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1.  Islands of non-essential genes, including a DNA translocation operon, in the genome of bacteriophage 0305ϕ8-36 
Bacteriophage  2012;2(1):25-35.
We investigate genes of lytic, Bacillus thuringiensis bacteriophage 0305ϕ8-36 that are non-essential for laboratory propagation, but might have a function in the wild. We isolate deletion mutants to identify these genes. The non-permutation of the genome (218.948 Kb, with a 6.479 Kb terminal repeat and 247 identified orfs) simplifies isolation of deletion mutants. We find two islands of non-essential genes. The first island (3.01% of the genomic DNA) has an informatically identified DNA translocation operon. Deletion causes no detectable growth defect during propagation in a dilute agarose overlay. Identification of the DNA translocation operon begins with a DNA relaxase and continues with a translocase and membrane-binding anchor proteins. The relaxase is in a family, first identified here, with homologs in other bacteriophages. The second deleted island (3.71% of the genome) has genes for two metallo-protein chaperonins and two tRNAs. Deletion causes a significant growth defect. In addition, (1) we find by “in situ” (in-plaque) single-particle fluorescence microscopy that adsorption to the host occurs at the tip of the 486 nm long tail, (2) we develop a procedure of 0305ϕ8-36 purification that does not cause tail contraction, and (3) we then find by electron microscopy that 0305ϕ8-36 undergoes tail tip-tail tip dimerization that potentially blocks adsorption to host cells, presumably with effectiveness that increases as the bacteriophage particle concentration increases. These observations provide an explanation of the previous observation that 0305ϕ8-36 does not lyse liquid cultures, even though 0305ϕ8-36 is genomically lytic.
PMCID: PMC3357382  PMID: 22666654
bacteriophage; deletion mutant; DNA sequencing; electron microscopy; fluorescence microscopy; informatics; long-genome; microbial biofilm
2.  Characterization of Pseudomonas chlororaphis myovirus 201φ2-1 via genomic sequencing, mass spectrometry, and electron microscopy 
Virology  2008;376(2):330-338.
Pseudomonas chlororaphis phage 201φ2-1 is a relative of Pseudomonas aeruginosa myovirus φKZ. Phage 201φ2-1 was examined by complete genomic sequencing (316,674 bp), by a comprehensive mass spectrometry survey of its virion proteins and by electron microscopy. Seventy-six proteins, of which at least 69 have homologues in φKZ, were identified by mass spectrometry. Eight proteins, in addition to the major head, tail sheath and tail tube proteins, are abundant in the virion. Electron microscopy of 201φ2-1 revealed a multitude of long, fine fibers apparently decorating the tail sheath protein. Among the less abundant virion proteins are three homologues to RNA polymerase β or β′ subunits. Comparison between the genomes of 201φ2-1 and φKZ revealed substantial conservation of the genome plan, and a large region with an especially high rate of gene replacement. The φKZ-like phages exhibited a two-fold higher rate of divergence than for T4-like phages or host genomes.
PMCID: PMC2577825  PMID: 18474389
3.  Complete Genomic Sequence and Mass Spectrometric Analysis of Highly Diverse, Atypical Bacillus thuringiensis phage 0305φ8-36 
Virology  2007;368(2):405-421.
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 0305φ8-36. Phage 0305φ8-36 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 0305φ8-36 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 0305φ8-36 has twice the virion protein coding sequence of T4. Based on its genomic isolation, 0305φ8-36 is a resource for future studies of vertical gene transmission.
PMCID: PMC2171028  PMID: 17673272
myovirus; Bacillus thuringiensis; pyrosequencing; virion protein; mass spectrometry

Results 1-3 (3)