ACTIVITY OF MYCOBACTERIAL PROMOTERS DURING INTRACELLULAR AND EXTRACELLULAR GROWTH
ACTIVITY OF MYCOBACTERIAL PROMOTERS DURING INTRACELLULAR AND EXTRACELLULAR GROWTH
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DOI：
10.1099/13500872-141-8-1785
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发表时间：
1995-08-01
期刊：
影响因子：
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通讯作者：
MCFADDEN, J
中科院分区：
文献类型：
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作者：
DELLAGOSTIN, OA;ESPOSITO, G;MCFADDEN, J
pUS933是一种双功能分枝杆菌-大肠杆菌翻译融合载体，含有氨基末端截短的大肠杆菌lacZ报告基因。构建含有牛分枝杆菌BCG hsp60基因、麻风分枝杆菌28kDa基因和麻风分枝杆菌18kDa基因的启动子、RBS和起始密码子的pUS933衍生物，并将其引入大肠杆菌、耻垢分枝杆菌和牛分枝杆菌BCG中。测量液体培养物中生长的分枝杆菌的β-半乳糖苷酶活性。引物延伸分析用于确定耻垢分枝杆菌中 18 kDa 启动子的转录起始点。用含有 pUS933 衍生物的重组 BCC 感染鼠巨噬细胞，并通过荧光显微镜和荧光测定法检查 BCG 细胞内生长期间的表达水平。 BCG hsp60 基因启动子和麻风分枝杆菌 28 kDa 基因启动子在所有检查的情况下均产生高水平的 β-半乳糖苷酶表达。相比之下，麻风分枝杆菌 18 kDa 启动子片段在耻垢分枝杆菌和液体培养物中生长的 BCG 中表达水平非常低，但在巨噬细胞内生长的 BCG 中，它被诱导至几乎与其他启动子一样高的水平。这表明18 kDa基因在细胞内生长期间被特异性激活，因此可能参与麻风分枝杆菌在巨噬细胞内的存活。这种调节模式可能有助于控制重组 BCG 菌株中外源基因的表达。
pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCC containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.