Pitt Logo LinkContact Us

Genetic and Biochemical Investigation of Bxb1 gp47, An Unusual Recombination Directionality Factor

Savinov, Andrew (2011) Genetic and Biochemical Investigation of Bxb1 gp47, An Unusual Recombination Directionality Factor. Undergraduate Thesis, University of Pittsburgh.

[img] PDF - Primary Text
Restricted to University of Pittsburgh users only until 06 May 2016.

Download (3071Kb) | Request a copy

    Abstract

    The temperate mycobacteriophage Bxb1 infects and forms lysogens of Mycobacterium smegmatis, a fast-growing relative of and model for M. tuberculosis. In Bxb1, as in other bacteriophages, switching between the lysogenic cycle and lytic cycle depends on a site-specific DNA recombinase called an integrase. In Bxb1 the directionality of the DNA recombination process depends on the phage-encoded gp47 protein, which acts as the Recombination Directionality Factor (RDF). A number of lines of evidence suggest that Bxb1 gp47 has some additional biological role as well, however. First, very close homologues are found in 15 other mycobacteriophages, including phage L5, whose system for phage DNA integration / excision does not include the Bxb1 gp47 homologue. Second, Bxb1 gp47 and homologues are found clustered with genes for phage DNA replication.Here we present our investigation into the putative multi-functionality of Bxb1 gp47. To begin, we performed a bioinformatic analysis which predicts Bxb1 gp47 and its homologues to contain a calcineurin-like phosphoesterase domain; this domain is predicted to confer a metal-dependent phosphatase activity on these proteins. Further, we analyzed the phenotypic repercussions of altering the Bxb1 gp47 gene by site-specific phage mutagenesis, performed using Bacteriophage Recombineering by Electroporation of DNA (BRED). Results from this work were consistent with the hypothesis that Bxb1 gp47 has an essential function in the lytic-cycle replication of Bxb1, and also showed that gp47 RDF activity is separable from the lytic-cycle function inferred for gp47. Finally, a number of variants of Bxb1 gp47 protein were overexpressed and purified for studies of RDF and phosphatase activity. RDF activity assays suggested that Motif I of the calcineurin-like phosphoesterase domain has no role in RDF activity, but that Motif V may be involved in both RDF and phosphatase activities. The phosphatase activity assays we have performed provide support for the hypothesis that Bxb1 gp47 is a manganese-dependent phosphatase enzyme. If this result is confirmed, Bxb1 gp47 will be revealed as a highly novel RDF with a secondary phosphatase activity.


    Share

    Citation/Export:
    Social Networking:

    Details

    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairHatfull, Graham Fgfh@pitt.edu
    Committee MemberArndt, Karen Marndt@pitt.edu
    Committee MemberGrindley, Nigel D Fnigel.grindley@yale.edu
    Committee MemberHendrix, Roger Wrhx@pitt.edu
    Title: Genetic and Biochemical Investigation of Bxb1 gp47, An Unusual Recombination Directionality Factor
    Status: Unpublished
    Abstract: The temperate mycobacteriophage Bxb1 infects and forms lysogens of Mycobacterium smegmatis, a fast-growing relative of and model for M. tuberculosis. In Bxb1, as in other bacteriophages, switching between the lysogenic cycle and lytic cycle depends on a site-specific DNA recombinase called an integrase. In Bxb1 the directionality of the DNA recombination process depends on the phage-encoded gp47 protein, which acts as the Recombination Directionality Factor (RDF). A number of lines of evidence suggest that Bxb1 gp47 has some additional biological role as well, however. First, very close homologues are found in 15 other mycobacteriophages, including phage L5, whose system for phage DNA integration / excision does not include the Bxb1 gp47 homologue. Second, Bxb1 gp47 and homologues are found clustered with genes for phage DNA replication.Here we present our investigation into the putative multi-functionality of Bxb1 gp47. To begin, we performed a bioinformatic analysis which predicts Bxb1 gp47 and its homologues to contain a calcineurin-like phosphoesterase domain; this domain is predicted to confer a metal-dependent phosphatase activity on these proteins. Further, we analyzed the phenotypic repercussions of altering the Bxb1 gp47 gene by site-specific phage mutagenesis, performed using Bacteriophage Recombineering by Electroporation of DNA (BRED). Results from this work were consistent with the hypothesis that Bxb1 gp47 has an essential function in the lytic-cycle replication of Bxb1, and also showed that gp47 RDF activity is separable from the lytic-cycle function inferred for gp47. Finally, a number of variants of Bxb1 gp47 protein were overexpressed and purified for studies of RDF and phosphatase activity. RDF activity assays suggested that Motif I of the calcineurin-like phosphoesterase domain has no role in RDF activity, but that Motif V may be involved in both RDF and phosphatase activities. The phosphatase activity assays we have performed provide support for the hypothesis that Bxb1 gp47 is a manganese-dependent phosphatase enzyme. If this result is confirmed, Bxb1 gp47 will be revealed as a highly novel RDF with a secondary phosphatase activity.
    Date: 06 May 2011
    Date Type: Completion
    Defense Date: 04 April 2011
    Approval Date: 06 May 2011
    Submission Date: 21 April 2011
    Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Undergraduate Thesis
    Refereed: Yes
    Degree: BPhil - Bachelor of Philosophy
    URN: etd-04212011-223422
    Uncontrolled Keywords: Bxb1; calcineurin; DNA recombination; integrase; lysogeny; lytic cycle; mycobacteriophage; phosphatase; RDF; secondary function
    Schools and Programs: University Honors College
    Date Deposited: 10 Nov 2011 14:40
    Last Modified: 29 May 2012 16:22
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-04212011-223422/, etd-04212011-223422

    Actions (login required)

    View Item

    Document Downloads