Hybrid Vigor: Importance of Hybrid λ Phages in Early Insights in Molecular Biology

Abstract

Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ imm21, λ imm434, and λ h434 imm21. These sequences show that the replacements of λ DNA by nonhomologous segments of 21 or 434 DNA occurred through homologous recombination in adjacent sequences that are nearly identical in the parental phages. These five genome sequences correct a number of errors in published sequence fragments of the 21 and 434 genomes, and they point out nine nucleotide differences from Sanger's original λ sequence that are likely present in most extant λ strains in laboratory use today. We discuss the historical importance of these hybrid phages in the development of fundamental tenets of molecular biology and in some of the earliest gene cloning vectors. The 434 and 21 genomes reinforce the conclusion that the genomes of essentially all natural λ-like phages are mosaics of sequence modules from a pool of exchangeable segments.

Keywords: bacteriophage; hybrid phage; lysogeny; phage; phage 21; phage 434; phage genetics; phage lambda; temperate phage.

FIG 1

Genome maps of phages 21 and 434. Maps of phages 21, λ, and 434 are shown with selected λ genes indicated; homologs in 21 and 434 are marked by the λ gene name. At the bottom, the major transcription units are indicated by horizontal arrows: red, early left and right operons; black, late operons; green, repressor transcript expressed in the lysogen. The universal order of gene functions in the λ-like phages is shown at the bottom. Gray areas between maps indicate regions of homology, with the percent identity values shown.

FIG 2

Phage 434B genome structure. The region of difference between 434 and 434B is shown, with putative genes indicated as pointed boxes, where the points indicate the direction of transcription. Selected 434 gene names are indicated on the map, and λ gene names are shown above the maps. Asterisks mark nonfunctional genes. The solid horizontal black bar at the top indicates the extent of DLP12 DNA in 434B. Red vertical bars mark the regions of identity where homologous recombination occurred between 434 and DLP12 to create 434B (see the text). A kilobase pair scale is shown below the 434 map.

FIG 3

Original λ imm434 construction. The five crosses used in the original construction of λ imm434 by Kaiser and Jacob (99) are shown in rounded boxes. λ DNA is shown as a red line, and 434 DNA is shown as a green line. A question mark indicates the initial boundary, whose location was uncertain. Selection used after each cross is shown between the boxes.

FIG 4

Structures of the λ imm434 and λ imm21 genomes. Maps of the immunity regions of λ, 434, and 21 are shown, with genes indicated by pointed boxes that denote the direction of transcription. Light yellow-shaded areas with percent identity indications represent regions of similarity. Red connecting trapezoids between genomes mark the identical regions within these regions of similarity, where the homologous recombination events that created λ imm434 and λ imm21 occurred. Above and below the maps, thick red horizontal bars denote the locations of the nonhomologous 434 and 21 immunity mosaic sections or modules, thin horizontal red lines mark the further extent that 434 or 21 DNA replaced λ DNA in λ imm434 and λ imm21, and dashed red lines mark the regions in which the homologous recombination events occurred.

FIG 5

Structure of the λ h434 imm21 genome. The λ h434 imm21 genome is shown at the top, with DNAs from different parents indicated by different colors. See the text for discussion of the following unexpected sequences: 434 DNA in the left end terS gene region and a patch of λ DNA in the imm21 region. The exchange that generated the h434 allele is shown at the bottom, with the genes in the region indicated.

FIG 6

Phage-specific protein domain interactions deduced through use of hybrid phages. Pointed boxes represent the genes involved and their encoded proteins with their N termini on the left (not drawn to scale). The points on the boxes indicate the direction of transcription. Vertical bars within the boxes denote the boundaries between protein functional domains, and arrows above each map indicate species-specific protein-protein or protein-DNA interactions. (A) Specificity domains in λ O protein. The black rectangle indicates the location of the replication origin (ori). The N-terminal portion of λ O protein specifically binds to the phage replication origin, ori, and the C-terminal part interacts with λ P protein, which in turn interacts with the host’s replication machinery (adapted from Furth et al. [104]). (B) Specificity domains in λ terminase. The TerS N-terminal portion contains a winged helix-turn-helix DNA binding domain (HTHw) that interacts with the DNA packaging recognition site cosB. The TerS C-terminal domain interacts in a species-specific manner with the N-terminal region of TerL, and the C terminus of TerL, in turn, is a specificity domain for interaction with the procapsid’s portal protein (190–192). (C) Specificity domains of the P22 terminase. The black rectangle indicates the packaging recognition site pac (~20 bp, codons 90 to 96 of the 162-codon gene). The interactions parallel those of λ, except that the pac site is inside the terS gene (, ; E. Gilcrease and S. Casjens, unpublished results).