Determining DNA packaging strategy by analysis of the termini of the chromosomes in tailed-bacteriophage virions

Abstract

Tailed-bacteriophage virions contain a single linear dsDNA chromosome which can range in size from about 18 to 500 kbp across the known tailed-phage types. These linear chromosomes can have one of several known types of termini as follows: cohesive ends (5'- or 3'-single-strand extensions), circularly permuted direct terminal repeats, short or long exact direct terminal repeats, terminal host DNA sequences, or covalently bound terminal proteins. These different types of ends reflect differing DNA replication strategies and especially differing terminase actions during DNA packaging. In general, complete genome sequence determination does not by itself elucidate the nature of these ends, so directed experimental analysis is usually required to understand the nature of the virion chromosome ends. This chapter discusses these methods.

Fig. 7.1

Four tailed-phage DNA packaging strategies. Packaging strategies of phages λ, P22, T7, and Mu are shown diagrammatically. Thick black horizontal lines represent phage concatemeric DNAs, or, in the case of Mu, phage genomes that are integrated into the host bacteria’s chromosome (the latter represented by thick gray line). Black circles mark the packaging recognition sites and horizontal black arrows represent individual packaging events. Vertical black lines indicate precise terminase cleavages and vertical gray lines indicate imprecise cleavages (see text). In each case except Mu, sequential series of packaging events occur, in which subsequent events (event 2 in figure) on the same concatemer molecule begin at the concatemer end created by the previous event (event 1); although only two successive events are shown, packaging series can in some cases be up to 10 or more events long. In phages λ and T7, each event begins and ends at a packaging recognition site, and in phage T7 the white rectangles show the region (the direct terminal repeat) that is duplicated in concert with packaging. In phage P22, the increasing width of the vertical gray boxes to the right, denotes the increased range of cleavage site locations as events proceed rightward. The small gray horizontal rectangle below the first P22 event is the optimal location of the Southern probe used to analyze pac fragments (see text).

Fig. 7.2

Restriction enzyme generated fragments from tailed-phage virion DNA. DNA fragments were separated by 0.8% agarose gel electrophoresis and visualized by staining with ethidium bromide. The phage virion source of the DNA is indicated above each panel, and the restriction enzymes used are indicated above each lane. A. Phage λ DNA after normal isolation and storage. DNA in the second and third lanes was heated to 75 °C for 15 min and then fast or slow cooled to room temperature, respectively, as described in the Methods. Terminal DNA fragments are indicated as follows: white square, left end fragment; gray square, right end fragment; black square, left and right end fragments annealed together by their cohesive ends. B. Phage P22 DNA. The pac fragments generated by PstI and EcoRI are indicated by black circles on the left and right, respectively. C. Phage Sf6 DNA. The locations of the diffuse pac fragment bands generated by imprecise packaging series initiation are indicated by white rectangles (see also ref. (31)). D. Phage SF6 DNA. Terminal DNA fragments are indicated as follows: white square, left end fragment; gray square, right end fragment.

Fig. 7.3

Neighbor-joining tree of large terminase subunit amino acid sequences. A neighbor-joining tree of 123 tailed-phage terminase amino acid sequences was generated by CLUSTAL X (59). The numbers near bifurcations are bootstrap values for 1,000 trials. Short bifurcating branches linking the major groups shown here were manually merged, since all had low bootstrap values, and the major deep branches are all shown as radiating from a single source. The names of the phages or prophages are shown at the right of each terminal branch (some prophage names are those proposed by Casjens (13)). Major robust, related groups of terminases are highlighted with gray boxes, and the packaging strategy and a prototype phage for each group is given at the far right. Those phages whose virion DNA termini structures have been experimentally determined are indicated as follows: #, 5′-cohesive ends in lambdoid phages;^, 5′-cohesive ends in P2-like phages; *, 3′-cohesive ends; $, T7-like phages with direct terminal repeats and no circular permutation; , phages with long direct terminal repeats and no circular permutation; @, phages with host DNA at termini; †, headful packaging in non-T4-like phages, including P22 and the gene transfer agents (GTA); %, headful packaging in T4-like phages; ●, headful packaging phages for which no obvious pac fragment band has been identified in ethidium stained electrophoresis gels of restricted DNA (see text; the darker orange boxed “headful/Sf6” subgroup within the “headful/P22” group appear to be one branch of this type of terminase). This figure is modified from Fig. 7.6 of Casjens et al. (32).