Dehio M, Knorre A, Lanz C, Dehio C
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Spemannstrasse 34, D-72076, Tübingen, Germany.
Gene. 1998 Jul 30;215(2):223-9. doi: 10.1016/s0378-1119(98)00319-9.
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.
基于可移动的广宿主范围（IncQ）质粒pMMB206构建了适合在汉赛巴尔通体中指导高水平蛋白质合成的表达载体。它们赋予卡那霉素抗性，并在多克隆位点前具有taclac（串联的tac-lacUV5）启动子，随后是rrnB转录终止点。虽然在一个载体中与taclac启动子融合的基因的表达是组成型的，但另一个载体携带的lacIq基因允许可控的、IPTG诱导的基因表达。通过将编码来自维多利亚水母绿色荧光蛋白（GFP）的突变gfp基因亚克隆到多克隆位点，并将所得质粒导入大肠杆菌和汉赛巴尔通体中来测试这些载体。通过流式细胞术测量荧光或直接通过免疫印迹来确定GFP表达。与大肠杆菌相比，汉赛巴尔通体中GFP的表达受lacIq的控制更严格，并且导致IPTG诱导型和组成型基因表达水平都高得多。内皮细胞的体外感染表明，GFP表达不会对汉赛巴尔通体与宿主细胞的相互作用产生不利影响。这些数据表明：（i）已建立的表达载体可用于在汉赛巴尔通体中指导可控的或组成型的高水平蛋白质合成；（ii）GFP是一种有价值的表达标记物，可能在研究这种新出现的人类病原体的细菌遗传学和细胞相互作用方面具有重要应用。
