The development of a genetic exchange system for Coxiella burnetii

Abstract

C. burnetii is a gram-negative bacterium placed in the family rickettsiaceae. It is the etiological agent of Q (query) fever in humans. C. burnetii is an obligate intracellular pathogen that is able to gain entry into and replicate within the host cell phagolysosome. How the bacterium is able to adapt to this harsh, acidic environment is not well understood, and is the focus of our laboratory. Many of the methods used for the elucidation of genetic information about organisms, including homologous recombination, transposon mutagenesis, and the use of reporter expression systems have not been feasible for intracellular bacteria because of the lack of genetic exchange techniques. Because of these limitations, researchers have used E. coli as a surrogate host for gene expression to characterize C. burnetii proteins. The generation of efficient and effective methods to introduce DNA into C. burnetii would enable definition and characterization of various genetic factors, such as key virulence mechanisms, gene expression and regulation, and promoter function. First, we attempted to construct a shuttle vector using the putative origin of replication for the endogenous plasmid, QpH1. The second aspect of this work was to adapt the published methods of transformation and selection into our lab in order to test the applicability of this method. Third, to improve this transformation strategy, and to provide a more efficient shuttle vector, we tried to add a visual screenable marker to the plasmid, pSKO(+)1000, to facilitate early detection and selection of transformed organisms. The fourth component was to develop a practical application of the transformation system. A means of manipulating the genome of C. burnetii would be of major importance in the identification and characterization of genes necessary for virulence and for phagolysosomal survival. The successful creation of this system proffers a potentially revolutionizing genetic tool for use with Coxiella burnetii, and perhaps, with other intracellular bacteria.