Capsids and Genomes of Jumbo-Sized Bacteriophages Reveal the Evolutionary Reach of the HK97 Fold

Hua, Jianfei; Huet, Alexis; López, Carlos A.; Toropova, Katerina; Pope, Welkin H.; Duda, Robert L.; Hendrix, Roger W.; Conway, James F.

doi:10.1128/mbio.01579-17

Cited by 70 publications

(89 citation statements)

References 69 publications

(88 reference statements)

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“…One can roughly estimate phage size from a plot of plaque diameter vs. supporting agarose gel concentration. Phage size increases as the slope of this plot increases, as seen by comparing the plot for near-jumbo myophage T4 (168.903 Kb genome [35]; Figure 1 [36]) to the plot for myophage G (626 Kb genome [6]; Figure 1) and podophage T3 (38.208 Kb genome [37]); the latter plot is horizontal (not shown in Figure 1). However, when comparing jumbo myophage G to jumbo myophage 0305phi8-36 (218.948 Kb genome [38]; Figure 1 [36]), this relationship is lost in that one visually observes no significant difference in slope, even though (1) the surface area of phage G (cryo-EM data in [6]) is over 2x the surface area of phage 0305phi8-36 (cryo-EM data in [39]) and (2) gel sieving is best correlated with particle surface area [40].…”

Section: Plaque Diameter Vs Amentioning

confidence: 96%

“…Jumbo phage isolations are also needed to answer questions of phage evolution, such as the following. Did phages begin as cells that have subsequently undergone reductive genomic evolution [63,64] or are jumbo phages the products of accretive evolution [6,65] (or both)? In addition, do phages have genes that promote phage evolution to provide fitness of the ecosystem of which the phage is a part [18]?…”

Section: Conclusion/perspectivesmentioning

confidence: 99%

“…What is the effect of changing the radius of the effective gel pore (P E ) on phage plaque formation? These questions achieved increased significance during our work [1,2] with the largest known phage, phage G [3][4][5][6][7]. Phage G did not propagate well either in liquid culture or in the traditional agar gels (typically 0.5-0.7%) used as the top layer, plaque-supporting gel during phage infectivity assays.…”

Section: Introductionmentioning

confidence: 99%

“…These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses.Here, we both review and augment data projected to help answer the above questions. We propose that data of this type are critical to efficient isolation of large phages, which include those with genomes longer than 200 Kb (jumbo phages [4][5][6]19]) and even larger phages with genome longer than 520 Kb (mega-phages [20,21]). The importance of these answers is illustrated by the fact that no mega-phage has ever been isolated [20,21], although even larger eukaryotic viruses have been isolated [22].…”

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confidence: 99%

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In-Gel Isolation and Characterization of Large (and Other) Phages

Serwer

Wright

2020

Viruses

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We review some aspects of the rapid isolation of, screening for and characterization of jumbo phages, i.e., phages that have dsDNA genomes longer than 200 Kb. The first aspect is that, as plaque-supporting gels become more concentrated, jumbo phage plaques become smaller. Dilute agarose gels are better than conventional agar gels for supporting plaques of both jumbo phages and, prospectively, the even larger (>520 Kb genome), not-yet-isolated mega-phages. Second, dilute agarose gels stimulate propagation of at least some jumbo phages. Third, in-plaque techniques exist for screening for both phage aggregation and high-in-magnitude, negative average electrical surface charge density. The latter is possibly correlated with high phage persistence in blood. Fourth, electron microscopy of a thin section of a phage plaque reveals phage type, size and some phage life cycle information. Fifth, in-gel propagation is an effective preparative technique for at least some jumbo phages. Sixth, centrifugation through sucrose density gradients is a relatively non-destructive jumbo phage purification technique. These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses.Here, we both review and augment data projected to help answer the above questions. We propose that data of this type are critical to efficient isolation of large phages, which include those with genomes longer than 200 Kb (jumbo phages [4][5][6]19]) and even larger phages with genome longer than 520 Kb (mega-phages [20,21]). The importance of these answers is illustrated by the fact that no mega-phage has ever been isolated [20,21], although even larger eukaryotic viruses have been isolated [22]. The existence of mega-phages is surmised from assembly of metagenomic sequences [20,21]. Host Bacteria In-Gel: Values of P E for Agarose Gels FundamentalsThe following data indicate that Gram-negative and Gram-positive phage hosts do not fit in the spaces of the 0.5-0.7% agar gels typically used to form phage plaques. This conclusion is derived, first, from the measurement of P E for agarose gels. This measurement begins by determining vs. sphere radius the minimal agarose gel concentration that excludes a sphere during electrophoresis [23]. These P E values are then used to anchor P E values determined from the sieving (gel-induced retardation) of spheres during gel electrophoresis. The most advanced study of the latter [24] yields the following relationship for underivatized, low-electro-osmosis agarose gels cast in 0.025 M sodium phosphate, pH 7.4, 0.001 M MgCl 2 at 25 • C: P E = 148A −0.87 nm; A is the weight percentage of agarose.A 0.7% agarose gel, if characterized by this relationship, has a P E of 202 nm, not large enough to admit a bacterial cell, such as the host for phage G. Our phage G host is~500 nm in radius and 2000-5000 nm in length, as is Escherichia coli [25], a representative of the ...

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Section: Plaque Diameter Vs Amentioning

confidence: 96%

Section: Conclusion/perspectivesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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In-Gel Isolation and Characterization of Large (and Other) Phages

Serwer

Wright

2020

Viruses

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“…These phages have an immense diversity in terms of size and complexity. The highly ubiquitous HK97-like fold is the building block for virtually all dsDNA containing phages, and allows for enormous versatility in icosahedral geometry, that can lead to differences in biophysical properties [5]. To withstand environmental stresses and the internal pressure that amasses as a result of dsDNA genome packaging, some dsDNA phages encode additional “decoration” proteins that bind to the exterior of their capsids and stabilize the virions.…”

Section: Introductionmentioning

confidence: 99%

The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy

Newcomer

Schrad

Gilcrease

et al. 2018

Preprint

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37The major coat proteins of dsDNA tailed phages and herpesviruses form capsids by a 38 mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, 39 followed by expansion and stabilization of the capsid. These viruses have evolved diverse 40 strategies to fortify their capsids, such as non-covalent binding of auxiliary "decoration" 41(Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding 42 strategy that precisely distinguishes between nearly identical three-fold and quasi-three-fold 43 sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image 44 reconstruction were employed to determine the structure of native phage L particles. NMR 45 was used to determine the structure/dynamics of Dec in solution. Lastly, the NMR structure 46 and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec 47 trimer. Key regions that modulate the binding interface were verified by site-directed 48 mutagenesis. 50Viral icosahedral capsids are formed from multiple copies of a single or a few types of 52 highly structurally-conserved coat proteins that encapsidate the genome [1]. The minimum 53 number of subunits needed to build an icosahedral capsid is 60 coat proteins, and the result 54 is a T=1 icosahedral geometry. There is a direct correlation between genome size and T-55 number for dsDNA containing phages. Building a bigger capsid necessitates the use of 56 more coat protein subunits, and requires that chemically identical proteins assemble into well-studied double stranded DNA (dsDNA) containing phages, the capsids have a T=7 59 geometry (see Figure 1A). These have 11 vertices formed by coat protein pentons and an 60 additional 60 hexons to create the capsid. The 12 th vertex breaks the icosahedral symmetry 61 and is occupied by a tail that specifies host binding. Estimates predict that 10 31 viruses are 62 in Earth's biosphere [3,4], with dsDNA containing bacteriophages being the most abundant. 63These phages have an immense diversity in terms of size and complexity. The highly 64 ubiquitous HK97-like fold is the building block for virtually all dsDNA containing phages, and 65 allows for enormous versatility in icosahedral geometry, that can lead to differences in 66 biophysical properties [5]. To withstand environmental stresses and the internal pressure 67 that amasses as a result of dsDNA genome packaging, some dsDNA phages encode 68 additional "decoration" proteins that bind to the exterior of their capsids and stabilize the 69 virions. How various decoration proteins recognize and bind to specific sites on capsids with 70 different icosahedral geometries is, however, still poorly understood. 71The tight packing of the dsDNA genomes into the virions of many phages and 72 herpesviruses creates enormous internal pressure (10 -60 atm) within the capsids, resulting 73 in high-energy states that prime the particles for infection, and facilitate delivery of the 74 majority of the viral genomes into the hosts [6]...

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