Giant Viruses Called 'Jumbo Phages'

Giant Viruses Called 'Jumbo Phages'

https://reasonandscience.catsboard.com/t3256-giant-viruses-called-jumbo-phages

TESSA KOUMOUNDOUROS (2022): As antibiotic-resistant bacteria become increasingly challenging to defeat, consulting those who've been in battle with our ancient foe long before us just makes a whole lot of sense. Viruses called bacteriophages have been clashing with bacteria since long before we even existed. So researchers from the University of California, San Diego, took a closer look at some of the lesser studied jumbo phages, specifically 201phi2-1, which infects Pseudomonas chlororaphis bacteria.

Named for their large genomes of over 200,000 base pairs long (most phages that infect Pseudomonas have genomes less than 100,000 base pairs), jumbo phages have an arsenal of techniques to counter bacterial defense mechanisms. Previously research found growing a shield around its genetic material was one of these tactics, making these viruses' life cycle unique.


The jumbo phage compared to T4 phage virus.

Separation of genomic material from the rest of the cell contents by encasing it "was previously thought to have evolved only once in the history of life", molecular biologist Thomas Laughlin and colleagues write in their new paper. But it turns out these tiny giants beat us to it.

"It's a different kind of compartment – unlike anything we have ever seen in nature," says biophysicist Elizabeth Villa.

Usually, phages inject their genetic material into the bacteria where it floats freely in the cell's inner goo (cytoplasm) as the virus hijacks the bacteria's equipment to replicate itself. But these jumbo phages construct a separating compartment around their DNA soon after it enters its host, a bit like how our cells have a nucleus to protect our DNA.

This physically prevents the bacteria's CRISPR 'immune system' and other defensive enzymes from messing with the viral DNA. Laughlin and their team used cryo-electron microscopy and tomography to examine this compartment down to the atomic scale. The protective casing was constructed from just a single type of protein, which the researchers called chimallin, after an ancient Aztec shield.


Micrograph of jumbo phage-infected bacterial cell (left) and nucleus-like compartment outlined in blue (right).

With the help of computer modeling, the researchers found the phage nucleus selectively allows molecules to pass through tiny pores – again similar to how cells control the environment around our genetic material, making this a remarkable example of convergent evolution – when completely unrelated organisms end up with a similar solution to the same biological problem.

Comment: 

Convergence, another problem for evolution
https://reasonandscience.catsboard.com/t2014-convergence-another-problem-for-evolution

Biologists are uncovering numerous examples of organisms that cluster together morphologically (structurally), and yet are genetically distinct. Frogs, lizards, or herbs that appear to be identical are actually different at the genetic level. An evolutionary interpretation of this data, then, demands that the morphologically identical organisms must have evolved independently of one another in a “repeatable” fashion.


Stephen J. Gould, Wonderful Life: The Burgess Shale and the Nature of History (1989), 51.
“…No finale can be specified at the start, none would ever occur a second time in the same way, because any pathway proceeds through thousands of improbable stages. Alter any early event, ever so slightly, and without apparent importance at the time, and evolution cascades into a radically different channel.1

Gould’s metaphor of “replaying life’s tape” asserts that if one were to push the rewind button, erase life’s history, and let the tape run again, the results would be completely different.2 The very essence of the evolutionary process renders evolutionary outcomes as nonreproducible (or nonrepeatable). Therefore, “repeatable” evolution is inconsistent with the mechanism available to bring about biological change.

Paleontologist J. William Schopf, one of the world’s leading authorities on early life on Earth, has made this very point in the book Life’s Origin.
Because biochemical systems comprise many intricately interlinked pieces, any particular full-blown system can only arise once…Since any complete biochemical system is far too elaborate to have evolved more than once in the history of life, it is safe to assume that microbes of the primal LCA cell line had the same traits that characterize all its present-day descendants.

"The nuclear pore in eukaryotes is a gigantic, complex structure with very distinctive ways of keeping most proteins out but specifically importing others. What we're probably looking at with the jumbo phage is a dramatically simpler method of solving the same problem," explains biochemist Kevin Corbett. "It's an amazingly creative solution – similar but simpler – to protecting its genome from the outside world by building a wall to separate it from bacterial defenses."

Incredibly, this shield can also grow as the phage's genome replicates. The researchers aren't quite sure how phages manage this yet but suspect the compartment is probably cracking open to allow more chimallin units to join it, which are produced in abundance on infection.

Meiying Gao (2017): Only 93 phages with genomes larger than 200 kbp have been isolated during the past 100 years since the discovery of phages (up to 30 June 2016). More than 80% of these were isolated during the past 3 years, which might be because of the revitalization of phage research and the progress in next-generation genome sequence technology in recent years. Tailed phages with genomes larger than 200 kbp are classified as “jumbo phages,” and phages of this kind usually harbor large virions. The jumbo phages are evolutionarily distant from the small genome phages. Despite the common features that differentiate them from smaller-genome phages, jumbo phages show more divergent characteristics among each other, such as low genome similarity, individual virion substructure, and different propagation mechanisms. 3

Thomas G. Laughli et.al.,(2022): Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors.  The bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

Bacteria have an array of defensive systems to target and destroy foreign nucleic acids. In turn, phages have mechanisms, including anti-restriction and anti-CRISPR proteins, that counter specific bacterial defence systems. A family of ‘jumbo phages’—named for their large genomes (typically over 200 kb) and large virion size assemble a selectively permeable, protein-based shell that encloses the replicating viral genome and is associated with a unique phage life cycle (Fig. 1a). 


a, Schematic of the jumbo phage infection cycle. 
b, Fluorescence microscopy of a 201phi2-1-infected P. chlororaphis cell at 45 mpi (n = 5 independent experiments). Phage nucleus shell component gp105 (green) is tagged with GFP, phage DNA (blue) is stained with DAPI and the outer cell membrane (red) is stained with FM4-64. 
c, Tomographic slice of a phage nucleus in a 201phi2-1-infected P. chlororaphis cell at 50–60 mpi. 
d, Segmentation of the tomogram in c. Outer and inner bacterial membranes are shown in burgundy and pink, respectively. The phage nucleus is coloured blue. Phage capsids and tails are green and cyan, respectively. PhuZ and RecA-like protein filaments are light purple and white, respectively. A subset of 500 host ribosomes is shown in pale yellow. 
e, Enlarged view of the boxed region in c. Yellow arrows point to the repetitive feature of the phage nucleus perimeter. 
f, Slice of the cytosolic face of the subtomogram average of the repetitive feature in the phage nucleus perimeter with a comma-shaped subunit outlined in yellow. 
g, Cytosolic and side views of the shell subtomogram average isosurface with a single subunit outlined in yellow. h, Schematic representation of the p442-like arrangement of chimallin protomers. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. Scale bars: 1 μm (b), 250 nm (c), 25 nm (e) and 10 nm (f).

This micrometre-scale nucleus-like compartment, termed the ‘phage nucleus’, forms de novo upon infection and grows with the replicating viral DNA. Meanwhile, phage proteins are synthesized in the host cell cytoplasm. These include PhuZ, a phage-encoded tubulin homologue that assembles into filaments that treadmill to transport empty capsids from their assembly sites at the host cell membrane to the surface of the phage nucleus. Capsids dock to the phage nuclear shell and are filled with viral DNA before detaching and completing assembly with phage tails. Mature particles are released by host cell lysis. In contrast to other characterized anti-restriction systems, the phage nuclear shell renders jumbo phages broadly immune to DNA-targeting host restriction systems, including CRISPR–Cas, throughout infection by serving as a physical barrier between the viral DNA and host nucleases.

Chimallin is the principal shell protein
We previously showed that the abundant and early-expressed 201phi2-1 protein gp105 becomes integrated into the nuclear shell2. Furthermore, 201phi2-1 gp105 has a molecular weight of 69.5 kDa (631 amino acids). Along with the high apparent compositional homogeneity of the shell, these data support 201phi2-1 gp105 as the principal component of the phage nuclear shell. Homologues of 201phi2-1 gp105 are encoded by a large set of jumbo phage that infect diverse bacteria including Pseudomonas, Vibrio, Salmonella, and Escherichia coli, but these proteins bear no detectable sequence homology to any other proteins. 

Comment:   This is one of the certainly many examples showing the lack of sequence homology, and refutes Hong Zhou's  (2015) claim that: Viruses likely originated from ancient cells through encapsulating cellular plasmids or  genome fragments by cellular protein 1 Consider as well its size, 631 amino acids are composing a protein of considerable length and enormous sequence space.  

Because of its role in protecting the phage genome against host defences, we named this protein chimallin (ChmA) after the chimalli, a shield carried by ancient Aztec warriors10. To understand the structure and assembly mechanisms of chimallin, we expressed and purified 201phi2-1 chimallin from E. coli. Size-exclusion chromatography coupled to multi-angle light scattering (SEC–MALS) of purified chimallin indicated a mixture of oligomeric states including monomers, well-defined assemblies of approximately 1.2 MDa, and larger heterogeneous species ranging from 4 to 13 MDa (mean of 6.9 MDa; Fig. 2a).


a, SEC–MALS analysis of purified 201phi2-1 chimallin. The measured molar masses of the three peaks are 6.9 MDa (range 4–13 MDa), 1.2 MDa and 87 kDa (left to right). dRI, differential refractive index. 
b,c, Z-slices from tomograms of samples from the correspondingly labelled SEC–MALS peaks in a. 
d, Top left, O-symmetrized reconstruction of the chimallin cubic assembly viewed along the four-fold axis. The protomers of one four-fold face are coloured. Top right, surface representation of the chimallin cubic assembly model viewed along the four-fold axis. Bottom right and bottom left, views of the model along the two-fold and three-fold axes, respectively. Red arrowheads point to the C-terminal segments of the yellow protomer. The green square, pink triangle and black oval indicate that the corresponding panels are viewed down the particle’s four-fold, three-fold and two-fold rotational symmetry axes, respectively. 
e, Localized asymmetric reconstruction of the chimallin protomer (left) and cartoon model (right). Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). Resolved core protomer termini are shown as spheres. 
f, Rainbow-coloured cartoon model of the chimallin protomer conformation in the cubic assembly. Resolved N and C termini are shown as spheres. Domains and segments are labelled. Unresolved linkers are shown as dashed lines. 
g, A rainbow-coloured fold diagram of chimallin (blue at N terminus, red at C terminus) with α-helices labelled alphabetically and β-strands labelled numerically. The N- and C-terminal domains are highlighted in blue and red, respectively. Dashed lines indicate unresolved loops. Scale bars, 50 nm.

Cryo-ET of the largest species revealed pleomorphic, closed compartments with near-identical morphology to the phage nuclear shell that we observe in situ (Fig. 2b). Analysis of the smaller, more defined assemblies by cryo-ET revealed a near-homogeneous population of cubic assemblies with a diameter of around 22 nm, and a minor population of rectangular assemblies with dimensions of approximately 22 × 33.5 nm (Fig. 2c). 2

Giant Viruses Called 'Jumbo Phages' and chimallin, its principal shell protein
https://www.youtube.com/watch?v=zCs-sgcHWVc



Emerging Aspects of Jumbo Bacteriophages

1. TESSA KOUMOUNDOUROS Giant Viruses Called 'Jumbo Phages' Could Help Us Fight Antibiotic Resistance 05 August 2022
2. Thomas G. Laughlin: Architecture and self-assembly of the jumbo bacteriophage nuclear shell 03 August 2022
3. Meiying Gao Jumbo Bacteriophages: An Overview 14 March 2017

Giant Viruses Called 'Jumbo Phages'

https://reasonandscience.catsboard.com/t3256-giant-viruses-called-jumbo-phages

TESSA KOUMOUNDOUROS (2022): As antibiotic-resistant bacteria become increasingly challenging to defeat, consulting those who've been in battle with our ancient foe long before us just makes a whole lot of sense. Viruses called bacteriophages have been clashing with bacteria since long before we even existed. So researchers from the University of California, San Diego, took a closer look at some of the lesser studied jumbo phages, specifically 201phi2-1, which infects Pseudomonas chlororaphis bacteria.

Named for their large genomes of over 200,000 base pairs long (most phages that infect Pseudomonas have genomes less than 100,000 base pairs), jumbo phages have an arsenal of techniques to counter bacterial defense mechanisms. Previously research found growing a shield around its genetic material was one of these tactics, making these viruses' life cycle unique.

Separation of genomic material from the rest of the cell contents by encasing it "was previously thought to have evolved only once in the history of life", molecular biologist Thomas Laughlin and colleagues write in their new paper. But it turns out these tiny giants beat us to it.

"It's a different kind of compartment – unlike anything we have ever seen in nature," says biophysicist Elizabeth Villa.

Usually, phages inject their genetic material into the bacteria where it floats freely in the cell's inner goo (cytoplasm) as the virus hijacks the bacteria's equipment to replicate itself. But these jumbo phages construct a separating compartment around their DNA soon after it enters its host, a bit like how our cells have a nucleus to protect our DNA.

This physically prevents the bacteria's CRISPR 'immune system' and other defensive enzymes from messing with the viral DNA. Laughlin and their team used cryo-electron microscopy and tomography to examine this compartment down to the atomic scale. The protective casing was constructed from just a single type of protein, which the researchers called chimallin, after an ancient Aztec shield.

With the help of computer modeling, the researchers found the phage nucleus selectively allows molecules to pass through tiny pores – again similar to how cells control the environment around our genetic material, making this a remarkable example of convergent evolution – when completely unrelated organisms end up with a similar solution to the same biological problem.

Comment: 

Convergence, another problem for evolution
https://reasonandscience.catsboard.com/t2014-convergence-another-problem-for-evolution

Biologists are uncovering numerous examples of organisms that cluster together morphologically (structurally), and yet are genetically distinct. Frogs, lizards, or herbs that appear to be identical are actually different at the genetic level. An evolutionary interpretation of this data, then, demands that the morphologically identical organisms must have evolved independently of one another in a “repeatable” fashion.


Stephen J. Gould, Wonderful Life: The Burgess Shale and the Nature of History (1989), 51.
“…No finale can be specified at the start, none would ever occur a second time in the same way, because any pathway proceeds through thousands of improbable stages. Alter any early event, ever so slightly, and without apparent importance at the time, and evolution cascades into a radically different channel.1

Gould’s metaphor of “replaying life’s tape” asserts that if one were to push the rewind button, erase life’s history, and let the tape run again, the results would be completely different.2 The very essence of the evolutionary process renders evolutionary outcomes as nonreproducible (or nonrepeatable). Therefore, “repeatable” evolution is inconsistent with the mechanism available to bring about biological change.

Paleontologist J. William Schopf, one of the world’s leading authorities on early life on Earth, has made this very point in the book Life’s Origin.
Because biochemical systems comprise many intricately interlinked pieces, any particular full-blown system can only arise once…Since any complete biochemical system is far too elaborate to have evolved more than once in the history of life, it is safe to assume that microbes of the primal LCA cell line had the same traits that characterize all its present-day descendants.

"The nuclear pore in eukaryotes is a gigantic, complex structure with very distinctive ways of keeping most proteins out but specifically importing others. What we're probably looking at with the jumbo phage is a dramatically simpler method of solving the same problem," explains biochemist Kevin Corbett. "It's an amazingly creative solution – similar but simpler – to protecting its genome from the outside world by building a wall to separate it from bacterial defenses."

Incredibly, this shield can also grow as the phage's genome replicates. The researchers aren't quite sure how phages manage this yet but suspect the compartment is probably cracking open to allow more chimallin units to join it, which are produced in abundance on infection.

Meiying Gao (2017): Only 93 phages with genomes larger than 200 kbp have been isolated during the past 100 years since the discovery of phages (up to 30 June 2016). More than 80% of these were isolated during the past 3 years, which might be because of the revitalization of phage research and the progress in next-generation genome sequence technology in recent years. Tailed phages with genomes larger than 200 kbp are classified as “jumbo phages,” and phages of this kind usually harbor large virions. The jumbo phages are evolutionarily distant from the small genome phages. Despite the common features that differentiate them from smaller-genome phages, jumbo phages show more divergent characteristics among each other, such as low genome similarity, individual virion substructure, and different propagation mechanisms. 3

Thomas G. Laughli et.al.,(2022): Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors.  The bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

Bacteria have an array of defensive systems to target and destroy foreign nucleic acids. In turn, phages have mechanisms, including anti-restriction and anti-CRISPR proteins, that counter specific bacterial defence systems. A family of ‘jumbo phages’—named for their large genomes (typically over 200 kb) and large virion size assemble a selectively permeable, protein-based shell that encloses the replicating viral genome and is associated with a unique phage life cycle (Fig. 1a). 

This micrometre-scale nucleus-like compartment, termed the ‘phage nucleus’, forms de novo upon infection and grows with the replicating viral DNA. Meanwhile, phage proteins are synthesized in the host cell cytoplasm. These include PhuZ, a phage-encoded tubulin homologue that assembles into filaments that treadmill to transport empty capsids from their assembly sites at the host cell membrane to the surface of the phage nucleus. Capsids dock to the phage nuclear shell and are filled with viral DNA before detaching and completing assembly with phage tails. Mature particles are released by host cell lysis. In contrast to other characterized anti-restriction systems, the phage nuclear shell renders jumbo phages broadly immune to DNA-targeting host restriction systems, including CRISPR–Cas, throughout infection by serving as a physical barrier between the viral DNA and host nucleases.

Chimallin is the principal shell protein
We previously showed that the abundant and early-expressed 201phi2-1 protein gp105 becomes integrated into the nuclear shell2. Furthermore, 201phi2-1 gp105 has a molecular weight of 69.5 kDa (631 amino acids). Along with the high apparent compositional homogeneity of the shell, these data support 201phi2-1 gp105 as the principal component of the phage nuclear shell. Homologues of 201phi2-1 gp105 are encoded by a large set of jumbo phage that infect diverse bacteria including Pseudomonas, Vibrio, Salmonella, and Escherichia coli, but these proteins bear no detectable sequence homology to any other proteins. 

Comment:   This is one of the certainly many examples showing the lack of sequence homology, and refutes Hong Zhou's  (2015) claim that: Viruses likely originated from ancient cells through encapsulating cellular plasmids or  genome fragments by cellular protein 1 Consider as well its size, 631 amino acids are composing a protein of considerable length and enormous sequence space.  

Because of its role in protecting the phage genome against host defences, we named this protein chimallin (ChmA) after the chimalli, a shield carried by ancient Aztec warriors10. To understand the structure and assembly mechanisms of chimallin, we expressed and purified 201phi2-1 chimallin from E. coli. Size-exclusion chromatography coupled to multi-angle light scattering (SEC–MALS) of purified chimallin indicated a mixture of oligomeric states including monomers, well-defined assemblies of approximately 1.2 MDa, and larger heterogeneous species ranging from 4 to 13 MDa (mean of 6.9 MDa; Fig. 2a).

Cryo-ET of the largest species revealed pleomorphic, closed compartments with near-identical morphology to the phage nuclear shell that we observe in situ (Fig. 2b). Analysis of the smaller, more defined assemblies by cryo-ET revealed a near-homogeneous population of cubic assemblies with a diameter of around 22 nm, and a minor population of rectangular assemblies with dimensions of approximately 22 × 33.5 nm (Fig. 2c). 2

Giant Viruses Called 'Jumbo Phages' and chimallin, its principal shell protein
https://www.youtube.com/watch?v=zCs-sgcHWVc


1. TESSA KOUMOUNDOUROS Giant Viruses Called 'Jumbo Phages' Could Help Us Fight Antibiotic Resistance 05 August 2022
2. Thomas G. Laughlin: Architecture and self-assembly of the jumbo bacteriophage nuclear shell 03 August 2022
3. Meiying Gao Jumbo Bacteriophages: An Overview 14 March 2017

Robert Sanders: Huge bacteria-eating viruses narrow gap between life and non-life FEBRUARY 12, 2020

Scientists have discovered hundreds of unusually large, bacteria-killing viruses with capabilities normally associated with living organisms, blurring the line between living microbes and viral machines.

These phages — short for bacteriophages, so-called because they “eat” bacteria — are of a size and complexity considered typical of life, carry numerous genes normally found in bacteria and use these genes against their bacterial hosts.

The team identified 351 phage genomes that were more than 200 kilobases long, four times the average phage genome length of 50 kilobytes (kb). They were able to establish the exact length of 175 phage genomes; the others could be much larger than 200 kb. One of the complete genomes, 735,000 base-pairs long, is now the largest known phage genome.

While most of the genes in these huge phages code for unknown proteins, the researchers were able to identify genes that code for proteins critical to the machinery, called the ribosome, that translates messenger RNA into protein. Such genes are not typically found in viruses, only in bacteria or archaea.

“Typically, what separates life from non-life is to have ribosomes and the ability to do translation; that is one of the major defining features that separate viruses and bacteria, non-life and life,” Sachdeva said. “Some large phages have a lot of this translational machinery, so they are blurring the line a bit.”

Question:  In face of the fact that viruses and bacteriophages are not derivative from living cells, but had a separate origin, the question arises how did these giant phages obtain ribosome machineries?

B.Al-Shayeb et.al., (2020):Clades of huge phages from across Earth’s ecosystems 12 February 2020

Huge phages likely use these genes to redirect the ribosomes to make more copies of their own proteins at the expense of bacterial proteins. Some huge phages also have alternative genetic codes, the nucleic acid triplets that code for a specific amino acid, which could confuse the bacterial ribosome that decodes RNA.

In addition, some of the newly discovered huge phages carry genes for variants of the Cas proteins found in a variety of bacterial CRISPR systems, such as the Cas9, Cas12, CasX and CasY families. Some of the huge phages also have CRISPR arrays, which are areas of the bacterial genome where snippets of viral DNA are stored for future reference, allowing bacteria to recognize returning phages and to mobilize their Cas proteins to target and cut them up. “The high-level conclusion is that phages with large genomes are quite prominent across Earth’s ecosystems, they are not a peculiarity of one ecosystem,” Banfield said. “And phages which have large genomes are related, which means that these are established lineages with a long history of large genome size. Having large genomes is one successful strategy for existence, and a strategy we know very little about.”

Many phages have genes involved in the de novo biosynthesis of purines and pyrimidines, and the interconversion of nucleic and ribonucleic acids and nucleotide phosphorylation states. These gene sets are intriguingly similar to those of bacteria with very small cells and putative symbiotic lifestyles.  Notably, many phages have genes with predicted functions in transcription and translation (Supplementary Table 6). Complete phage genomes encode up to 67 tRNAs, with sequences that are distinct from those of their hosts


A family tree of huge phage, reconstructed using the sequence of the phages’ terminase genes. 
The inner to outer rings display the presence of the CRISPR-Cas system, host phylum, environmental sampling type and genome size. The 10 newly identified groups or clades of huge phage are listed on the left.  The phylogeny of phages was reconstructed using large terminase sequences from this study (n = 397) and similar matches from all RefSeq r92 proteins (n = 532). The tree also includes large terminase sequences from complete RefSeq phage, the Lak megaphage clade9 (n = 9) and non-artefactual phage genomes that are more than 200 kb, from a previous study14. Huge phage clades identified in this study were independently corroborated with a phylogenetic reconstruction of major capsid protein (MCP) genes and protein clustering. The tree was rooted using eukaryotic herpesvirus terminases (n = 7). The inner to outer rings display the presence of CRISPR–Cas in this study, host phylum, environmental sampling type and genome size. Host phylum and genome size were not included for RefSeq protein database matches for which the sequence may be from an integrated prophage or part of organismal genome projects. Scale bars show the number of substitutions per site (left) and number of base pairs (right).


a, Cell diagram of bacterium–phage and phage–phage interactions that involve CRISPR targeting during superinfection. Arrows indicate CRISPR–Cas targeting of the prophage and phage genomes. Phage names indicate related groups delineated by whole-genome alignment. We only included CRISPR interactions from samples of subjects of the same human cohort. 
b, Maximum likelihood phylogenetic tree of Cas12 subtypes a–i. Phage-encoded Cas12i and CasΦ, the new effector, are outlined in red, with bacteria-encoded proteins in blue. Bootstrap values >90 are shown on the branches (circles). Cas14 and type V-U trees are provided separately. Scale bars indicates the number of substitutions per site.
c, Top, alignment of the consensus repeats from the A9 phage array and predicted host bacterial arrays. Bottom, interaction network showing the targeting of bacteria-encoded (blue) and phage-encoded (red) CRISPR spacers. The number of edges indicate the number of spacers from the array with targets to the smaller node. Solid edges denote spacer targets with no or one mismatch, and dashed edges denote two to three mismatches (to account for degeneration in old-end phage spacers, diversity in different subjects or phage mutation to avoid targeting).

We show that phages with huge genomes are widespread across Earth’s ecosystems. We manually completed 35 genomes, distinguishing them from prophages, providing accurate genome lengths and complete inventories of genes, including those encoded in complex repeat regions that break automated assemblies. Even closely related phages have diversified across habitats. For instance, huge phages equipped with CRISPR–Cas systems might be tamed and used to modulate the functions of the bacterial microbiome or eliminate unwanted bacteria.

The huge phages comprise extensive clades, suggesting that a gene inventory comparable in size to those of many symbiotic bacteria is a conserved strategy for phage survival. Overall, their genes appear to redirect the protein production capacity of the host to favour phage genes by first intercepting the earliest steps of translation and subsequently ensuring the efficient production of proteins. These inferences are aligned with findings for some eukaryotic viruses, which control every phase of protein synthesis. Some phages acquired CRISPR–Cas systems with unusual compositions that may function to control host genes and eliminate competing phages.

Jingwen Guan: Intracellular Organization by Jumbo Bacteriophages 18 December 2020

Since their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been “jumbo phages,” with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. 


The bacteriophage nucleus-like shell combats antiphage defense mechanisms.
Jumbo phages assemble a proteinaceous shell that separates phage genomes from the bacterial cytoplasm and segregates proteins according to function during viral replication. DNA replication and transcription occur inside the shell, while translation and metabolic processes take place in the cytoplasm. The shell physically shields phage genomes from attack by DNA-targeting CRISPR-Cas systems and restriction enzymes. However, RNA-targeting CRISPR-Cas systems can cleave phage transcripts in the cytoplasm. It remains unknown how phage DNA is protected upon injection (depicted as a dashed curve and a question mark).


Intracellular development of jumbo phages after infection of a bacterial host.
Upon encountering a bacterial host, the phage injects its genomic DNA, accompanied by the translocation of virion RNA polymerases (vRNAPs) that are present inside the viral capsid. vRNAPs transcribe early phage genes, including genes encoding the shell protein and PhuZ. While the host chromosome is degraded, PhuZ forms filaments anchored at each cell pole and gradually establishes a bipolar spindle in the cell. Meanwhile, the shell proteins assemble a nucleus-like compartment to sequester phage DNA from the cytoplasm and protect phage DNA against host defense systems. Nonvirion RNAPs (nvRNAPs) appear later inside the compartment and are responsible for expression of the late phage genes, including viral structural genes. As phage DNA replicates inside the shell, the compartment grows and is pushed toward the cell center by dynamically unstable filaments of the PhuZ spindle. The cell becomes elongated and forms a bulge at midcell. After the phage nucleus is settled in the cell center, treadmilling PhuZ filaments rotate the shell locally and deliver empty phage capsids from the cell inner membrane to the phage nucleus for subsequent DNA encapsidation. Rotation of the phage nucleus facilitates even distribution of phage capsids around the shell surface, ensuring efficient DNA packaging. Eventually, filled capsids are released from the shell and assemble into mature phage particles together with other viral structural components that are generated in the cytoplasm, followed by cell lysis to release phage progeny to the environment.

Not only are viral factories able to concentrate replicase proteins, viral genomes, and host proteins required for viral replication, but they also function to protect against cellular antiviral defense systems, as in the case of the jumbo phage nucleus