Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria
Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria
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DOI：
10.1099/00221287-147-8-2065
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发表时间：
2001-08-01
期刊：
影响因子：
2.8
通讯作者：
Lidstrom, ME
中科院分区：
文献类型：
--
作者：
Marx, CJ;Lidstrom, ME
充分利用细菌基因组序列中可用的信息需要方便的工具来快速进行基因操作。一种需要新的遗传工具的细菌是甲基化菌AM1。IncQ和小IncP载体被证明不适合在这种细菌中使用，但一个小IncP质粒的自发突变体被分离出来，在敲诈氏芽孢杆菌AMI中有效地起作用。对该质粒进行了测序，并作为开发改良的广宿主克隆载体的基础。这些载体被发现可以在多种细菌中复制，并具有以下优点：(1)在大肠杆菌中拷贝数高；(2)小尺寸（7.2和8.0 kb）；(3)完全序列；(4)多种独特的限制性位点；(5) lacZ α筛选蓝白；(6)菌种间的共轭动员；(7)易于适应于物种特异性启动子探针和表达载体。以lacZ和xylE为报告基因的克隆载体构建了两个低背景启动子探针载体；这些基因在m.o resquens AM1中有效表达。我们开发了用于敲诈勒索芽孢杆菌AM1的特异性表达载体，这些芽孢杆菌可以大量表达外源基因，并概述了一种用于其他细菌的特异性表达载体的简单策略。强mxaF启动子被用于表达，因为大肠杆菌lacc衍生的启动子表达水平很低。这一套遗传工具将能够对M.敲诈勒索AM1的生理进行更复杂的分析，这些载体也应该是研究各种细菌物种的有价值的工具。
Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AMI. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue-white screening via lacZ alpha; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.