Expression of β-lactamase inCoxiella burnetiitransformants

Abstract

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid 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 beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-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 beta-lactamase by antibody blotting done to confirm transformants.

Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria

“…This plasmid appeared to both autonomously replicate and, in some instances, integrate into the chromosomal copy of the ARS by homologous recombination. Four years later, the presence of the bla gene product b-lactamase in transformed C. burnetii was further confirmed using immunoblot analysis (Suhan and Thompson, 2000). These seminal findings provided hope for future genetic modification of C. burnetii in showing that ampicillin could be used as a selectable marker and that homologous recombination was possible.…”

Advances in Genetic Manipulation of Obligate Intracellular Bacterial Pathogens

“…A commonly used method is microscopic visualization of green or red fluorescent proteins encoded by introduced transgenes (Lukacova et al, 1999; Troyer et al, 1999; Renesto et al, 2002; Baldridge et al, 2005, 2010b; Felsheim et al, 2006, 2010; Liu et al, 2007; Beare et al, 2009; Chen et al, 2010). RT-PCR, northern blotting and immunoblotting have been employed by several investigators to confirm expression of fluorescent and antibiotic resistance proteins (Suhan and Thompson, 2000; Baldridge et al, 2005, 2010b; Felsheim et al, 2010). …”