DNA Requirementsin Vivofor Phage T4 Packaging

Abstract

Phage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 terminase gene sequences were introduced into prophage lambda imm434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles. Multiple copy prophage lambda imm434 genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total. Deletion or base change of the 24-bp gene 16 3' region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac sequence. Moreover, a specific gene 16-containing pac fragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion. We conclude that both the copy number of homologous sequences and the DNA pac sequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4.

Divergence and Mosaicism among Virulent Soil Phages of the Burkholderia cepacia Complex

“…These results are inconsistent with a packaging mechanism involving initial cleavage at or near a pac site, followed by subsequent rounds of headful packaging. However, the findings do not discriminate between random initiation, initiation at multiple pac sites, or terminase recognition of pac followed by movement and cleavage at distant sites (7,33).…”

The Generalized TransducingSalmonellaBacteriophage ES18: Complete Genome Sequence and DNA Packaging Strategy

“…5A), it is almost certain that ES18, like P22, packages additional headfuls of DNA in a sequential manner along the concatemeric product of rollingcircle DNA replication. We note that in P22, Sf6, SPP1, T4, and P1 the region where the packaging series initiates is within or overlaps the gene that encodes the small terminase subunit (19,54,80,96), the protein that carries the specificity for recognition of the DNA to be packaged (15,16,19). In P22 this site has been genetically identified, and it lies near the center of the 120-bp region within which P22 initiates DNA packaging (96); similarly, the Sf6 pac site also lies near the center of the 1,800-bp region within which it initiates packaging (E. Gilcrease and S. Casjens, unpublished results).…”

“…The presence of such a submolar fragment is considered to be diagnostic of this type of headful DNA packaging, as opposed to mechanisms like those of phages or T7, which generate ends at precisely the same location in all virion DNA molecules. However, the virion DNAs of some apparently headful packaging phages do not exhibit an obvious pac fragment, e.g., 933W (68), A118 (55), HK620 (21), APSE-1 (88), HSIC (J. Paul, personal communication), Sf6 (17), 11 (56), 42 (64), Aa23 (94), and probably T4 (54). We have recently presented evidence that Sf6 packages DNA in a manner that is very similar to that of the phages that do exhibit a pac fragment; however, the DNA cut that initiates the packaging series occurs not at a precise location but at scattered locations within an 1,800-bp region such that the pac fragment electrophoresis band, although present, is so diffuse as to be essentially invisible to staining (17).…”

Molecular architecture of tailed double-stranded DNA phages

“…The first cut of it's DNA concatemer can be made at different points. 37,38 Bacteriophage φ29 synthetize monomeric genomes which have covalently linked gp3 proteins attached to the 5′-ends of both DNA strands. 39 φ29 does not have a small terminase, and its large terminase does not have nuclease activity.…”