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Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis.

Stockdale SR, Collins B, Spinelli S, Douillard FP, Mahony J, Cambillau C, van Sinderen D - PLoS ONE (2015)

Bottom Line: Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled.Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production.Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork, Western Road, Cork, Ireland.

ABSTRACT
Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

No MeSH data available.


Related in: MedlinePlus

EM images of (A-N) TP901-1erm structural module mutants and (O) TP901-1erm control.(A) Few tails, but no capsids, produced by mutant PortalTP901-1::Ter. (B) Capsids, without attached tails, visualised for mutant MCP1TP901-1::Ter. (C) Full, but scarce, phage particles produced following mutation MCP2TP901-1::Ter. (D) Tails, but no capsids, produced for mutant SfpTP901-1::Ter. (E) Tails-only produced for mutant MHPTP901-1::Ter. (F) Mature-resembling phages produced for mutant MCP3TP901-1::Ter. (G) Unconnected capsids and tails observed in HTC1TP901-1::Ter. (H) Distinct capsids and tails observed in HTC2TP901-1::Ter. (I) Separated capsids and tails imaged from mutant TapTP901-1::Ter. (J) Capsids with long polytails isolated for mutant TtpTP901-1::Ter. (K) Capsids only are observed for mutant MTPTP901-1::Ter. (L) Capsids and insoluble aggregates, proposed to be the TMP, observed in preparations of mutant gpGTP901-1::Ter. (M) Capsids, but no tails, observed in preparations of mutant gpTTP901-1::BamHI. (N) Mature-resembling phage produced by mutant gpGfsTTP901-1. (O) Control TP901-1erm mature infectious phages. Refer to text for a more detailed description characterizing each mutant. Scale bars are 50 nm long.
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pone.0131676.g003: EM images of (A-N) TP901-1erm structural module mutants and (O) TP901-1erm control.(A) Few tails, but no capsids, produced by mutant PortalTP901-1::Ter. (B) Capsids, without attached tails, visualised for mutant MCP1TP901-1::Ter. (C) Full, but scarce, phage particles produced following mutation MCP2TP901-1::Ter. (D) Tails, but no capsids, produced for mutant SfpTP901-1::Ter. (E) Tails-only produced for mutant MHPTP901-1::Ter. (F) Mature-resembling phages produced for mutant MCP3TP901-1::Ter. (G) Unconnected capsids and tails observed in HTC1TP901-1::Ter. (H) Distinct capsids and tails observed in HTC2TP901-1::Ter. (I) Separated capsids and tails imaged from mutant TapTP901-1::Ter. (J) Capsids with long polytails isolated for mutant TtpTP901-1::Ter. (K) Capsids only are observed for mutant MTPTP901-1::Ter. (L) Capsids and insoluble aggregates, proposed to be the TMP, observed in preparations of mutant gpGTP901-1::Ter. (M) Capsids, but no tails, observed in preparations of mutant gpTTP901-1::BamHI. (N) Mature-resembling phage produced by mutant gpGfsTTP901-1. (O) Control TP901-1erm mature infectious phages. Refer to text for a more detailed description characterizing each mutant. Scale bars are 50 nm long.

Mentions: In order to further assess the functionality of the mutant derivatives of TP901-1, and to ascertain if negative polar downstream effects were the cause of loss/reduction of infectivity of these mutants, a dual approach of Western blotting (employing antibodies raised against capsid and tail-associated proteins) and electron microscopic (EM) analysis of sucrose or CsCl gradient purified particles was applied (Figs 2 and 3, respectively). EM analysis of purified phage lysates of TP901-1erm mutants was performed to visualize structural assemblages. For each phage mutant, approximately 50 particles were observed to establish the reproducibility of the analysis. Through these observations, complete phage particles or separated head (DNA-filled or empty) and tail structures were discerned, depending on the mutant.


Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis.

Stockdale SR, Collins B, Spinelli S, Douillard FP, Mahony J, Cambillau C, van Sinderen D - PLoS ONE (2015)

EM images of (A-N) TP901-1erm structural module mutants and (O) TP901-1erm control.(A) Few tails, but no capsids, produced by mutant PortalTP901-1::Ter. (B) Capsids, without attached tails, visualised for mutant MCP1TP901-1::Ter. (C) Full, but scarce, phage particles produced following mutation MCP2TP901-1::Ter. (D) Tails, but no capsids, produced for mutant SfpTP901-1::Ter. (E) Tails-only produced for mutant MHPTP901-1::Ter. (F) Mature-resembling phages produced for mutant MCP3TP901-1::Ter. (G) Unconnected capsids and tails observed in HTC1TP901-1::Ter. (H) Distinct capsids and tails observed in HTC2TP901-1::Ter. (I) Separated capsids and tails imaged from mutant TapTP901-1::Ter. (J) Capsids with long polytails isolated for mutant TtpTP901-1::Ter. (K) Capsids only are observed for mutant MTPTP901-1::Ter. (L) Capsids and insoluble aggregates, proposed to be the TMP, observed in preparations of mutant gpGTP901-1::Ter. (M) Capsids, but no tails, observed in preparations of mutant gpTTP901-1::BamHI. (N) Mature-resembling phage produced by mutant gpGfsTTP901-1. (O) Control TP901-1erm mature infectious phages. Refer to text for a more detailed description characterizing each mutant. Scale bars are 50 nm long.
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pone.0131676.g003: EM images of (A-N) TP901-1erm structural module mutants and (O) TP901-1erm control.(A) Few tails, but no capsids, produced by mutant PortalTP901-1::Ter. (B) Capsids, without attached tails, visualised for mutant MCP1TP901-1::Ter. (C) Full, but scarce, phage particles produced following mutation MCP2TP901-1::Ter. (D) Tails, but no capsids, produced for mutant SfpTP901-1::Ter. (E) Tails-only produced for mutant MHPTP901-1::Ter. (F) Mature-resembling phages produced for mutant MCP3TP901-1::Ter. (G) Unconnected capsids and tails observed in HTC1TP901-1::Ter. (H) Distinct capsids and tails observed in HTC2TP901-1::Ter. (I) Separated capsids and tails imaged from mutant TapTP901-1::Ter. (J) Capsids with long polytails isolated for mutant TtpTP901-1::Ter. (K) Capsids only are observed for mutant MTPTP901-1::Ter. (L) Capsids and insoluble aggregates, proposed to be the TMP, observed in preparations of mutant gpGTP901-1::Ter. (M) Capsids, but no tails, observed in preparations of mutant gpTTP901-1::BamHI. (N) Mature-resembling phage produced by mutant gpGfsTTP901-1. (O) Control TP901-1erm mature infectious phages. Refer to text for a more detailed description characterizing each mutant. Scale bars are 50 nm long.
Mentions: In order to further assess the functionality of the mutant derivatives of TP901-1, and to ascertain if negative polar downstream effects were the cause of loss/reduction of infectivity of these mutants, a dual approach of Western blotting (employing antibodies raised against capsid and tail-associated proteins) and electron microscopic (EM) analysis of sucrose or CsCl gradient purified particles was applied (Figs 2 and 3, respectively). EM analysis of purified phage lysates of TP901-1erm mutants was performed to visualize structural assemblages. For each phage mutant, approximately 50 particles were observed to establish the reproducibility of the analysis. Through these observations, complete phage particles or separated head (DNA-filled or empty) and tail structures were discerned, depending on the mutant.

Bottom Line: Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled.Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production.Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork, Western Road, Cork, Ireland.

ABSTRACT
Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

No MeSH data available.


Related in: MedlinePlus