Organization of a contractile injection system and its membrane-attacking spike.

A, A schematic showing the main components of R-type pyocin, one of the simplest CIS [16]. B, Two most common architectures of central spike complexes. For clarity, the diagram does not show facultative enzymatic or partner-attachment domains. The type of targeted bacterial cell wall is indicated. C, Gene organization of central spike complexes. The diagram depicts only two of the most common occurrences of facultative domains, which are shown semitransparent. In type II complexes, the tip gene is not necessarily immediately downstream of the spike gene.

Structure and properties of spike-tip complexes.

A. Ribbon diagrams of the PAAR repeat tip protein gp5.4 with key residues labeled. The structural elements equivalent to PAAR motif sequences are colored magenta. The corresponding amino acid sequences are given below in the magenta box. The prolines are colored gray. The cyclically permuted element is labeled with a red arrow. B. The crystal structure of gp5β-gp5.4 is shown as a ribbon diagram. One of the three chains of gp5β is colored in pink while the other two are in semitransparent gray. The gp5.4 protein is colored in a rainbow pattern along the length of the polypeptide chain with the N-terminus in blue and the C-terminus in red. The molecular surfaces are colored according to their electrostatic potential and Kyle-Doolittle hydrophobicity, respectively. C. Hydrogen bond network between main chain gp5β and gp5.4 that seals the hydrophobic interface between two proteins.

Location of spike-tip complexes in phages.

A and B, Cryo-EM reconstructions of the baseplate regions of T4 5.4am (A) and RB43 (B). Isosurfaces colored by cylinder radius with rainbow palette. C and D, Sliced view of cryo-EM reconstruction of the T4 5.4am (C) and RB43 (D) baseplates. The rigid-body fitted (gp27)3-(gp5)3-gp5.4 protein complex structure, is shown as a ribbon representation fitted into the T4 5.4am cryo-EM map (C). The RB43 baseplate with the rigid-body fitted spike (orf204). The structures predicted by AlphaFold are in a ribbon representation.

Localization of the P2 phage spike protein in the infected cell.

A, Flowchart of the fractionation procedure. The same nomenclature (colors and abbreviations) is used to label the cellular fractions in panels C and D. B, The two top rows in the left panel show titration of P2 vir1 Vam46 on either C-520 (permissive, supD) or C-2 (non-permissive) E. coli lawns. The two bottom rows in the left panel show titration of P2 vir1 Vam46 that remained in solution after a 15 min incubation with cells. These cells were subsequently used in the fractionation procedure. The right panel shows quantitative representation (mean ± SD) of the titration-presented in the left panel. The experiment was repeated six times with similar outcomes. C, Western blot analysis of cell fractions from cultures which were either uninfected (left section of the blot) or infected with P2 Vir1 Vam46 (right section). Each column is a separate fraction of the fractionation procedure shown in panel A. The rows correspond to different antibodies used against cellular proteins with known localizations: GroEL is a soluble cytoplasmic protein, MalE (MBP, maltose binding protein) is a soluble periplasmic protein, OmpF is the outer membrane porin F. GpV co-localizes with MalE. The blot is representative of four biological replicates. D, A Coomassie stained polyacrylamide SDS gel showing a purified P2 Vir1 phage sample (labeled P2) and fractionated lysates of uninfected C-2 cells (labeled N) or infected with P2 Vir1 Vam (labeled I). The red arrow points to the P2 sheath protein; its identity was confirmed by LC/MS/MS analysis. The P2 capsid protein is partially masked by a cellular membrane component with a similar electrophoretic mobility. The inset in a black box (lower right) shows qPCR analysis of P2 genomic DNA (mean ± SD) found in different cellular fractions. The fluorescent signal was converted to the number of plaque forming units (pfus) using a calibration curve as described in the methods section. The experiment was repeated three times with similar outcomes. The significance was determined by Student’s two-tailed t-test with one, two, and three stars (*, **, ***) corresponding to p-values of less than than 0.05, 0.001, and 0.0001, respectively.

Comparative properties of T4+ and T4 (5.4am).

A, Single growth cycle of T4+ and T4 5.4am phages. E. coli BE cells (non-permissive = sup+) were infected at a multiplicity of infection (MOI) of 0.05, and intracellular phage accumulation was measured at the indicated time points. Phage stocks were produced on the sup+ E. coli strain BE and the progenies were analyzed on the supD permissive suppressor strain CR63. Data points represent the mean and range of two independent experiments normalized to the input titer. The inset shows the plaque morphology on BE bacteria. B, Evolution of the T4+ to T4 5.4am ratio when mixed phage inoculums were grown on E. coli BE (sup+) or CR63 (supD) cultures over eight successive growth cycles. The initial T4+/T4 5.4am ratio was 0.08 and the MOI of each growth cycle was less than 0.1. The percentage of 5.4+ phages was determined by PCR of genomic DNA and restriction. The data points represent the mean and range of two independent experiments. The dashed and dotted lines are mathematical simulations in which the fitness of the T4 5.4am mutant phage was 1.55 (sup+) and 1.14 (supD) times lower than that of the WT. C, Comparison of the ratio of the infectious to physical virions for T4+ and T4 5.4am. The relative ratios of pfu/ml to A260nm are plotted for three independent biological samples of T4+ and T4 5.4am which were grown on E. coli BE and purified on CsCl gradients (Materials and Methods). The titers were determined on E. coli BE, and the 260nm absorbances were measured by UV spectroscopy. The error bars are SDs which were derived from errors associated with titer and absorbance measurements (each parameter was measured three times). The significance was determined by two-way ANOVA with a p-value of less than 0.0001 (****). D, E, F, G. Characterization of infection of sup+ E. coli K-12 B178 and its transposon mutant hldD::Tn10 by T4+ and T4 5.4am. Efficiency of plating (EOP), adsorption, phage yield, and efficiency of center of infection (ECOI) were determined as described in Material and Methods. The cells used for infection were grown to OD600 = 0.5 in LB medium supplemented with tryptophan (50µg/ml) and glucose (0.4 %). Phage stocks were produced on non-permissive sup+ strain BE and purified using CsCl gradients in all experiments except for the phage yield determination studies in which the phage was produced on a permissive supF strain (T4-(5.4am)-gp5.4 particles). The results represent the mean of three independent experiments.

Gp5.4 is essential for T4 growth on deep-rough LPS mutants.

A, Growth of T4+, T4 5.4am, and T4 K10 38am 51am on B178 (WT) and hldD::Tn10 bacteria. Phage stocks produced on sup+ (BE), supF (NM538) or supD (CR63) bacterial strains were diluted and spotted onto the indicated bacterial lawns. Results are representative of three independent experiments. B, The hldD-waaL operon of E. coli K12 and the biosynthesis pathway of the core LPS, adapted from [61]. C, Silver-stained Tris-tricine SDS-PAGE analysis of LPS extracted from the indicated strains. D, Growth of T4+ and T4 5.4am on hldD::Tn10 transformed with an empty vector (pCA24N) or plasmids (p-) expressing hldD (pCA24N-hldD) and rfaC (pCA24N-rfaC). Top agar plates were inoculated with exponentially growing cultures induced with 100µM IPTG for 1 hour and spotted with dilutions of phage stocks produced on a sup+ E. coli strain. Results are representative of four independent experiments. E, Adsorption and growth of T4+ and T4 5.4am on B178 (WT) and hldD::Tn10 b bacteria infected at a low MOI (between 0.025 and 0.05) at 37°C (see Materials and Methods). Aliquots were withdrawn at indicated times, treated with chloroform, and plated on E. coli BE at appropriate dilutions.

The 5.4am null mutation can be complemented by providing gp5.4 either in vivo during phage growth or in vitro to a phage stock.

T4+ and T4 5.4am phage stocks were either produced on a supF strain (E. coli K-12, NM538) or on sup+ strain (E. coli B strain, Rosetta DE3) harboring either an empty vector (pET23d) or a plasmid expressing 5.4 (pET23d-5.4) induced with 1 mM IPTG. Phage stocks 5 and 6 were complemented with recombinant gp5.4 in vitro by incubation for 45 min at 37°C with a fresh cell extract made from uninfected bacteria expressing gp5.4 (see Materials and Methods for details). Phage stocks were titrated on E. coli BE and equal amounts were spotted onto the hldD::Tn10 harboring pBSPL0+, a plasmid expressing a supF suppressor tRNA under the control of a T4 late promoter (p-supF). The phage stocks were also spotted on the hldD::Tn10 mutant harboring pBR322 ΔTc (empty vector) and on B178 (WT) harboring pBSPL0+ (p-supF). The experiment is representative of two independent experiments.