Ronchel M C, Ramos J L
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain.
Appl Environ Microbiol. 2001 Jun;67(6):2649-56. doi: 10.1128/AEM.67.6.2649-2656.2001.
Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Deltaasd P. putida background. A P. putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a P. putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the P. putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P. putida CMC4 was still detectable in soils after 100 days.
活性生物遏制（ABC）系统的设计目的是随意控制细菌群体的存活或死亡。这些系统基于一种杀伤基因的使用，例如一种孔蛋白诱导蛋白，如大肠杆菌gef基因编码的蛋白，以及一个调节回路，该回路根据环境信号的存在与否来控制杀伤基因的表达。基于恶臭假单胞菌TOL质粒间位裂解调节回路，设计了一种用于降解模型污染物的重组微生物ABC系统。该系统由Pm启动子与lacI的融合体组成，其表达由3 - 甲基苯甲酸通过XylS控制，以及合成的P（lac）启动子与gef的融合体。在存在模型污染物的情况下，细菌细胞存活并降解目标化合物，而在没有芳香羧酸的情况下则诱导细胞死亡。该系统有两个主要缺点：（i）在土壤中细菌细胞死亡缓慢，而在实验室测定的液体培养物中杀伤速率较快；（ii）出现突变体，其发生率约为每细胞和每代10^(-8)，在污染物耗尽后不会死亡。我们通过将其置于恶臭假单胞菌Δasd背景中来强化ABC系统。恶臭假单胞菌Δasd突变体仅在补充了二氨基庚二酸、蛋氨酸、赖氨酸和苏氨酸的复杂培养基中才能存活。我们构建了一种恶臭假单胞菌Δasd菌株MCR7，其宿主染色体中有Pm::asd融合体。该菌株在3 - 甲基苯甲酸存在下能够存活，因为必需代谢物的合成是通过XylS依赖性诱导实现的。在恶臭假单胞菌MCR7菌株中，将ABC系统（Pm::lacI，xylS，P（lac）::gef）整合到宿主染色体中，得到菌株MCR8。逃避杀伤的MCR8突变体数量低于我们的检测限（每细胞和每代<10^(-9)个突变体）。MCR8菌株在含有3 - 甲基苯甲酸的根际土壤中存活并定殖，其水平与野生型菌株相似。然而，在没有污染物的土壤中，它在不到20至25天内就消失了，而一个asd(+)、具有生物遏制作用的对应菌株，如恶臭假单胞菌CMC4，在100天后在土壤中仍可检测到。
