Isolation of novel large and aggregating bacteriophages

Abstract

Viruses are detected via either biological properties such as plaque formation or physical properties. The physical properties include appearance during microscopy and DNA sequence derived from community sequencing. The assumption is that these procedures will succeed for most, if not all, viruses. However, we have found that some bacteriophages are in a category of viruses that are not detected by any of these classical procedures. Given that the data already indicate viruses to be the "largest reservoir of unknown genetic diversity on earth," the implied expansion of this reservoir confirms the belief that the genome project has hardly begun. The first step is to fill gaps in our knowledge of the biological diversity of viruses, an enterprise that will also help to determine the ways in which (a) viruses have participated in evolution and ecology and (b) viruses can be made to participate in disease control and bioremediation. We present here the details of procedures that can be used to cultivate previously undetectable viruses that are either comparatively large or aggregation-prone.

Fig. 6.1.

Effect on plaque diameter of the temperature of agarose gelation before growth of bacteriophage 0305φ8–36. Host cell-containing molten agarose was added to a Petri plate previously equilibrated at either (a) 42°C, (b) 37°C, (c) 25°C, or (d) 4°C. The plates were then placed at room temperature (25 ±3°C) and inoculated after the agarose gelled. The plates were then incubated in thermal contact with each other for 8 h at room temperature.

Fig. 6.2.

Isolation of hosts. The procedure for bacteriophage isolation in Section 2.1 was performed with the host cells omitted from the molten agarose. The Petri plate was incubated for 20 h at 25°C and then photographed.

Fig. 6.3.

Analysis of aggregation by analytical ultracentrifugation with fluorescence detection. The following were subjected to centrifugation at 3000 rpm, 20°C in a Beckman XLA analytical ultracentrifuge with fluorescence detection optics (36): bacteriophage 0305φ8–36 (filled circles) and bacteriophage T7 (empty squares). Data processing was performed by the procedures in ref. (37).