Article Figures & Data
Figures
- FIG 1
Map of the SPN3US genome highlighting genes with homologs. Homologs identified in both phages PhiEaH2 and CR5 are shown in purple and homologs of ϕKZ in black. Newly identified essential genes are indicated by red-shaded Es, and genes whose essential status requires confirmation are indicated by question marks. In the SPN3US track, genes colored dark blue have a homolog of a head protein in ϕKZ, genes colored light blue have homology with a tail protein in ϕKZ, and genes colored yellow have homology with a virion protein in ϕKZ whose head/tail location is unassigned. Subunit names of the vRNAP are shaded in green. Subunit names of the nvRNAP are shaded in yellow.
- FIG 2
SDS-PAGE profiles of SPN3US, ϕKZ, and 201ϕ2-1 phages. (A) Profiles of CsCl step gradient-purified P. aeruginosa phage ϕKZ, P. chlororaphis phage 201ϕ2-1, and S. enterica serovar Typhimurium phage SPN3US. (B) Profiles of wild-type (WT) and amber mutant phages of SPN3US after propagation on nonpermissive S. enterica serovar Typhimurium (strain TT9079).
- FIG 3
Cross-plating of SPN3US amber mutants in the nonpermissive S. enterica serovar Typhimurium strain (TT9079). Approximately 107 particles of 29(am22) (A) and 64_112(am27) (B) were seeded into the bacterial overlay (the boxed areas show no plaques/lysis in the lawn). Mutant phages were spotted onto the overlay, with approximate numbers of PFU in each spot indicated. Note that when 29(am22) and 64_112(am27) were spotted onto the overlays containing themselves, there was only clearing caused by lysis from without in the presence of a large number of particles.
- FIG 4
SDS-PAGE gels used for mass spectrometry of SPN3US amber mutants. Shown are 241(am11) (A) and 64_112(am27) (B). sup+, mutant propagation on the permissive host; sup−, mutant propagation on the nonpermissive host.
- FIG 5
Map of SPN3US genome comparing proteomes of mutant 64_112(am27) when grown on permissive (sup+) and nonpermissive (sup−) hosts. The proteome of 64_112(am27) propagated on the sup+ or permissive host is indicated in green, and its proteome when propagated on the sup− or nonpermissive mutant host is indicated in blue. The gene encoding gp64 (marked with a red cross) is essential, as determined by sequencing of a mutant (am2) with a single amber mutation in its genome, in the gp64 gene. gp112 is not a virion protein, and its essential status requires clarification. In the SPN3US track, genes colored dark blue have homology with a head protein in ϕKZ, genes shaded light blue have homology with a tail protein in ϕKZ, and genes colored yellow have homology with a virion protein in ϕKZ whose head/tail location in unassigned.
- FIG 6
Transmission electron microscopy of SPN3US wild type and amber mutant phage 64_112(am27). (A) Wild-type phage in a nonpurified lysate stained with PTA. (B) CsCl-purified wild-type phage stained with ammonium molybdate. (C and D) CsCl gradient-purified particles of 64_112(am27) grown on the nonpermissive host stained with ammonium molybdate (C) and uranyl formate (D).
- FIG 7
Scheme showing the homologous vRNAP subunits of SPN3US, ϕKZ, and 201ϕ2-1 (2-1). Subunits detected by mass spectrometry in purified virions are bracketed in green. The newly identified C-terminal subunit of vRNAP β′ (SPN3US gp244) is boxed in red. The orange triangles indicate introns, and the blue square indicates a homing nuclease. The SPN3US nvRNAP subunits are also included. Cellular RNAP conserved regions and archaeal and cyanobacterial subunit split sites in β and β′ are indicated (56, 61). Note that the ∼300-residue lineage-specific insert in T. thermophilus β′ located between the Lane all regions a5 and a6 is not indicated.
Tables
- TABLE 1
Identification of mutations resulting in amber codons in the SPN3US genome in a mixture of 50 mutant candidatesa
Genome positionb Gene location Reference base Alternate base No. for reference basec No. for alternate based Alternate frequency (%) 215999 241 C T 28,278 3,645 12.9 23427 29 C T 17,315 1,776 10.3 195232 225 C T 33,887 2,181 6.4 169242 186 C T 22,789 1,374 6.0 186856 214 G A 22,375 1,240 5.5 37455 39 C T 31,428 1,439 4.6 192318 222 G A 25,506 995 3.9 33261 35 C T 35,090 1,295 3.7 30811 34 C T 37,702 1,370 3.6 63849 64 C T 40,330 1,415 3.5 46330 47 C T 32,816 1,092 3.3 201073 235 C T 36,575 1,189 3.3 66658 70 C T 28,668 916 3.2 153240 169 C T 25,241 785 3.1 23982 30 C T 34,630 1,054 3.0 230169 258 G A 17,353 499 2.9 13997 19 C T 24,577 597 2.4 203975 238 G A 21,902 513 2.3 159924 171 C T 18,225 420 2.3 20159 25 C T 28,444 612 2.2 190698 219 C T 34,501 731 2.1 146047 168 C T 31,761 633 2.0 159099 171 C T 48,284 898 1.9 63957 64 C T 41,325 638 1.5 170989 186 G A 30,758 416 1.4 179577 203 C T 26,162 322 1.2 179287 203 G A 29,504 352 1.2 77022 78 C T 33,542 239 0.7 81829 82 G A 37,167 219 0.6 229576 257 C T 33,223 183 0.6 170544 186 C T 30,232 126 0.4 ↵a Mutations subsequently identified in individual mutant phage genomes (Tables 2 and 3) are in boldface.
↵b Position relative to that of GenBank accession number JN641803.1.
↵c Number of times reference base observed.
↵d Number of times alternate base observed.
- TABLE 2
Amber mutation sites in mixtures containing two SPN3US amber mutants whose particles were combined in amounts differing by 6.25-fold
Mixture Mutant Mutation positiona SNP % SPN3US ORF DNA changeb Amino acid changec 1 am21 186856 85.70 214 c.682C→T p.Q228. am28 203975 13.80 238 c.1186C→T p.Q396. 2 am24 159099 82.30 171 c.361C→T p.Q121. am30 190698 15.70 219 c.271C→T p.Q91. ↵a Position relative to that of GenBank accession number JN641803.1. Amber mutation positions were confirmed in each mutant by Sanger sequencing.
↵b “c.” indicates the coordinate of the mutation within the open reading frame and its reference to called base change.
↵c Periods after position numbers indicate that the glutamine at that position in the polypeptide chain is not replaced, as its codon has been mutated to a nonsense codon and the protein product truncated.
- TABLE 3
Amber mutations identified in SPN3US individual mutant phage genomes
Mutant Genome reference positiona Reference base Called base SNP % SPN3US ORF DNA changec Amino acid changed am1b 46630 C T 99.8 47 c.1432C→T p.Q478. am2 63849 C T 99.6 64 c.763C→T p.Q255. am3 77022 C T 99.6 78 c.1459C→T p.Q487. am6b 20159 C T 99.5 25 c.184C→T p.Q62. am11 215999 C T 100.0 241 c.2011C→T p.Q671. am13 66658 C T 99.7 70 c.181C→T p.Q61. am18 179577 C T 99.8 203 c.377G→A p.W126. am19 179287 G A 99.8 203 c.667C→T p.Q223. am22 23427 C T 100.0 29 c.358C→T p.Q120. am26b 13997 C T 99.5 19 c.70C→T p.Q24. 146047 C T 99.8 168 c.4673G→A p.W1558. am27 63957 C T 99.5 64 c.871C→T p.Q291. 100963 C T 99.9 112 c.100C→T p.Q34. am39 169242 C T 99.5 186 c.343C→T p.Q115. am43 215999 C T 100.0 241 c.2011C→T p.Q671. am50 170544 C T 99.5 186 c.1645C→T p.Q549. ↵a Position relative to GenBank accession number JN641803.1.
↵b Mutant sequenced on an Illumina Hi-Seq machine.
↵c “c.” indicates the coordinate of the mutation within the open reading frame and its reference to called base change.
↵d Periods after position numbers indicate that the glutamine in that position in the polypeptide chain is not replaced, as its codon has been mutated to an amber stop codon and the protein product truncated.
- TABLE 4
Features of SPN3US proteins encoded by essential genes
Gene product Mass (kDa) Essentiala ϕKZ homolog Function/commentb 19 10.6 ND 25 14.6 Yes ORF62 STR; low-copy-number tail protein 29 24.5 Yes ORF67 47 62.8 Yes STR; head/neck protein 64 48.9 Yes ORF101 STR; head, possible neck protein 70 32.2 Yes 78 60.3 Yes 112 10.9 ND 168 188.1 Expected ORF145 STR; baseplate/fiber protein 171 47.1 Yes ORF130 STR; possible baseplate/fiber protein 186 95.7 Yes ORF165 SbcC subunit 203 51.9 Yes ORF157 STR; tail protein 214 28.1 Yes ORF153 STR; head protein 219 28.6 Yes ORF147 238 82.1 Yes ORF182 STR; tail protein, possible host membrane-targeting function (predicted N-terminal transmembrane domain residues 152 and 174) 241 159.1 Yes ORF178 STR; vRNAP βN - TABLE 5
Mass spectral counts detected for 64_112(am27) and 241(am11) propagated on permissive (sup+) and nonpermissive (sup−) hostsa
Gene product Length (aa) Total spectrum count vRNAP subunit am27 am11 sup+ sup− sup+ sup− 37 127 6 0 6 0 42 431 37 36 27 0 β′M 158 171 3 0 2 0 218 222 23 28 24 0 βC 240 519 56 38 14 0 β′N 241 1,401 203 152 110 0 βN 244 240 12 13 10 0 β′Cb - TABLE 6
Proteins in giant phages with similarity to SPN3US proteins that are encoded by genes identified as having an amber mutation in this study or expected to be essential
Phage % identity of match to SPN3US proteina: gp75b gp256b gp260b gp19 gp25 gp29 gp47 gp64 gp70 gp78 gp112 gp168 gp171 gp186 gp203 gp214 gp219 gp238 gp241 gp244c PhiEaH2 88 85 87 69 78 56 71 29 49 77 44 81 63 80 78 65 75 78 88 CR5 63 67 70 51 42 27 38 26 31 46 43 57 47 44 53 59 71 ϕKZ 24 26 34 20 24 16 20 22 30 21 24 22 29 30 201ϕ2-1 23 25 34 21 23 10 23 24 29 14 23 21 34 28 ϕPA3 23 27 33 15 25 15 20 23 30 14 22 21 31 29 Ea35-70 28 26 34 24 25 23 16 22 35 25 22 22 30 30 PhiEaH1 22 27 31 17 26 23 15 20 22 29 20 17 24 30 27 RSL2 23 28 31 18 21 16 16 24 24 13 22 25 32 30 JM-2012 22 25 31 17 22 14 ? 24 23 18 22 20 28 26 VP4B 21 21 28 20 22 12 20 22 24 18 17 26 24 OBP 23 20 29 22 25 13 19 22 24 18 20 26 18 EL 21 22 25 26 25 13 18 21 24 18 18 25 16 ϕR1-37 28 25 21 17 AR9 28 13 23 21 15 BpSp 26 14 24 20 19 ↵a Similarity was determined using PSI-BLAST with a maximum of three iterations.
↵b Three proteins were included as positive controls, as they must be essential in all myoviruses and are expected to be detectable in related phages. They are gp75 (major capsid), gp256 (sheath protein), and gp260 (terminase).
↵c The newly identified candidate vRNAP subunit, gp244, in SPN3US and its homologs are also included.
Supplemental material
- Supplemental file 1 -
Table S1 (Homologous proteins found in SPN3US, Erwinia phage phiEaH2, phage CR5, and Pseudomonas phage ϕKZ.)
Table S2 (Titers and reversion rates of SPN3US amber mutant candidates.)
Table S3 (Mutations detected in SPN3US amber mutants.)
Table S4 (Mass spectral counts obtained by GelCMS for the two amber mutants, 241am11 and 64_112am27.)
PDF, 374K
- Supplemental file 1 -
