TY - JOUR
T1 - Identification of essential genes in the Salmonella phage SPN3US reveals novel insights into giant phage head structure and assembly
AU - Thomas, Julie A.
AU - Quintana, Andrea Denisse Benítez
AU - Bosch, Martine A.
AU - De Peña, Adriana Coll
AU - Aguilera, Elizabeth
AU - Coulibaly, Assitan
AU - Wu, Weimin
AU - Osier, Michael V.
AU - Hudson, André O.
AU - Weintraub, Susan T.
AU - Black, Lindsay W.
N1 - Funding Information:
We are grateful to Stephen C. Hardies for invaluable discussions and for allowing use of his bioinformatics resources. We have enjoyed our interactions with Alasdair C. Steven and Naiqian Cheng throughout this work. We also thank the following individuals: Sangryeol Ryu, for providing bacteriophage SPN3US; John Roth, Natalie Duleba, and Sherwood Casjens for Salmonella strains; David Peabody for suppressor plasmids; Qin Dan for technical support; Ru-Ching Hsia (UMB Electron Microscopy Core Imaging Facility) for TEM analyses; Kevin Hakala, Sammy Pardo, and Dana Molleur (University of Texas Health Science Center at San Antonio [UTHSCSA]) for mass spectrometry analyses; Michelle Zanache, John Ashton, and Jason Myers (University of Rochester Genomics Research Center) for sequencing of mutants and helpful advice; and Borries Demeler and the UTHSCSA Bioinformatics Center for assistance with computational aspects of the project. Mass spectrometry analyses were conducted at the UTHSCSA Institutional Mass Spectrometry Laboratory. J.A.T. and L.W.B. conceived and supervised the project. J.A.T., A.D.B.Q., M.A.B., A.C.D.P., E.A., A.C., W.W., and L.W.B. performed experiments and data analyses. S.T.W. supervised the MS analysis and performed the MS data analysis. M.V.O. assisted with the bioinformatics analysis. A.O.H. assisted with data interpretation and preparation of the manuscript. We all read and approved the final manuscript. This study was supported by the Thomas H. Gosnell School of Life Sciences and the College of Science at RIT (J.A.T.), NIH grant AI11676 (L.W.B.), and NIH grant 1S10RR025111-01 (S.T.W.). This work, including the efforts of Julie A. Thomas, was funded by Thomas H. Gosnell School of Life Sciences. This work, including the efforts of Lindsay W. Black, was funded by HHS | National Institutes of Health (NIH) (AI11676). This work, including the efforts of Susan T. Weintraub, was funded by HHS | National Institutes of Health (NIH) (1S10RR025111-01).
PY - 2016
Y1 - 2016
N2 - Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage φKZ, have long genomes packaged into large, atypical virions. Many aspects of φKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with φKZ and related phages,~61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" β' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution.
AB - Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage φKZ, have long genomes packaged into large, atypical virions. Many aspects of φKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with φKZ and related phages,~61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" β' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution.
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U2 - 10.1128/JVI.01492-16
DO - 10.1128/JVI.01492-16
M3 - Article
C2 - 27605673
AN - SCOPUS:84995470800
VL - 90
SP - 10284
EP - 10298
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 22
ER -