To investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479-bp terminal repeat), and identified the virion proteins (55), of Bacillus thuringiensis bacteriophage 0305phi8-36. Phage 0305phi8-36 is an atypical myovirus with three large curly tail fibers. An accurate mode of DNA pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey. Advanced informatic techniques were used to identify classical morphogenesis genes. The 0305phi8-36 genes were highly diverged; 19% of 247 closely spaced genes have similarity to proteins with known functions. Genes for virion-associated, apparently fibrous proteins in a new class were found, in addition to strong candidates for the curly fiber genes. Phage 0305phi8-36 has twice the virion protein coding sequence of T4. Based on its genomic isolation, 0305phi8-36 is a resource for future studies of vertical gene transmission.

Isolation and characterization of new environmental bacteriophages are performed for (1) analyzing microbial evolution and ecology and (2) delivering biological therapy. The sampling of environmental bacteriophages appears, however, to be limited by the procedure (usually liquid enrichment culture) used to propagate them. An alternative, less competitive procedure is developed here for the purpose of isolating new bacteriophages. This procedure involves extraction directly into and then propagation in a dilute agarose gel. Adaptations of this procedure are used to avoid repeated isolation of the same bacteriophage. Some newly isolated bacteriophages grow so poorly that they appear inaccessible to liquid enrichment culture. Four comparatively high titer bacteriophages were isolated and characterized by a genomic sequence survey. Some had genomes with extremely distant relationships to those of other bacteriophages, based on a tree built from the large terminase genes. These methods find novel genomes by rapidly isolating and screening diverse bacteriophages.

The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.

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