Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker.

Abstract

Expression vectors suitable for directing high levels of protein synthesis in Bartonella henselae have been constructed based on the mobilisable broad-host-range (IncQ) plasmidpMMB206. They confer kanamycin resistance and feature the taclac (tac-lacUV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. While expression of genes fused to the taclac promoter is constitutive in one vector, the lacIq gene carried by the other vector allows a controlled, IPTG-inducible gene expression. These vectors were tested by subcloning a mutated gfp gene coding for the green fluorescent protein (GFP) from Aequorea victoria into the multiple cloning site and introducing the resulting plasmids into Escherichia coli and B. henselae. GFP expression was determined by measuring fluorescence via flow cytometry or directly by immunoblotting. Compared to E. coli, expression of GFP in B. henselae was more tightly controlled by lacIq and resulted in much higher levels of both IPTG-induced and constitutive gene expression. In vitro infection of endothelial cells indicated that GFP expression does not adversely affect the interaction of B. henselae with host cells. These data demonstrate that (i) the established expression vectors are useful for directing controlled or constitutive high-level protein synthesis in B. henselae and (ii) that GFP is a valuable expression marker which may has important applications in studying the bacterial genetics and cellular interactions of this emerging human pathogen.