Barker L P, Brooks D M, Small P L
Microscopy Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA.
Mol Microbiol. 1998 Sep;29(5):1167-77. doi: 10.1046/j.1365-2958.1998.00996.x.
Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.
海分枝杆菌与结核分枝杆菌一样,是一种生长缓慢的致病性分枝杆菌,能够在巨噬细胞中存活和复制。利用启动子捕获载体pFPV27,我们构建了一个海分枝杆菌基因组DNA的200 - 1000 bp片段文库,这些片段插入到无启动子的绿色荧光蛋白(GFP)基因上游。只有那些含有活性启动子的质粒才能表达GFP。用这个融合文库感染巨噬细胞,并分离出含有荧光细菌的吞噬体。用荧光激活细胞分选仪分离出细胞内活性更高的启动子构建体,并对插入片段进行部分测序。在细胞内表达的启动子融合体与编码膜蛋白和生物合成酶等的分枝杆菌基因具有同源性。GFP在细胞内的表达是在培养基中生长的相同克隆的2 - 20倍。将几个启动子构建体转化到耻垢分枝杆菌、牛分枝杆菌卡介苗和结核分枝杆菌中。在所有测试的分枝杆菌菌株中,这些构建体的GFP表达均为阳性。分选吞噬体中的荧光细菌避免了从可能含有混合细菌群体的巨噬细胞中分离单个克隆的问题。这种方法使我们能够分离出12个含有在巨噬细胞中差异表达的启动子构建体的海分枝杆菌克隆。