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

Abstract

BackgroundThe 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.ResultsHere 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.ConclusionThis 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.

Generating genomic platforms to studyCandida albicanspathogenesis

“…Sanger sequencing of both 5' and 3' ends was performed to confirm ORF identity and exclude clones containing primer or recombination errors. Previous studies have reported mutation rates in primers of 3-10% (26,29). Similar rates were observed in this work.…”

“…Large-scale cloning projects, with the goal of cloning all predicted ORFs into flexible recombinational vectors, have been described for several model organisms, including Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli K-12, Arabidopsis thaliana, Xenopus laevis and Drosophila melanogaster, as well as infectious microorganisms such as Brucella melitensis, Plasmodium falciparum, Helicobacter pylori, Chlamydia pneumonia, Staphylococcus aureus and viruses (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

The Xenopus ORFeome: A resource that enables functional genomics

“…ORFeomes using the Gateway recombination cloning system have previously been constructed for a range of species, including: human ( Lamesch et al, 2007 , Rual et al, 2004 , Yang et al, 2011 ), Caenorhabditis elegans ( Reboul et al, 2003 ), Saccharomyces cerevisiae ( Gelperin et al, 2005 ), Brucella melitensis ( Dricot et al, 2004 ), Chlamydophila pneumoniae ( Maier et al, 2012 ), Staphylococcus aureus ( Brandner et al, 2008 ), Escherichia coli ( Rajagopala et al, 2010 ), as well as being partially available for Drosophila melanogaster ( Bischof et al, 2013 ), and mouse ( Temple et al, 2009 ). See Table 1 for a summary of methods and coverage.…”

Generating genomic platforms to study Candida albicans pathogenesis

“…Sanger sequencing of both 5′ and 3′ ends was performed to confirm ORF identity and exclude clones containing primer or recombination errors. Previous studies have reported mutation rates in primers of 3–10% ( 27 , 30 ). Similar rates were observed in this work.…”