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.
Highlights
Neglect was typical of the following questions about gel supported plaques used for isolation, cloning and preparative propagation of phages
Is a bacterial host cell smaller than the spaces within the supporting gel, typically an agar gel, in which bacteria were propagating for plaque formation? What is the effect of changing the radius of the effective gel pore (PE ) 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]
We propose that data of this type are critical to efficient isolation of large phages, which include those with genomes longer than 200 Kb and even larger phages with genome longer than 520 Kb
Summary
Neglect was typical of the following questions about gel supported plaques used for isolation, cloning and preparative propagation of phages. The more rapidly one can (1) obtain plaques of environmental phages and (2) characterize phage particles in a plaque, the more effective is screening of newly isolated phages for several purposes These purposes include phage therapy of infectious disease [8,9,10,11] and genomic sequence-analysis of the evolution of both phages and their hosts [12,13,14]. 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], even larger eukaryotic viruses have been isolated [22]. The existence of mega-phages is surmised from assembly of metagenomic sequences [20,21]
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