Cebolla A, Vázquez M E, Palomares A J
Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, Spain.
Appl Environ Microbiol. 1995 Feb;61(2):660-8. doi: 10.1128/aem.61.2.660-668.1995.
An easy way to identify microorganisms is to provide them with gene markers that confer a unique phenotype. Several genetic constructions were developed to use eukaryotic luciferase genes for bacterial tagging. The firefly and click bettle luciferase genes, luc and lucOR, respectively, were cloned under constitutive control and regulated control from different transcriptional units driven by P1, lambda PR, and Ptrc promoters. Comparison of the expression of each gene in Escherichia coli cells from identical promoters showed that bioluminescence produced by luc could be detected luminometrically in a more sensitive manner. In contrast, luminescence from intact lucOR-expressing cells was much more stable and resistant to high temperatures than that from luc-expressing cells. To analyze the behavior of these constructions in other gram-negative bacteria, gene fusions with luc genes were cloned on broad-host-range vectors. Measurements of light emission from Rhizobium meliloti, Agrobacterium tumefaciens, and Pseudomonas putida cells indicated that both luciferases were poorly expressed from P1 in most bacterial hosts. In contrast, the lambda promoter PR yielded constitutively high levels of luciferase expression in all bacterial species tested. PR activity was not regulated by temperature when the thermosensitive repressor cI857 was present in the bacterial species tested, except for E. coli. In contrast, the regulated lacIq-Ptrc::lucOR fusion expression system behaved in a manner similar to that observed in E. coli cells. After IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, this system produced the highest levels of lucOR expression in all bacterial species tested.(ABSTRACT TRUNCATED AT 250 WORDS)
鉴定微生物的一种简单方法是为它们提供赋予独特表型的基因标记。已开发出几种基因构建体，用于将真核荧光素酶基因用于细菌标记。萤火虫荧光素酶基因和叩头虫荧光素酶基因，分别为luc和lucOR，在由P1、λPR和Ptrc启动子驱动的不同转录单元的组成型控制和调控控制下进行克隆。比较相同启动子在大肠杆菌细胞中每个基因的表达，结果表明，luc产生的生物发光可以通过发光测定法以更灵敏的方式检测到。相比之下，表达完整lucOR的细胞发出的光比表达luc的细胞发出的光更稳定，且耐高温。为了分析这些构建体在其他革兰氏阴性菌中的行为，将与luc基因的基因融合体克隆到广宿主范围载体上。对苜蓿根瘤菌、根癌土壤杆菌和恶臭假单胞菌细胞发光的测量表明，在大多数细菌宿主中，两种荧光素酶在P1启动子下的表达都很差。相比之下，λ启动子PR在所有测试的细菌物种中都能持续产生高水平的荧光素酶表达。当在所测试的细菌物种中存在热敏阻遏物cI857时，除了大肠杆菌外，PR活性不受温度调节。相比之下，受调控的lacIq-Ptrc::lucOR融合表达系统的行为与在大肠杆菌细胞中观察到的相似。在异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，该系统在所有测试的细菌物种中产生了最高水平的lucOR表达。（摘要截短于250字）
