Cloning and functional expression of the Coxiella burnetii citrate synthase gene in Escherichia coli.

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RESUMO

The citrate synthase gene from the obligate intracellular rickettsial parasite Coxiella burnetii was cloned and expressed in Escherichia coli. Transduction into E. coli with a C. burnetii gene library constructed in the cosmid vector pHK17 resulted in the functional complementation of the gltA mutation of E. coli MOB154. A GltA+ clone carrying 16.4 kilobase pairs of C. burnetii DNA and designated pJCC959 was isolated and characterized. Southern hybridization analysis confirmed that the pJCC959 cloned insert consists of C. burnetii DNA and that homology exists with the Rickettsia prowazekii citrate synthase gene. Subcloning analysis with the multicopy expression vector pUC8 revealed that citrate synthase expression was under control of a C. burnetti promoter. In vitro transcription-translation of subclones pLPM20 and pLPM30 established a molecular weight of ca. 46,000 for the monomer form of the cloned enzyme. Transposon Tn5 mutagenesis of pLPM30 defined the coding region to approximately 1.2 kilobase pairs of C. burnetii DNA. Maxicell analysis of selected pLPM30::Tn5 insertion derivatives identified the direction of transcription and the relative translational start and stop sites and substantiated the molecular weight value calculated from the in vitro analysis. Inhibition studies showed that citrate synthase activity in crude cell extracts obtained from strain MOB154 transformed with the cloned C. burnetii gene was markedly inhibited by 4 mM ATP, while 4 mM alpha-ketoglutarate had virtually no effect. These data indicate that the C. burnetii enzyme displays regulatory behavior characteristic of the small gram-positive bacterial and eucaryotic enzyme.

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