The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages

Chaikeeratisak, Vorrapon; Nguyen, Katrina; Egan, MacKennon E.; Erb, Marcella L.; Vavilina, Anastasia; Pogliano, Joe

doi:10.1016/j.celrep.2017.07.064

Cited by 110 publications

(251 citation statements)

References 22 publications

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“…4d). Although this shell-like structure has only been documented among the jumbo phages of Pseudomonas 28,29 , we consider that physical occlusion of phage genomic DNA through this and other mechanisms may comprise a novel route to immune system evasion in bacteria.…”

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confidence: 90%

“…As a control, a protein previously shown to be internalized in the shell, ORF152, was imaged revealing co-localization with the DAPI-positive phage nucleus. While the rules for protein internalization in the shell are currently unknown, these data and work from the initial shell studies 28,29 suggest that the default localization for large host-encoded proteins is to be excluded from the shell.…”

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confidence: 99%

“…Recently it was shown that ΦKZ and ΦKZ-like phages infecting Pseudomonas sp. construct an elaborate nucleus-like, proteinaceous compartment where phage DNA replicates, with PhuZ, a phage tubulin homologue, centering the compartment within the host cell 25-29 . Proteins involved in DNA replication and transcription localize inside the shell, while proteins mediating translation and nucleotide synthesis are relegated to the cytoplasmic space, akin to the eukaryotic nucleus.…”

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confidence: 99%

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A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

Mendoza

Berry

Nieweglowska

et al. 2018

Preprint

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13All viruses require strategies to inhibit or evade the immunity pathways of cells they 14 infect. The viruses that infect bacteria, bacteriophages (phages), must avoid nucleic-acid 15 targeting immune pathways such as CRISPR-Cas and restriction endonucleases to 16 replicate efficiently 1 . Here, we show that a jumbo phage infecting Pseudomonas 17 aeruginosa, phage ΦKZ, is resistant to many immune systems in vivo, including CRISPR-18 Cas3 (Type I-C), Cas9 (Type II-A), Cas12 (Cpf1, Type V-A), and Type I restriction-19 modification (R-M) systems. We propose that ΦKZ utilizes a nucleus-like shell to protect 20 its DNA from attack. Supporting this, we demonstrate that Cas9 is able to cleave ΦKZ 21 DNA in vitro, but not in vivo and that Cas9 is physically occluded from the shell 22 assembled by the phage during infection. Moreover, we demonstrate that the Achilles 23 heel for this phage is the mRNA, as translation occurs outside of the shell, rendering the 24 phage sensitive to the RNA targeting CRISPR-Cas enzyme, Cas13a (C2c2, Type VI-A). 25Collectively, we propose that the nucleus-like shell assembled by jumbo phages enables 26 potent, broad spectrum evasion of DNA-targeting nucleases. 28Phage infection and replication can cause bacterial death via cell lysis, necessitating protective 29 immune systems to negate this great threat to bacterial viability 2,3 . Restriction-modification (R-30 M) and adaptive CRISPR-Cas (clustered regularly interspaced short palindromic repeats and 31 CRISPR-associated genes) immunity detect and degrade phage nucleic acids to protect the 32 host 1 . Recent CRISPR-Cas discovery efforts have led to the characterization of six distinct 33 CRISPR-Cas types (I-VI) with independent mechanisms for CRISPR RNA (crRNA) biogenesis, 34 surveillance complex assembly, substrate selection, and target degradation 4 . Type III and VI 35CRISPR-Cas systems target RNA 5,6 , while Types I, II, and V predominantly target DNA 7-9 . 36Despite these differences, all characterized CRISPR-Cas systems function via crRNA-guides 37 derived from the DNA-based CRISPR array, which stores the memory of past infections 10,11 . 39Phages that infect Pseudomonas aeruginosa avoid CRISPR-mediated destruction by encoding 40 "anti-CRISPR" (Acr) proteins that inhibit the Type I-E and I-F CRISPR-Cas systems [12][13][14] . We 41 sought to determine whether any P. aeruginosa phages are also resistant to the Type I-C 42 CRISPR-Cas subtype, also present in P. aeruginosa 15 . This subtype is notable as it is the most 43 minimal Type I system identified to date, is highly abundant in bacterial genomes 16 , and is 44 understudied relative to other subtypes. We identified an isolate in our lab encoding a Type I-C 45 system, and designed and expressed a crRNA targeting related phages JBD30 and DMS3m, 48 phages did not possess functional Type I-C Acr proteins ( Fig. 1a). To test the sensitivity of other 49 phage families against the Type I-C system, we transferred the necessary cas genes (cas3, 50 cas5, cas8, cas7) into the chromosome of a...

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confidence: 90%

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confidence: 99%

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A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

Mendoza

Berry

Nieweglowska

et al. 2018

Preprint

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“…We identified proteins encoded in huge phage genomes that cluster with AcrVA5, AcrVA2, and AcrIIA7 and may function as Acrs. Also, identified were tubulinhomologs (PhuZ) and proteins (SI) that create a proteinaceous phage "nucleus" (Chaikeeratisak, Nguyen, Egan, et al , 2017) . The phage nucleus was recently shown to protect https://docs.google.com/document/d/1ZV8fmYx0e8oQT1UmuGD3a03pU_not9Y61tjsYPR6Gcg/edit# 11/31 the phage genome against host defense by physically blocking CRISPRCas degradation (Mendoza et al , 2018) .…”

Section: Crisprcas Mediated Interactionsmentioning

confidence: 99%

Clades of huge phage from across Earth’s ecosystems

Al-Shayeb

Sachdeva

Chen

et al. 2019

Preprint

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Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirtyfour genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPRCas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPRCas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phageencoded functions. In addition, some phage may repurpose bacterial CRISPRCas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earth's ecosystems.

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“…128 129 Deepening similarities between the eukaryotic nucleus and the viral factories of phage 201 Φ2-1, 130 201 Φ2-1 possesses homologues of eukaryotic tubulin (PhuZ), and this tubulin polymerises via 131 6 dynamic instability, positioning the factory in the centre of the infected cell (Chaikeeratisak et al 132 2017). The PhuZ spindle is the only known example of a cytoskeletal structure that shares three key 133 properties with the eukaryotic spindle: dynamic instability, a bipolar array of filaments, and central 134 positioning of DNA (Chaikeeratisak et al 2017). This is a significant parallel since eukaryotic nuclei 135 are positioned in the cell by microtubule-dependent motors during development and differentiation 136 (Star 2009).…”

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Evidence supporting a viral origin of the eukaryotic nucleus

Bell¹

2019

Preprint

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29The defining feature of the eukaryotic cell is the possession of a nucleus that uncouples transcription 30 from translation. This uncoupling of transcription from translation depends on a complex process 31 employing hundreds of eukaryotic specific genes acting in concert and requires the 7-32 methylguanylate (m7G) cap to prime eukaryotic mRNA for splicing, nuclear export, and cytoplasmic 33 translation. The origin of this complex system is currently a paradox since it is not found or needed 34 in prokaryotic cells which lack nuclei, yet it was apparently present and fully functional in the Last 35Eukaryotic Common Ancestor (LECA). According to the Viral Eukaryogenesis (VE) hypothesis the 36 abrupt appearance of the nucleus in the eukaryotic lineage occurred because the nucleus descends 37 from the viral factory of a DNA phage that infected the archaeal ancestor of the eukaryotes. 38 Consequently, the system for uncoupling of transcription from translation in eukaryotes is predicted 39 by the VE hypothesis to be viral in origin. In support of this hypothesis it is shown here that m7G 40 capping apparatus that primes the uncoupling of transcription from translation in eukaryotes is 41 present in viruses of the Mimiviridae but absent from bona-fide archaeal relatives of the eukaryotes 42 such as Lokiarchaeota. Furthermore, phylogenetic analysis of the m7G capping pathway indicates 43 that eukaryotic nuclei and Mimiviridae obtained this pathway from a common ancestral source that 44 predated the origin of LECA. These results support the VE hypothesis and suggest the eukaryotic 45 nucleus and the Mimiviridae descend from a common First Eukaryotic Nuclear Ancestor (FENA). 46 47 48 49 50 51 52 53 54 55 A membrane-bound nucleus defines the eukaryotic domain, and all cellular organisms without nuclei 56 are prokaryotic (Sapp 2005; Stanier and Van Niel 1962). Its presence contributes to a great divide 57 between eukaryotes and prokaryotes defined by features such as linear chromosomes, telomeres, 58 nuclear pores, the spliceosome, mitosis, meiosis, the sexual cycle, and the endoplasmic reticulum. 59 Since the nucleus separates the eukaryotic genome from the ribosomal apparatus, its presence also 60 introduces an uncoupling of transcription from translation unique to the eukaryotic domain. 61 62 Lokiarchaeota reportedly 'bridges the gap between prokaryotes and eukaryotes' and are proposed 63 to be bona-fide archaeal relative of the eukaryotes (Spang et al. 2015). Lokiarchaeota and related 64 Asgardians encode Crenactins, the ESCRT-III complex, a family of small Ras-like GTPases and a 65 ubiquitin system, making them a plausible direct descendent of an archaeal ancestor of the 66 eukaryotes (Koonin 2015). This discovery supports the Eocyte tree of life where eukaryotes evolved 67 from a specific archaeon rather than representing a sister group to the archaea (Riviera and Lake 68 1992). If Lokiarchaeota are bona-fide archaeal relatives of the eukaryotes, the last common 69 ancestor of the Asgard archaea and the eukaryotes ...

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The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages

Cited by 110 publications

References 22 publications

A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

Clades of huge phage from across Earth’s ecosystems

Evidence supporting a viral origin of the eukaryotic nucleus

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