Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154
Thesis (PhD)--University of Pretoria, 2010.
Saved in:
| Other Authors: | |
|---|---|
| Format: | Thesis |
| Published: |
University of Pretoria
2013
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613452763136000 |
|---|---|
| access_status_str | Open Access |
| author2 | Louw, Maureen E. |
| author_browse | Louw, Maureen E. |
| author_facet | Louw, Maureen E. |
| collection | Thesis |
| dc_rights_str_mv | © 2008 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD)--University of Pretoria, 2010. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/29540 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:36:22.807Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/29540 Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 Louw, Maureen E. Theron, Jacques upetd@up.ac.za Du Plessis, Erika Margarete Acid phosphatase Staphylococcus aureus Prokaryotic cells UCTD Thesis (PhD)--University of Pretoria, 2010. Reporter gene technology has facilitated greatly the analysis of gene expression and the study of individual promoters and their regulation. Although various reporter gene systems are available, none of them are universally applicable and consequently, studies aimed at screening of new reporters are continuing. Toward this end, an acid phosphatase, designated SapS, was identified and characterized from the culture supernatant of a Staphylococcus aureus strain isolated from vegetables. Biochemical characterization of the 30-kDa monomeric enzyme indicated that it displayed optimum activity at 40°C and pH 5, using p-nitrophenyl phosphate (pNPP) as substrate. The enzymatic activity was enhanced by Mg2+, but was inhibited by EDTA and molybdate. Based on its properties and amino acid sequence analyses, SapS was classified as a new member of the bacterial class C family of non-specific acid phosphatases. The S. aureus SapS enzyme was subsequently evaluated as a reporter for host strain evaluation and cell surface display. Bacillus halodurans of which the major cell wall protease gene (wprA) was inactivated was used as expression host, and the cell wall-binding domain of the cwlC gene from B. halodurans was used as an anchoring motif for cell surface display. The results from in vitro enzyme activity assays indicated that extracellular production of the SapS reporter enzyme was improved 3.5-fold in the mutant compared to wild-type B. halodurans strain. Zymographic detection of SapS activity showed that the SapS-CwlC fusion protein was localized in the B. halodurans cell wall fraction, thus demonstrating the potential of SapS as a reporter for cell surface display of heterologous proteins. The versatility of the SapS enzyme as a reporter for gene expression and protein secretion in both Gram-positive and Gram-negative bacteria was also investigated. Transcriptional and translational fusions of the sapS gene with selected heterologous promoters and signal sequences were constructed, and expressed in Escherichia coli, B. subtilis and B. halodurans. The strongest promoter for heterologous protein production in each of the host strains was identified, i.e. the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis, and the B. halodurans σD promoter in B. halodurans. Microbiology and Plant Pathology unrestricted 2013-09-07T15:53:10Z 2008-12-17 2013-09-07T15:53:10Z 2008-09-03 2010-12-17 2008-11-18 Thesis Du Plessis, EM 2008, Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylocuccus aureus strain 154, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/29540 > D556/ag http://hdl.handle.net/2263/29540 http://upetd.up.ac.za/thesis/available/etd-11182008-090253/ © 2008 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Acid phosphatase Staphylococcus aureus Prokaryotic cells UCTD Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 |
| title | Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 |
| title_full | Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 |
| title_fullStr | Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 |
| title_full_unstemmed | Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 |
| title_short | Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154 |
| title_sort | development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from staphylococcus aureus strain 154 |
| topic | Acid phosphatase Staphylococcus aureus Prokaryotic cells UCTD |
| url | http://hdl.handle.net/2263/29540 http://upetd.up.ac.za/thesis/available/etd-11182008-090253/ |