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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

Immunological detection of TP901-1erm structural proteins precipitated from phage lysates by PEG8000.Negative control represents PEG8000 precipitation of mitomycin C induced strain NZ9000-Crot712. The protein detected by the primary antibody is depicted at the right hand side of the image, with black-filled triangles highlighting the TP901-1 protein sizes and a black-unfilled triangle indicating the MCP1Tuc2009 protein in the Tuc2009 control lane that is ~25 kDa smaller than MCP1TP901-1. Western blots against MCP1TP901-1 resulted in two non-specific bands, at approximately 50 and 40 kDa, that could not be removed during optimisation.
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pone.0131676.g002: Immunological detection of TP901-1erm structural proteins precipitated from phage lysates by PEG8000.Negative control represents PEG8000 precipitation of mitomycin C induced strain NZ9000-Crot712. The protein detected by the primary antibody is depicted at the right hand side of the image, with black-filled triangles highlighting the TP901-1 protein sizes and a black-unfilled triangle indicating the MCP1Tuc2009 protein in the Tuc2009 control lane that is ~25 kDa smaller than MCP1TP901-1. Western blots against MCP1TP901-1 resulted in two non-specific bands, at approximately 50 and 40 kDa, that could not be removed during optimisation.

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)

Immunological detection of TP901-1erm structural proteins precipitated from phage lysates by PEG8000.Negative control represents PEG8000 precipitation of mitomycin C induced strain NZ9000-Crot712. The protein detected by the primary antibody is depicted at the right hand side of the image, with black-filled triangles highlighting the TP901-1 protein sizes and a black-unfilled triangle indicating the MCP1Tuc2009 protein in the Tuc2009 control lane that is ~25 kDa smaller than MCP1TP901-1. Western blots against MCP1TP901-1 resulted in two non-specific bands, at approximately 50 and 40 kDa, that could not be removed during optimisation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493119&req=5

pone.0131676.g002: Immunological detection of TP901-1erm structural proteins precipitated from phage lysates by PEG8000.Negative control represents PEG8000 precipitation of mitomycin C induced strain NZ9000-Crot712. The protein detected by the primary antibody is depicted at the right hand side of the image, with black-filled triangles highlighting the TP901-1 protein sizes and a black-unfilled triangle indicating the MCP1Tuc2009 protein in the Tuc2009 control lane that is ~25 kDa smaller than MCP1TP901-1. Western blots against MCP1TP901-1 resulted in two non-specific bands, at approximately 50 and 40 kDa, that could not be removed during optimisation.
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