen Flavobacterium columnare.
Many opportunistic pathogens can alternate between inside- and outside-host environments during their life cycle. The opportunistic fish pathogen Flavobacterium columnare is an inhabitant of the natural microbial community and causes significant yearly losses in aquaculture worldwide. The bacterium grows in varying colony morphotypes that are associated with either virulence (rhizoid type) or resistance to starvation and phages (rough type). Rough type strains can arise spontaneously or can be induced by phage infection. To identify the determinants of morphotype fitness, we measured virulence, growth parameters, biofilm-forming ability and resistance to amoeba and ciliate predation of both…
Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome
Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5'-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we…
Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77.
The major capsid proteins VP16 and VP17 of bacteriophage P23-77 have been crystallized using both recombinant and purified virus and preliminary diffraction analyses have been performed.
On the astrobiological relevance of viruses in extraterrestrial ecosystems
AbstractViruses are the dominant form of genetically reproducing entities on Earth. Yet, viruses are mostly neglected in the context of astrobiology due to their non-living nature. In this discussion it is considered whether viruses are likely to be transferred within bacterial endospores to other planetary bodies through lithopanspermia. Interestingly, it seems possible that ecosystems of panspermial origin might yield biospheres in which viruses are absent. The evolutionary pathway of life in these systems might differ significantly from the path observed on Earth. We hypothesize that the difference in the two potential ways for the emergence of life, those being panspermial or local orig…
Phage Specificity of the Freshwater Fish Pathogen Flavobacterium columnare
ABSTRACT Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages.
High Nutrient Concentration Can Induce Virulence Factor Expression and Cause Higher Virulence in an Environmentally Transmitted Pathogen
Environmentally transmitted opportunistic pathogens shuttle between two substantially different environments: outside-host and within-host habitats. These environments differ from each other especially with respect to nutrient availability. Consequently, the pathogens are required to regulate their behavior in response to environmental cues in order to survive, but how nutrients control the virulence in opportunistic pathogens is still poorly understood. In this study, we examined how nutrient level in the outside-host environment affects the gene expression of putative virulence factors of the opportunistic fish pathogen Flavobacterium columnare. The impact of environmental nutrient concen…
Subcellular localization of bacteriophage PRD1 proteins in Escherichia coli
Bacteria possess an intricate internal organization resembling that of the eukaryotes. The complexity is especially prominent at the bacterial cell poles, which are also known to be the preferable sites for some bacteriophages to infect. Bacteriophage PRD1 is a well-known model serving as an ideal system to study structures and functions of icosahedral internal membrane-containing viruses. Our aim was to analyze the localization and interactions of individual PRD1 proteins in its native host Escherichia coli. This was accomplished by constructing a vector library for production of fluorescent fusion proteins. Analysis of solubility and multimericity of the fusion proteins, as well as their …
Protist predation can select for bacteria with lowered susceptibility to infection by lytic phages
Background: Consumer-resource interactions constitute one of the most common types of interspecific antagonistic interaction. In natural communities, complex species interactions are likely to affect the outcomes of reciprocal co-evolution between consumers and their resource species. Individuals face multiple enemies simultaneously, and consequently they need to adapt to several different types of enemy pressures. In this study, we assessed how protist predation affects the susceptibility of bacterial populations to infection by viral parasites, and whether there is an associated cost of defence on the competitive ability of the bacteria. As a study system we used Serratia marcescens and i…
Recent advances in the expression, evolution, and dynamics of prokaryotic genomes
Work on the molecular and cellular biology of prokaryotic microorganisms and their phage continues to be at the cutting edge in many areas of fundamental research in the life sciences. An important stimulus has been the large number of complete bacterial and archaeal genome sequences that have appeared over the past decade and that now are being produced at a rate of several hundred per year. This information is the foundation for all of the “-omics” leading to a global evaluation of the state of the cell under various conditions of growth or stress. An overview of the sequenced prokaryotic genomes has also greatly influenced our thinking about the evolution of life on earth. The EMBO Confe…
Intensive aquaculture selects for increased virulence and interference competition in bacteria
Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare , change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003–2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbre…
Fluorescence Properties of the Chromophore-Binding Domain of Bacteriophytochrome from Deinococcus radiodurans
Fluorescent proteins are versatile tools for molecular imaging. In this study, we report a detailed analysis of the absorption and fluorescence properties of the chromophore-binding domain from Deinococcus radiodurans and its D207H mutant. Using single photon counting and transient absorption techniques, the average excited state lifetime of both studied systems was about 370 ps. The D207H mutation slightly changed the excited state decay profile but did not have a considerable effect on the average decay time of the system or the shape of the absorption and emission spectra of the biliverdin chromophore. We confirmed that the fluorescence properties of both samples are very similar in vivo…
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 geneti…
Selenomethionine labeling of large biological macromolecular complexes: probing the structure of marine bacterial virus PM2.
There is a need for improved tools for labeling protein species within large macromolecular assemblies. Here we describe a method for the efficient selenomethionine labeling of the membrane-containing bacterial virus PM2 for structural studies. By examining potential host cells a strain was found which was auxotrophic for methionine, and by performing a multiparameter search of conditions it was possible to derive a robust protocol which simultaneously minimized the toxic effects of the selenomethionine, so that a reasonable virus yield was maintained, whilst still achieving essentially complete labeling. This has allowed us to fingerprint the protein constituents of the virus in a relative…
Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1.
AbstractBacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diff…
Efficient DNA Packaging of Bacteriophage PRD1 Requires the Unique Vertex Protein P6
ABSTRACT The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infecti…
New enveloped dsRNA phage from freshwater habitat.
Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a tri-layered virion structure. Here, we present a new putative member of the Cystoviridae family, bacteriophage ϕNN. ϕNN was isolated from a Finnish lake in contrast to the previously identified cystoviruses, which originate from various legume samples collected in the USA. The nucleotide sequence of the virus reveals a strong genetic similarity (~80 % for the L-segments, ~55 % for the M-segments and ~84 % for the S-segments) to Pseudomonas phage ϕ6, the type member of the virus family. However, the relationship between ϕNN and other cystoviruses is more distant. In general, proteins located in the int…
Molecular Characterization of a Variant of Bacillus anthracis-Specific Phage AP50 with Improved Bacteriolytic Activity▿ †
ABSTRACT The genome sequence of a Bacillus anthracis -specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.
Evolutionary rescue of bacteria via horizontal gene transfer under a lethal β-lactam concentration
β-Lactams are a commonly used class of bactericidal antibiotics. The number of β-lactam-resistant pathogens is constantly increasing in hospitals around the world. Interestingly, most of the β-lactam-resistant bacteria carry mobile genetic elements, such as conjugative plasmids, that render the pathogen resistant. These elements mediate their own transfer from one bacterium to another, producing new resistant strains via horizontal gene transfer. Here we investigated whether it is possible that transfer of the resistance element from another bacterium may evolutionarily rescue a susceptible bacterium exposed to a lethal concentration of the β-lactam ampicillin. Indeed, the rescuing occurs e…
The use of low-resolution phasing followed by phase extension from 7.6 to 2.5 Å resolution with noncrystallographic symmetry to solve the structure of a bacteriophage capsid protein
P2, the major capsid protein of bacteriophage PM2, adopts the double β-barrel fold characteristic of the PRD1-adenoviral lineage. The 2.5 Å resolution X-ray data obtained by analysis of the two major lattices of a multiple crystal of P2 were phased by molecular replacement, using as a search model structure factors to 7.6 Å resolution obtained from electron density cut from the map of the entire PM2 virion. Phase extension to 2.5 Å resolution used solely sixfold cycling averaging and solvent flattening. This represents an atypical example of an oligomeric protein for which the structure has been determined at high resolution by bootstrapping from low-resolution initial phases.
A Unique Group of Virus-Related, Genome-Integrating Elements Found Solely in the Bacterial Family Thermaceae and the Archaeal Family Halobacteriaceae
ABSTRACT Viruses SH1 and P23-77, infecting archaeal Haloarcula species and bacterial Thermus species, respectively, were recently designated to form a novel viral lineage. In this study, the lineage is expanded to archaeal Halomicrobium and bacterial Meiothermus species by analysis of five genome-integrated elements that share the core genes with these viruses.
Probing protein interactions in the membrane-containing virus PRD1.
PRD1 is a Gram-negative bacteria infecting complex tailless icosahedral virus with an inner membrane. This type virus of the family Tectiviridae contains at least 18 structural protein species, of which several are membrane associated. Vertices of the PRD1 virion consist of complexes recognizing the host cell, except for one special vertex through which the genome is packaged. Despite extensive knowledge of the overall structure of the PRD1 virion and several individual proteins at the atomic level, the locations and interactions of various integral membrane proteins and membrane-associated proteins still remain a mystery. Here, we demonstrated that blue native PAGE can be used to probe pro…
Scoping the effectiveness and evolutionary obstacles in using plasmid-dependent phages to fight antibiotic resistance
Aim: To investigate the potential evolutionary obstacles in the sustainable therapeutic use of plasmid-dependent phages to control the clinically important conjugative plasmid-mediated dissemination of antibiotic resistance genes to pathogenic bacteria. Materials & methods: The lytic plasmid-dependent phage PRD1 and the multiresistance conferring plasmid RP4 in an Escherichia coli host were utilized to assess the genetic and phenotypic changes induced by combined phage and antibiotic selection. Results & conclusions: Resistance to PRD1 was always coupled with either completely lost or greatly reduced conjugation ability. Reversion to full conjugation efficiency was found to be rare…
Phage-borne factors and host LexA regulate the lytic switch in phage GIL01.
ABSTRACT The Bacillus thuringiensis temperate phage GIL01 does not integrate into the host chromosome but exists stably as an independent linear replicon within the cell. Similar to that of the lambdoid prophages, the lytic cycle of GIL01 is induced as part of the cellular SOS response to DNA damage. However, no CI-like maintenance repressor has been detected in the phage genome, suggesting that GIL01 uses a novel mechanism to maintain lysogeny. To gain insights into the GIL01 regulatory circuit, we isolated and characterized a set of 17 clear plaque ( cp ) mutants that are unable to lysogenize. Two phage-encoded proteins, gp1 and gp7, are required for stable lysogen formation. Analysis of …
High temperature and bacteriophages can indirectly select for bacterial pathogenicity in environmental reservoirs
The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial…
Comparing the different morphotypes of a fish pathogen - implications for key virulence factors in Flavobacterium columnare
Background: Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067 in rainbow trout ( Onconrhynchus mykiss ) and used high-resolution scanning electron microscopy to identify the fine structures of the cells grown in liquid and on agar. We also analysed the proteins secreted extracellularly and in membrane vesicles to identify possible virulence factors. Results: Only the Rhizoid morphotype was virulent in rain…
The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions
ABSTRACT Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrost…
Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage
The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the ke…
Bacteriophage P23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.
Summary It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid archi…
Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2
ABSTRACT The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 ( Corticoviridae ) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect an…
Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2.
Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane protein…
The closest relatives of icosahedral viruses of thermophilic bacteria are among viruses and plasmids of the halophilic archaea.
We have sequenced the genome and identified the structural proteins and lipids of the novel membranecontaining, icosahedral virus P23-77 of Thermus thermophilus. P23-77 has an 17-kb circular double-stranded DNA genome, which was annotated to contain 37 putative genes. Virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. Analysis of the bacteriophage genome revealed it to be evolutionarily related to another Thermus phage (IN93), archaeal Halobacterium plasmid (pHH205), a genetic element integrated into Haloarcula genome (designated here as IHP for integrated Ha…
Phage-driven loss of virulence in a fish pathogenic bacterium
Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria) co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four s…
Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.
Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in bothEscherichia coliandSalmonella entericain the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction i…
Data from: Intensive aquaculture selects for increased virulence and interference competition in bacteria
Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare, change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003-2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbreak…