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首页> 外文期刊>Applied and Environmental Microbiology >Development of a Gene Cloning and Inactivation System for Halorespiring Desulfitobacterium dehalogenans
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Development of a Gene Cloning and Inactivation System for Halorespiring Desulfitobacterium dehalogenans

机译：卤吸脱硫脱硫杆菌基因克隆和灭活系统的开发

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Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 103 to 104transformants per μg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG+host9 was shown to replicate at a permissive temperature of 30°C, whereas the replicon was not functional at 40°C. The D. dehalogenans frdCAB operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG+host9 carrying a 0.6-kb internal frdA fragment. Single-crossover integration at the frdA locus occurred at a frequency of 3.3 × 10?4 per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in D. dehalogenans and allow for its further exploitation as a dedicated degrader.
机译：已开发出用于多功能，严格厌氧，可吸入卤素和富马酸的革兰氏阳性细菌 Desulfitobacterium dehalogenans 的高效宿主载体系统。开发并验证了基于电穿孔的转化程序，该程序可产生每μg克隆载体pIL253约10 3 至10 4 转化子。宽宿主范围的载体pG + host9被显示在30°C的允许温度下复制，而复制子在40°C时不起作用。 D。克隆，表征和预测了预测编码富马酸还原酶的脱卤素人frdCAB 操纵子，并通过携带0.6kb内部 frdA 的pG + host9使其插入失活分段。在 frdA 位点的单交换整合发生频率为每个细胞3.3×10 ？4 ，导致富马酸酯还原酶活性部分受损。本文所述的基因克隆和失活系统为进一步阐明 D中的呼吸道网络提供了坚实的基础。 dehalogenans ，并允许其进一步开发用作专门的降解剂。

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