Novel DNA packaging recognition in the unusual bacteriophage N15

Abstract

Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos(N15)) is closely related to cos(λ), but whereas the cosB(N15) subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB(λ). A bioinformatic study of N15-like phages indicates that cosB(N15) also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB-λ. The DNA binding domain of TerS-N15 is a dimer.

Keywords: Bacteriophage; DNA packaging; Terminase; Virus DNA packaging; Virus DNA recognition; Virus assembly; Virus evolution.

Figure A1. Sedimentation velocity experiment with gp1 NTD (1-93)

Purified gp1-E93 was loaded in 3 different concentrations into analytical ultracentrifuge cells and centrifuged at 50,000 RPM for 12 hours in 100 mM NaCl + 20 mM Tris, pH 8.22 at 20 C. A_o at 230 nm for cell 1= 1.2551, cell 2= 0.628 and cell 3= 0.2865. SEDFIT and NLLS programs were used to determine sedimentation rates and molecular weights (freeware, non-linear least squares). Left panel: A230 scans as a function of radial position taken at hourly intervals. Right panel: The data in left panel were analyzed by NLLS (non-linear least squares) to determine the molecular weight.

Figure A2. Sedimentation velocity experiment with DNA oligomers

1 μM N15 specific DNA (left panel, N15 bp 36-65) and non-specific DNA (right panel) and increasing concentrations of gp1-E93 in 100 mM NaCl, 20 mM Tris, pH8.22 at 20C. Centrifugation and analysis as described in legend to Figure A1.

Figure 1. Comparison of the coses and terminase small subunit genes of λ, 21 and N15

(A) cos^λ (above) and cos^N15 (below). (B) Alignment of left ends of λ (above) and N15 (below) DNAs. Vertical lines indicate sequence identity. Bp 1-15 (boxed) are cosN base pairs at the mature left chromosome ends. The large box (dotted) is I2, a sequence feature highly conserved in λ-like phages. The R3 and R2 segments of cos^λ are also boxed. (C) Alignment of the DNA binding segments of the λ and N15 terminase small subunits. The winged helix-turn-helix DNA binding motif of λ gpNu1 is formed by residues 5-12 (support helix), 13-15 (turn), 16-25 (recognition helix) and wing (residues 31-39) (de Beer et al., 2002).

Figure 2. Cross of λ cos2 with pJM0N15 to generate recombinant phages with chimeric terminases

The lethal cos2 deletion prevents DNA packaging by λ cos2. Recovery of plaque forming recombinants required that λ cos2 pick up a functional cosN from pJMN015 by a double crossover as indicated by the dotted lines. pJM0N15 contains λ sequence from 44,141 to an introduced XbaI site at 48442. To the right of the XbaI site is N15 sequence including cos and the terminase genes. To pick up cosN, the left crossover occurs in the long region of λ sequence homology to the left of the XbaI site. The right crossover takes place between segments of partial sequence identity between the λ and N15 segments of λ cos2 and pJM0N15, generating hybrid phages with a chimeric terminase gene. Recombinants were selected as Kn^R-transducing phages. The small black rectangle represents cos^N15, and the small white box represents the cos2 mutation.

Figure 3. Defining cosB^N15: effects of scramble mutations on N15 cosmid packaging

(A) Sequence of the N15 chromosome showing locations of the scramble mutations. The gene 1 ATG start codon is underlined. See text for additional details. (B) Effects of scramble mutations on packaging of N15 cosmids by helper phage λ–P1 N15^hy4. Numbers along the x axis are N15 base pair positions. The large blue rectangle represents the N15 1 gene. The y axis is the level of cosmid-containing Ap^R transducing phages/induced lysogen, normalized to 100% the yield the wild type cosmid (dotted line). Dashed lines represent the standard error of the mean (SEM) limits for yields of the wild type cosmids for the two sets of experiments. Vertical bars represent the yields of scramble mutations. Experiment 1 included the sc^42-47, sc^48-53, sc^54-59, sc^60-65, sc^66-71 mutations, and the yield of Ap^R-transducing phages for the cos⁺ cosmid was 7.5 [SEM=0.72]/induced cell. Experiment 2 included the sc^71-91 and sc^92-199 mutations, and the yield of Ap^R-transducing phages for the cos⁺ cosmid was 2.6 × 10⁻³ [SEM= 9.5 × 10⁻⁴]. Results are from three independent experiments.

Figure 4

Cosmid packaging experiment: Effects of R3 and rR2 mutations on cosmid packaging by N15^hy4 terminase.

Figure 5. Summary of proposed cosB structures for λ, Monarch, N15, and Puccoon

Zones highlighted in green are segments of significant sequence identity, ~50% or more. Blue zone is the region of structural, but not sequence, similarity between λ and Monarch. TerS binding sites (R sequences) are indicated by blue boxes, and IHF binding sites (I1) by red boxes. Alignments of cos and TerS sequences for N15-like phages and prophages with bipartite cosBs are in Figure S3A and S3B, respectively. Alignments of cos and TerS sequences for Monarch-like prophages with tripartite cosBs are in Figure S4A and S4B, respectively.

Figure 6. Unequal crossing over by a phage with a tripartite, λ-like cosB generates a cosB with a single recombinant R3/R1 sequence

See text for discussion.