RESEARCH ARTICLE Open AccessGenomic and phenotypic characterization ofin vitro-generated Chlamydia trachomatisrecombinantsBrendan M Jeffrey 1,2† , Robert J Suchland 3† , Steven G Eriksen 2 , Kelsi M Sandoz 1,2 and Daniel D Rockey 1,2*AbstractBackground: Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro andin vivo, although the molecular and cellular biology of this process is not well understood. In this study, wedetermined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitrounder antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp.,including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypeswith parental phenotypes and genotypes.Results: Each of the 190 examined recombination events was the product of homologous recombination, and nocandidate targeting motifs were identified at recombination sites. There was a single deletion event in onerecombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was noevidence for preference for any specific region of the chromosome for recombination, and analyses of a total ofover 200 individual recombination events do not provide any support for recombination hotspots in vitro. Twomeasurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in theabsence of centrifugation was examined, and this property segregated to regions of the chromosome that carrythe polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells variedamong recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome.Conclusions: These experiments examined the process of recombination in C. trachomatis and identified tools thatcan be used to associate phenotype with genotype in recombinant progeny. There were no data supporting thehypothesis that particular nucleotide sequences are preferentially used for recombination in vitro. Selectedphenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminaryanalysis of the relationship of genetic variation to phenotypic variation in the chlamydiae.Keywords: Chlamydia, Recombination, Hotspot, Attachment, Secondary inclusionsBackgroundChlamydia trachomatis is a Gram-negative obligateintracellular bacterium that is a leading cause of prevent-able blindness and sexually transmitted diseases world-wide [1]. Much of the biology of infection and diseaseremains unclear in this system, owing largely to the lackof a routine genetic system for these organisms. Whilemany aspects of these challenges have recently beenovercome [2,3], the use of genetic transformation in thissystem is just beginning to be exploited. One aspect ofchlamydial biology that is poorly understood involvesthe mechanism of lateral gene transfer among chlamy-dial strains both in the laboratory and, most likely, in pa-tients. Coinfection of host cells in vitro with chlamydialisolates encoding different drug resistance markers leadto generation of dual resistant recombinant progeny[4,5]. These results lend support to pre- and post-genomicanalyses demonstrating that chlamydiae recombine ininfected hosts [6-12], likely following infection of a patient* Correspondence: rockeyd@orst.edu† Equal contributors1 Molecular and Cellular Biology Program, Oregon State University, Corvallis,OR, USA2 Department of Biomedical Sciences, Oregon State University, Corvallis, OR,USAFull list of author information is available at the end of the article© 2013 Jeffrey et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Jeffrey et al. BMC Microbiology 2013, 13:142http://www.biomedcentral.com/1471-2180/13/142
