Expression of β-lactamase in Coxiella burnetii transformants

Abstract

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding L-lactamase. However, nonplasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for L-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the L-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of L-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of L-lactamase by antibody blotting done to confirm transformants. ß

Constitutive SOS expression and damage‐inducible AddAB‐mediated recombinational repair systems for Coxiella burnetii as potential adaptations for survival within macrophages

“…Initial successful transformation experiments of C. burnetii were based on the identification of an autonomous replication sequence ( ars ), used as a plasmid replicon for a Coxiella ‐specific shuttle vector (Chen et al ., 1990; Suhan et al ., 1994). Genetic analyses of potentially transformed bacteria revealed both extra‐ and intrachromosomal states of the shuttle vector, but selection was hampered by the appearance of spontaneously resistant bacteria after long‐term treatment with ampicillin (Suhan et al ., 1996; Suhan and Thompson, 2000). These initial experiments indicated the potential for future genetic manipulation strategies and generation of specific mutants of C. burnetii by allelic exchange.…”

Targeting the tick-pathogen interface for novel control strategies

“…Transformation and tick infection with recombinant bacteria has recently been reported for Escherichia coli (52,53), B. burgdorferi (54)(55)(56)(57), B. afzelii (58), and R. monacensis (59). Methods have been developed for the production and detection of recombinant organisms in cultured cells for the tick-borne rickettsial pathogens, R. prowazekii (60)(61)(62), C. burnetii (63,64), A. phagocytophilum (65), Ehrlichia muris (66), and F. tularensis (67-69). Transformation of the protozoan parasite, B. bovis, with potential applications for other Babesia species, has also been reported (70).…”