Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it>
<p>Abstract</p> <p>Background</p> <p>The Gram-negative bacterium <it>Chlamydia pneumoniae</it> (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen b...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2012-11-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1471-2164/13/632 |
| _version_ | 1818808241110384640 |
|---|---|
| author | Maier Christina J Maier Richard H Virok Dezso Peter Maass Matthias Hintner Helmut Bauer Johann W Önder Kamil |
| author_facet | Maier Christina J Maier Richard H Virok Dezso Peter Maass Matthias Hintner Helmut Bauer Johann W Önder Kamil |
| author_sort | Maier Christina J |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The Gram-negative bacterium <it>Chlamydia pneumoniae</it> (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis.</p> <p>Results</p> <p>Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (<it>Chlamydia pneumoniae</it> strain CWL029) in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in <it>Escherichia coli</it> and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of <it>E. coli</it> harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates.</p> <p>Conclusion</p> <p>This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.</p> |
| first_indexed | 2024-12-18T19:38:25Z |
| format | Article |
| id | doaj.art-2c3a6540c65443ba909c10972e9288f1 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-18T19:38:25Z |
| publishDate | 2012-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-2c3a6540c65443ba909c10972e9288f12022-12-21T20:55:31ZengBMCBMC Genomics1471-21642012-11-0113163210.1186/1471-2164-13-632Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it>Maier Christina JMaier Richard HVirok Dezso PeterMaass MatthiasHintner HelmutBauer Johann WÖnder Kamil<p>Abstract</p> <p>Background</p> <p>The Gram-negative bacterium <it>Chlamydia pneumoniae</it> (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis.</p> <p>Results</p> <p>Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (<it>Chlamydia pneumoniae</it> strain CWL029) in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage) and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in <it>Escherichia coli</it> and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of <it>E. coli</it> harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates.</p> <p>Conclusion</p> <p>This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.</p>http://www.biomedcentral.com/1471-2164/13/632ORFeomeChlamydia pneumoniaeOmicsPathogenSystems biologyInfectious diseases |
| spellingShingle | Maier Christina J Maier Richard H Virok Dezso Peter Maass Matthias Hintner Helmut Bauer Johann W Önder Kamil Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it> BMC Genomics ORFeome Chlamydia pneumoniae Omics Pathogen Systems biology Infectious diseases |
| title | Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it> |
| title_full | Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it> |
| title_fullStr | Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it> |
| title_full_unstemmed | Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it> |
| title_short | Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen <it>Chlamydia pneumoniae</it> |
| title_sort | construction of a highly flexible and comprehensive gene collection representing the orfeome of the human pathogen it chlamydia pneumoniae it |
| topic | ORFeome Chlamydia pneumoniae Omics Pathogen Systems biology Infectious diseases |
| url | http://www.biomedcentral.com/1471-2164/13/632 |
| work_keys_str_mv | AT maierchristinaj constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit AT maierrichardh constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit AT virokdezsopeter constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit AT maassmatthias constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit AT hintnerhelmut constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit AT bauerjohannw constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit AT onderkamil constructionofahighlyflexibleandcomprehensivegenecollectionrepresentingtheorfeomeofthehumanpathogenitchlamydiapneumoniaeit |