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

Abstract

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 ...

Characterization and Comparative Genomics Analysis of a New Bacteriophage BUCT610 against Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae

“…Crude phage was obtained after the supernatant was filtered by the sterile 0.22 μm filter. The crude phage was further purified and concentrated with 30% sterile sucrose solution by centrifugation at 30,000 rpm and 4 °C for 2 h [ 44 , 45 ]. The supernatant after centrifugation was discarded and the bottom phage precipitate was resuspended with phosphate buffer saline (PBS) to obtain pure phage BUCT610, which was then stored at 4 °C.…”

Basics for Improved Use of Phages for Therapy

“…Each one of these procedural aspects (i.e., 1–4) selects against phages of types known to have members. Most dramatically, (2) and (4) eliminate relatively large phages [ 19 , 20 , 21 , 24 ]. Membrane enveloped phages are also under-sampled [ 25 ].…”

“…This process has continued with the collaborative finding that (1) the bacterial host for phage G is not the relatively large Bacillus megaterium initially proposed [ 27 ], but an average-sized Lysinibacillus [ 29 ], and (2) anecdotally, phage G is not well preserved by freezing. In the Serwer laboratory’s experience, centrifugation in a sucrose gradient has been, so far, the least damaging to large environmental phages, including phage G [ 24 , 30 ]. To illustrate the benefits of the above information, the phage G preparation used for a 6.1 Å cryo-EM structure of the DNA-enclosing outer shell [ 29 ] started as a two-Petri plate, top-agarose lysate (plate stock) and ended with purification via rate zonal centrifugation in a sucrose gradient [ 24 ].…”

“…In the Serwer laboratory’s experience, centrifugation in a sucrose gradient has been, so far, the least damaging to large environmental phages, including phage G [ 24 , 30 ]. To illustrate the benefits of the above information, the phage G preparation used for a 6.1 Å cryo-EM structure of the DNA-enclosing outer shell [ 29 ] started as a two-Petri plate, top-agarose lysate (plate stock) and ended with purification via rate zonal centrifugation in a sucrose gradient [ 24 ].…”

“…However, to our knowledge, no coronavirus-like phage has ever been described in a publication. This inconsistency is possibly explained by the data that indicate that long tails assist large phage propagation [ 24 ] and that coronaviruses are as large, but without tails. Good reason exists to try to isolate “corona-phages” with in-dilute gel techniques [ 36 ].…”