Date of Graduation

1996

Document Type

Dissertation/Thesis

Abstract

A 5.8-kb EcoRI chromosomal fragment from Coxiella burnetii functions to initiate plasmid replication in Escherichia coli from a 403-bp minimal autonomous replication sequence (ars) (Chen, 1989). The minimal ars contains motifs found in bacterial origins, including two DnaA boxes, three AT-rich 21-mers, a transcriptional promoter leading rightwards, and potential IHF and FIS binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames adjacent to ars shared identity to genes often located near bacterial chromosomal origins. These were rpmH, rnpA, 9K, and 60K, in E. coli and Pseudomonas putida. To provide direct evidence for origin function in C. burnetii ars, replicative intermediates were examined by two dimensional gel agarose electrophoresis. However, replication initiations in chromosomal ars were not detected. Since gene transfer into C. burnetii is not available, the ability of plasmids containing ars to genetically transform C. burnetti was examined. Plasmid pSKO(+)1000 stably transformed C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 which contained the C. burnetii ars cloned into a ColE1-type replicon coding for {dollar}\\beta{dollar}-lactamase, was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected and survivors were examined for the transformed genotype by Southern blot hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination, via a single site crossover event. This resulted in the insertion of pSKO(+)1000 and duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located as free autonomous plasmid. An ampicillin resistance plasmid lacking the C. burnetii ars did not stably transform C. burnetii. A biological assay showed {dollar}\\beta{dollar}-lactamase protein in transformed C. burnetii that was not present in wild type cells. Futhermore, expression of bla was confirmed by the detection of the {dollar}\\beta{dollar}-lactamase protein in transformed C. burnetii by Western blot analysis. These results demonstrated for the first time genetic transformation of an obligate intracellular bacterium.

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