Figure 1.
Relationships among tail needle C-terminal domains of the P22-like phages.
A neighbor-joining tree (created with Clustal X2 [80]) is shown with selected branch lengths (numbers between 0. and 1) and bootstrap values out of 1000 trials (between 1 and 1000). The nodes far from the branch tips are not well-supported and are not shown. A scale in fractional difference is shown in the lower left. Branches A, B and C have many members and the splits at these branch tips show the regions within which the individual members diverge (the larger tree in figure S1 shows the placement of all the individual sequences). The “phage Sf6” branch is not related to the other branches and its inclusion here is to demonstrate this, and does not imply any phylogenetic relationship with the other branches. The branches not labeled “phage” are from P22-like prophages in the following bacterial genome sequences: Rett1, Providencia rettgeri DSM 1131; Ars1, Arsenophonus nasoniae; APSE-1/−2, Hamiltonella defensa; øSG1, Sodalis glossinidius; Cart1, Pectobacterium carotovorum PBR1692; Morg1, Morganella morganii KT; Serr1/2/3, Serratia plymuthica strains AS9, AS12 and AS13; Blatt1/2, Escherichia blattae strains DSM 4481 and 105725.
Table 1.
Crystallographic data collection and refinement statistics.
Figure 2.
Atomic structure of the phage HS1 tail needle knob.
A. Ribbon diagram of bacteriophage HS1 tail needle knob determined crystallographically to 1.1 Å resolution; the N-termini are at the bottom of the diagram. Helices are shown in red, ß-sheets in yellow, and random coil in green; the bound L-glutamate is shown as sticks-and-balls and phosphate is shown as a small red sticks. B. Magnified view of L-glutamate trapped at the HS1 needle knob dimeric protomer:protomer interface. L-glutamate (in stick-and-balls) is overlaid to the final 2Fo-Fc electron density map (gray) contoured at 1.5σ above background. C. Side chains (sticks) from protomer A (yellow) and protomer B (green) that interact with L-glutamate (stick-and-balls). The indicated HS1 needle amino acids correspond to Sf6 needle amino acids as follows with Sf6 residue numbers in parentheses: Glu181(146), Lys235(200), Ser283(248), Asp285(250), Leu290(255) and Lys312(277). D. Structural models of full length Sf6 and HS1 tail needles. The two amino acid differences (from the Sf6 needle) that lie at positions in or near the knob domain, Ser169 and Ala305 of the HS1 tail needle, are shown as blue spheres. The models were obtained by using the Robetta full-chain protein structure prediction server [81]; the N-terminal parts of the needle protein shafts whose structures are modeled from the homologous P22 tail needle have a light gray surface contour behind. In all the panels, α-helices, β-strands and loops are colored in red, yellow and green, respectively.
Figure 3.
Structural models of the P22 gp26 tail needle and chimeric tail needles.
A. Crystal structure of P22 tail needle gp26 (pdb 3C9I). B–D. Homology structural models of chimeric P22 needles with Sf6 knob (in phage UC-0911), HS1 knob (in phage UC-0926) and foldon tip (in phage UC-0927). Chimeric models were generated for illustration with align function of PyMol (Version 1.3, Schrodinger, LLC, San Carlos, CA), where C-terminal knob domains of Sf6 and HS1 tail needle (pdb 3RWN, 4K6B), C-terminal foldon domain from fibritin fiber of the bacteriophage T4 (pdb 1AA0) were fused downstream of P22 gp26 tail needle helical core residues 1–140 (pdb 3C9I), respectively. In all three models, arrow indicates point of fusion.
Table 2.
Phage and bacteria used in this study.
Table 3.
Effects of genetic modification the C-terminal needle domain.
Figure 4.
Potassium ion release by phage P22 infection.
A. Infection of Salmonella strain UB-0001 by P22 clear mutant phage UC-0011 at different multiplicities of infection (MOIs). Full strain genotypes at given in Table 2 of article text. B. Infection of Salmonella UB-0001 at 30° and 37°C by phage P22 UC-0011. C. Infection of Salmonella host strains that have (UB-0001) or do not have (UB-2130) P22’s O-antigen surface polysaccharide receptor by phage P22 UC-011; potassium ion release by uninfected Salmonella UB-0001 is also shown for comparison. D. Infection of a Salmonella host that has no P22 prophage (UB-0001) and a host that carries a P22 prophage that expresses its repressor (c2) gene but is missing the sieA and gtrABC genes (UB-0134) by P22 UC-0011. E. Infection of Salmonella UB-0001 by P22 phages that carry two mutations in the tail needle knob that should abrogate L-glutamate binding (see Table 2 of article text for amino acid changes). All infections were carried out at 30°C and MOI of 10 unless otherwise indicated. F. Infection of Salmonella UB-0001 by P22 UC-0011 with and without 10 mM L-glutamate added to the medium outside of the cells. Potassium ion measurements were performed as described in Materials and Methods of article text.
Figure 5.
Potassium ion release by P22 phages with modified tail needles.
A. Infection of Salmonella strain UB-0001 by P22 phages with altered needle proteins at MOI = 10. Potassium ion release was measured at 30°C as described in Materials and methods with a potassium electrode. Salmonella host strain UB-0001 was infected by the following phages: UC-0911 fully P22 tail needle (▴); UC-0918, needle has Sf6 C-terminal knob domain and part of the shaft (▵); UC-0926, needle has HS1 C-terminal knob domain (•); UC-0927, foldon replaces needle C-terminal domain (⧫); no phage infection (○). B. Infection of Salmonella strain UB-0001 by P22 phages with foldon-tipped needle at various MOIs as follows: P22 UC-0911 with fully P22 tail needle at MOI = 10 (○); P22 UC-0927 where foldon replaces needle’s C-terminal domain at MOI = 10 (•), MOI = 50 (▪) and MOI = 100 (⧫); no phage (□). The horizontal axis is time after infection.