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Structure and Assembly

Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly

Julie A. Thomas, Andrea Denisse Benítez Quintana, Martine A. Bosch, Adriana Coll De Peña, Elizabeth Aguilera, Assitan Coulibaly, Weimin Wu, Michael V. Osier, André O. Hudson, Susan T. Weintraub, Lindsay W. Black
R. M. Sandri-Goldin, Editor
Julie A. Thomas
aThomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, USA
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Andrea Denisse Benítez Quintana
aThomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, USA
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Martine A. Bosch
aThomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, USA
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Adriana Coll De Peña
aThomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, USA
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Elizabeth Aguilera
bNatural and Physical Sciences, Baltimore City Community College, Baltimore, Maryland, USA
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Assitan Coulibaly
bNatural and Physical Sciences, Baltimore City Community College, Baltimore, Maryland, USA
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Weimin Wu
cNational Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Michael V. Osier
aThomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, USA
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André O. Hudson
aThomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, New York, USA
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Susan T. Weintraub
dUniversity of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Lindsay W. Black
eUniversity of Maryland School of Medicine, Baltimore, Maryland, USA
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R. M. Sandri-Goldin
University of California, Irvine
Roles: Editor
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DOI: 10.1128/JVI.01492-16
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  • FIG 1
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    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
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    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
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    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
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    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
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    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
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    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
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    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

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  • TABLE 1

    Identification of mutations resulting in amber codons in the SPN3US genome in a mixture of 50 mutant candidatesa

    Genome positionbGene locationReference baseAlternate baseNo. for reference basecNo. for alternate basedAlternate frequency (%)
    215999241CT28,2783,64512.9
    2342729CT17,3151,77610.3
    195232225CT33,8872,1816.4
    169242186CT22,7891,3746.0
    186856214GA22,3751,2405.5
    3745539CT31,4281,4394.6
    192318222GA25,5069953.9
    3326135CT35,0901,2953.7
    3081134CT37,7021,3703.6
    6384964CT40,3301,4153.5
    4633047CT32,8161,0923.3
    201073235CT36,5751,1893.3
    6665870CT28,6689163.2
    153240169CT25,2417853.1
    2398230CT34,6301,0543.0
    230169258GA17,3534992.9
    1399719CT24,5775972.4
    203975238GA21,9025132.3
    159924171CT18,2254202.3
    2015925CT28,4446122.2
    190698219CT34,5017312.1
    146047168CT31,7616332.0
    159099171CT48,2848981.9
    6395764CT41,3256381.5
    170989186GA30,7584161.4
    179577203CT26,1623221.2
    179287203GA29,5043521.2
    7702278CT33,5422390.7
    8182982GA37,1672190.6
    229576257CT33,2231830.6
    170544186CT30,2321260.4
    • ↵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

    MixtureMutantMutation positionaSNP %SPN3US ORFDNA changebAmino acid changec
    1am2118685685.70214c.682C→Tp.Q228.
    am2820397513.80238c.1186C→Tp.Q396.
    2am2415909982.30171c.361C→Tp.Q121.
    am3019069815.70219c.271C→Tp.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

    MutantGenome reference positionaReference baseCalled baseSNP %SPN3US ORFDNA changecAmino acid changed
    am1b46630CT99.847c.1432C→Tp.Q478.
    am263849CT99.664c.763C→Tp.Q255.
    am377022CT99.678c.1459C→Tp.Q487.
    am6b20159CT99.525c.184C→Tp.Q62.
    am11215999CT100.0241c.2011C→Tp.Q671.
    am1366658CT99.770c.181C→Tp.Q61.
    am18179577CT99.8203c.377G→Ap.W126.
    am19179287GA99.8203c.667C→Tp.Q223.
    am2223427CT100.029c.358C→Tp.Q120.
    am26b13997CT99.519c.70C→Tp.Q24.
    146047CT99.8168c.4673G→Ap.W1558.
    am2763957CT99.564c.871C→Tp.Q291.
    100963CT99.9112c.100C→Tp.Q34.
    am39169242CT99.5186c.343C→Tp.Q115.
    am43215999CT100.0241c.2011C→Tp.Q671.
    am50170544CT99.5186c.1645C→Tp.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 productMass (kDa)EssentialaϕKZ homologFunction/commentb
    1910.6ND
    2514.6YesORF62STR; low-copy-number tail protein
    2924.5YesORF67
    4762.8YesSTR; head/neck protein
    6448.9YesORF101STR; head, possible neck protein
    7032.2Yes
    7860.3Yes
    11210.9ND
    168188.1ExpectedORF145STR; baseplate/fiber protein
    17147.1YesORF130STR; possible baseplate/fiber protein
    18695.7YesORF165SbcC subunit
    20351.9YesORF157STR; tail protein
    21428.1YesORF153STR; head protein
    21928.6YesORF147
    23882.1YesORF182STR; tail protein, possible host membrane-targeting function (predicted N-terminal transmembrane domain residues 152 and 174)
    241159.1YesORF178STR; vRNAP βN
    • ↵a ND, the “essential” status of proteins encoded by genes with amber mutations in double mutants was not determined.

    • ↵b STR, protein detected as part of the virion by mass spectrometry.

  • TABLE 5

    Mass spectral counts detected for 64_112(am27) and 241(am11) propagated on permissive (sup+) and nonpermissive (sup−) hostsa

    Gene productLength (aa)Total spectrum countvRNAP subunit
    am27am11
    sup+sup−sup+sup−
    371276060
    424313736270β′M
    1581713020
    2182222328240βC
    2405195638140β′N
    2411,4012031521100βN
    2442401213100β′Cb
    • ↵a 64_112(am27) grown on the nonpermissive host formed tailless particles.

    • ↵b gp244 was identified as a candidate for the C-terminal region of β′ in this study (see the text).

  • 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:
    gp75bgp256bgp260bgp19gp25gp29gp47gp64gp70gp78gp112gp168gp171gp186gp203gp214gp219gp238gp241gp244c
    PhiEaH288858769785671294977448163807865757888
    CR56367705142273826314643574744535971
    ϕKZ2426342024162022302124222930
    201ϕ2-12325342123102324291423213428
    ϕPA32327331525152023301422213129
    Ea35-702826342425231622352522223030
    PhiEaH1222731172623152022292017243027
    RSL22328311821161624241322253230
    JM-2012222531172214?24231822202826
    VP4B21212820221220222418172624
    OBP23202922251319222418202618
    EL21222526251318212418182516
    ϕR1-3728252117
    AR92813232115
    BpSp2614242019
    • ↵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.

Additional Files

  • Figures
  • Tables
  • 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

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Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly
Julie A. Thomas, Andrea Denisse Benítez Quintana, Martine A. Bosch, Adriana Coll De Peña, Elizabeth Aguilera, Assitan Coulibaly, Weimin Wu, Michael V. Osier, André O. Hudson, Susan T. Weintraub, Lindsay W. Black
Journal of Virology Oct 2016, 90 (22) 10284-10298; DOI: 10.1128/JVI.01492-16

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Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly
Julie A. Thomas, Andrea Denisse Benítez Quintana, Martine A. Bosch, Adriana Coll De Peña, Elizabeth Aguilera, Assitan Coulibaly, Weimin Wu, Michael V. Osier, André O. Hudson, Susan T. Weintraub, Lindsay W. Black
Journal of Virology Oct 2016, 90 (22) 10284-10298; DOI: 10.1128/JVI.01492-16
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