Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae

• Published in: BMC genomics Vol. 13; no. 1; p. 632
• Main Authors: Maier, Christina J, Maier, Richard H, Virok, Dezso Peter, Maass, Matthias, Hintner, Helmut, Bauer, Johann W, Önder, Kamil
• Format: Journal Article
• Language: English
• Published: London BioMed Central 16.11.2012; BioMed Central Ltd; BMC
• Subjects: Animal Genetics and Genomics; Bacterial genetics; Bacterial pneumonia; Biomedical and Life Sciences; Bronchitis; Chlamydia; Chlamydia pneumoniae; Chlamydophila pneumoniae; Chlamydophila pneumoniae - genetics; Chlamydophila pneumoniae - pathogenicity; Cloning, Molecular; Construction; Deoxyribonucleic acid; DNA; DNA sequencing; DNA, Bacterial; Drug development; E coli; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Gene Library; Genes; Genetic aspects; Genetic research; Genome, Bacterial; Genomes; Genomics; Health aspects; Humans; Infection; Infectious diseases; Life Sciences; Microarrays; Microbial Genetics and Genomics; Microbiology; Nucleotide sequencing; Omics; Open reading frames; Open Reading Frames - genetics; ORFeome; Pathogen; Pathogens; Physiological aspects; Plant Genetics and Genomics; Plasmids; Pneumonia; Prokaryote microbial genomics; Protein interaction; Proteins; Proteomics; Recombinant proteins; Recombinant Proteins - genetics; Research Article; Sequence Analysis, DNA; Sexually transmitted diseases; STD; Systems biology; Two-Hybrid System Techniques; Vectors; Virulence; virulence factors; Virulence Factors - genetics; Yeasts; Hungary; Austria; Chlamydia pneumoniae; Infectious diseases; Pathogen; Systems biology; ORFeome; Omics
• ISSN: 1471-2164; 1471-2164
• DOI: 10.1186/1471-2164-13-632

Background The Gram-negative bacterium Chlamydia pneumoniae (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. Results Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn ( Chlamydia pneumoniae 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 Escherichia coli 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 E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion 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.