The tail is also similar. Whatever the details, aggregating bacterio- that the volume of the internal cavity of the capsid’s shell deter- phages are likely to have genes that were selected for advantages mines the mature DNA length.18,19 Thus, in this case, deletion provided in the environment only. These genes will be non- mutants are not more physically stable than the wild type essential in the laboratory. In the current communication, we test bacteriophage. Unfortunately for deletion analysis, the genomes for such genes by isolating deletion mutants of 0305w8-36. We of all sufficiently characterized long-genome (. 200 Kb) bacterio- continue by exploring extracellular events that might explain the phages, other than 0305w8-36, are permuted to our knowledge. finding that 0305w8-36 is more virulent when forming plaques Thus, the work described here appears to be the first deletion than it is in liquid culture. analysis for any long-genome bacteriophage. Bacteriophage 0305w8-36 is an appropriate model virus for selecting deletion mutants, because the 0305w8-36 genome has Results been found8 to be non-permuted (all DNA molecules have the same ends) and terminally repetitious (the sequence at one end The first deletion mutant, D(118,397–124,982). After selection is repeated at the other). Based on work with other bacterio- for mutants stable to elevated temperature, we isolated three phages,14,15 a non-permuted, terminally repetitious genome is independent mutants, total, two of which were deletion mutants, produced by cleavage from a concatemer of mature genomes by a based on the restriction endonuclease analysis described in the sequence-specific nuclease. If a bacteriophage genome is non- Materials and Methods Section. In Figure 1A, a pair of vertical permuted, the genome becomes shorter when genes are deleted, red lines indicates each of the regions deleted, within a low- while the DNA-encapsulating shell remains the same size. The resolution map of the entire 218.948 Kb genome (6.479 Kb reasons are that (1) shell size is determined before packaging terminal repeat; 247 identified orfs). ©exerts and (2) red2uced no mechanism p0ressure 1on exists th2ecapsid, to add redundant Lthereabymanking genomic DNA ddeletioneD(s118,397B–124,9i82). to The first 0305w8-36 oOrfs deletion s200–c207 mutant wiere was edeletned, missing as nucleotides cshowenat . fill the shell.14,15 The shortened genome of a deletion mutant 118,397–124,982 (3.01% of the genome) and will be called mutants physically selectable because they are physically more higher resolution in Figure 1B. We found a single deletion and its stable (more resistant to elevated temperature) than the wild type approximate position by restriction endonuclease analysis that bacteriophage.16,17 detected a single deletion in the 110,000–130,000 area (not In contrast, elongation of a terminal repeat occurs when genes shown). We obtained confirmation and a more precise position are also cleaved and pDackaged ofrom a nconcatoemer. tThe redason isislocatetdthredeiletibonpreucisely tbySeanger .sequencing of the PCR are deleted from bacteriophages with permuted genomes, which by PCR amplification across the site of the deletion. Finally, we Figure 1. Maps of the bacteriophage 0305w8-36 genome and its deletions. (A) A low-resolution map of the entire genome. (B) A high-resolution map of the D(118,397–124,982) deletion with distance from the left end (Kb) at the top, orf number in the next line and homolog just below the corresponding arrow. (C) A high-resolution map of the D(181,412–189,531) deletion, with the same annotations. Orfs indicated by a green arrow were found8 by protein mass spectrometry to encode a protein of the mature bacteriophage capsid. Orfs indicated by a blue arrow were not found to encode a protein of the mature bacteriophage capsid. The direction of an arrow indicates the direction of transcription and translation. Red lines [vertical in (A); horizontal in (B and C)] indicate the span of a deletion. The numbers in parentheses are the numbers of the nucleotides deleted. The protein encoded by gene 205 is a minor capsid protein with location not yet identified in electron micrographs. 26 Bacteriophage Volume 2 Issue 1